Metalloproteins : Structural Aspects (Advances in Protein Chemistry; V. 42)

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ADVANCES IN PROTEIN CHEMISTRY Volume 42 Metalloproteins: Structural Aspects

This Page Intentionally Left Blank

ADVANCES IN PROTEIN CHEMISTRY EDITED BY C. 6. ANFINSEN

JOHN T. EDSALL

Department of Biology The Johns Hopkins University Baltimore, Maryland

Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts

FREDERIC M. RICHARDS

DAVID S. EISENBERG

Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut

Department of Chemistry and Biochemistry University of California, Los Angeles Los Angeles, California

VOLUME 42

MetaI Io proteins : St r uct u ral Aspects

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @

Copyright 0 1991 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. San Diego, California 92101 United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road. London NWI 7DX

Library of Congress Catalog Card Number:

ISBN

0-12-034242-1 (alk. paper)

PRIN'ED IN THE UNITED STATES OF AMERICA 9 1 9 2 9 3 9 4

9 8 7 6 5 4 3 2 1

44-8853

CONTENTS PREFACE

.

vii

Structural Aspects of

eta1 Liganding to Func.ional Groups in Proteins

JENNY

I. 11. 111. IV. V. VI. VII. VIII. IX.

X. XI. XII. XIII. XIV. XV. XVI.

P. GLUSKER

Metal-Binding Sites in Proteins . Polarizabilities of Metals and Ligands . Redox Behavior of Metal Ions . Number of Atoms Packed in First Coordination . Sphere around Metal Ion Metal-Ligand Bond Distances . Asymmetry in Ionic Shape . Strengths of Bonds from Metal Ions to Ligands . Selection of Metal Ions for Complexation . Metal Binding to Isolated Carboxylate Groups . Metal Binding to Imidazole Groups . Metal Binding to Sulfur-Containing Groups. . Metal Binding to Main-Chain Carbonyl Groups . Metal Binding to Two Groups . Metals in Protein Crystal Structures . Electron Transfer in Metalloproteins . Metal Competition and Replacement . References .

3 4 7 8

17 20 21 26 30 36 37 38 38 39 58 65 66

Calcium Binding Sites in Proteins: A Structural Perspective

CATHERINE A. MCPHALEN, NATALIE C. J. STRYNADKA, AND MICHAELN. G. JAMES I. Introduction . 11. Functional and Structural Overview of Protein Ca2+-Binding Sites . 111. Regularities and Recurrent Themes in Ca2+-Binding Sites . IV. Discussion and Summary . References . V

77 86 108 136 140

vi

CONTENTS

Copper Protein Structures

ELINOR T. ADMAN I. Introduction . 11. Cupredoxins: Proteins That Bind Only Type I Copper . 111. Proteins That Bind Only Type I1 Copper . IV. Proteins That Bind Only Type I11 Copper . V. Proteins That Bind More Than One Type of Copper VI. Summary and Conclusions . References .

145

.

148 168 172 178 190 192

Perspectives on Non-heme Iron Protein Chemistry JAMES

B. HOWARD AND DOUGLAS C. REES

I. Introduction . 11. Structures and Functions of Mononuclear Iron Proteins . 111. Structures and Mechanisms of Binuclear Octahedral Iron Proteins . IV. Tetrahedral Iron: Fe:S Proteins . V. Conclusions . References .

199 227 239 25 1 27 1 272

Structural Biology of Zinc

DAVIDW. CHRISTIANSON

I. Introduction . 11. Stereochemistry of Biological Zinc-Ligand Interactions . . 111. Long-Range Protein-Metal Interactions IV. Examples of Zinc in Biological Catalysis and Regulation . V. Protein Engineering of Zinc-Binding Sites . VI. Summary . References .

28 1 287 304 3 10 344 348 349

AUTHOR INDEX

.

357

SUBJECT INDEX

.

378

PREFACE

This volume of Advances in Protein Chemistry is the first in which all articles address a single specialized theme within protein science-metalprotein interactions from a structural perspective. Future volumes of Advances in Protein Chemistry will include other thematic volumes in which the reviews will cover different aspects of a single broad area and, as in the past, collections of reviews on a variety of major topics. The first article in this volume, by Jenny P. Glusker, treats general aspects of metal liganding to functional groups in proteins. This article presents a detailed summary of the geometry of interaction of metals with the various chemical groups of proteins. It also presents, in Sections I through VIII, a lucid development of the principles and terminology of the field of metal-protein interactions. It is with these sections that the newcomer to the field of metalloproteins should start. The second chapter, by Catherine A. McPhalen, Natalie C. J. Strynadka, and Michael N. G. James, discusses calcium-binding sites in proteins from a structural perspective. The authors describe the structures of over forty calcium-binding proteins and produce a detailed picture of a “regular” protein calcium-binding site with regard to types and geometries of ligands. In this chapter, and elsewhere in the volume, protein three-dimensional structures are frequently named by their deposition codes in the Brookhaven Protein Data Bank. These codes are an integer followed by three letters, for example, 3TLN for themolysin. In the third chapter, Elinor T. Adman presents a comprehensive view of the structures of copper-containing proteins. This view includes the topological folding of many of these proteins, as shown by ribbon drawings, as well as details of copper-ligand interactions. T h e fourth chapter offers the perspectives of James B. Howard and Douglas C. Rees on non-heme iron protein chemistry. Section I of this chapter presents a particularly broad and accessible summary of ironcontaining proteins, and subsection B gives a quite general discussion of experimental methods for characterizing metalloproteins which will be helpful to newcomers to the field. The last chapter, by David W. Christianson, on the structural biology of zinc analyzes the huge literature in this area. The discussion of the structural aspects of zinc-containing proteins, as the discussions of the other metalloproteins in the four preceding chapters, is at a level of structural detail that would not have been possible even five years ago. In vii

...

Vlll

PREFACE

fact, this entire volume is testimony to the importance of structural methods to our current level of understanding protein function. C. B. ANFINSEN JOHN T. EDSALL DAVID EISENBERG FREDERIC M. RICHARDS

STRUCTURAL ASPECTS OF METAL LlGANDlNG TO FUNCTIONAL GROUPS IN PROTEINS

.

By JENNY P GLUSKER institute for Cancer Research. Fox Chase Cancer Center. Philadelphia. Pennsylvania 19111

I . Metal-Binding Sites in Proteins . . . . . . . . . . . . . . . . . . I1. Polarizabilities of Metals and Ligands . . . . . . . . . . . . . . . . 111. Redox Behavior of Metal Ions . . . . . . . . . . . . . . . . . . IV . Number of Atoms Packed in First Coordination Sphere around Metal Ion . V . Metal-Ligand Bond Distances . . . . . . . . . . . . . . . . . . VI . Asymmetry in Ionic Shape . . . . . . . . . . . . . . . . . . . . VII . Strengths of Bonds from Metal Ions to Ligands . . . . . . . . . . . . VIII . Selection of Metal Ions for Complexation . . . . . . . . . . . . . . IX . Metal Binding to Isolated Carboxylate Groups . . . . . . . . . . . . X . Metal Binding to Imidazole Groups . . . . . . . . . . . . . . . . XI . Metal Binding to Sulfur-Containing Groups . . . . . . . . . . . . . XI1 . Metal Binding to Main-Chain Carbonyl Groups . . . . . . . . . . . XI11. Metal Binding to Two Groups . . . . . . . . . . . . . . . . . . XIV . Metals in Protein Crystal Structures . . . . . . . . . . . . . . . . A . Copper . . . . . . . . . . . . . . . . . . . . . . . . . . B. Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Manganese . . . . . . . . . . . . . . . . . . . . . . . . . D . Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Magnesium . . . . . . . . . . . . . . . . . . . . . . . . F. Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . G Copperandzinc . . . . . . . . . . . . . . . . . . . . . . H . Copper, Zinc, and Cadmium . . . . . . . . . . . . . . . . . . I . Zincandcalcium . . . . . . . . . . . . . . . . . . . . . . XV Electron Transfer in Metalloproteins . . . . . . . . . . . . . . . . XVI . Metal Competition and Replacement . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

3 4

7

8 17 20 21 26 30 36

37 38 38 39 39 42 45 45 50 52

.

55

56 57 58 65 66

.

Metal ions serve a variety of functions in proteins. the most important of which are to enhance the structural stability of the protein in the conformation required for biological function and/or to take part in the catalytic processes of enzymes . Metal ions can activate chemical bonds and make them more amenable to reaction . They can take part in trigger and control mechanisms by specifically altering o r stabilizing a macromolecular conformation on binding (see Rasmussen. 1990). Certain metals can also undergo redox reactions . T h e presence of metal ions in the active site may be crucial to the activity of an enzyme . Sometimes only one specific metal ion in a specific ADVANCES IN PROTEIN CHEMISTRY. Vol. 42

1

.

Copyright 0 1991 by Academic Press Inc. All rights of reproduction in any form reserved.

2

JENNY P. GLUSKER

TABLE I Concentrations of Ions in Seawater and Blood Plasma" Seawater lon

Na+ Mg2+ K+ Ca2+

cu2+ Fe Zn2+ co2+ Ni2+

Blood plasma

(d) 470 50 10 10 0.001 0.0001 0.0001 10-5.5

138 1

4 3 0.015

0.02 0.02 0.002 0

From Hay (1984).

oxidation state can be employed to aid in catalysis, while in other cases a variety of metal ions of similar sizes can be used. On the other hand, the role of the metal ion may be to bind various domains of the protein together. In this way the metal acts as a template, bringing reacting groups into the correct relative orientation for reaction (Hughes, 1981). A wide variety of metal ions is found in the environment and in the body so that there is a good selection available for use by proteins for biochemical processes (see Table I). However, of these metals, K + and Mg2+ are found inside the cell, while Na+ and Ca2+ are generally excluded. T h e properties of such metals that are useful in the structure and function of proteins, particularly enzymes, are reviewed here from a structural point of view. The atoms or groups of atoms that surround a metal ion and which are close enough to be chemically bonded are termed ligands (Kauffman et al., 1983). Ligands donate an electron pair to the bond and are generally negatively charged or neutral. The number of such liganding atoms surrounding a central metal ion is termed the coordination number of the metal ion. Important in a study of metal-ligand interactions are the polarizabilities of both the metal ion and the ligand, the number of the ligands around each metal ion, and the stereochemistry of the resulting arrangement (Williams, 1959, 1970). Metal ions, because they are positively charged, act as electrophiles; that is, they seek the possibility of sharing electron pairs so that a bond or a charge-charge interaction can be formed and electrical neutrality can thereby be attained. In these types of interactions, metal ions act like hydrogen ions (i.e., protons), except that they have a greater ionic volume

3

STRUCTURAL ASPECTS OF METAL LlCANDlNG

and may also have a higher charge. The hydrogen ion has been referred to as a “poor man’s metal.” However, a metal differs from a proton; unlike a proton, which is essentially a bare nucleus and orders of magnitude smaller than other cations, a metal ion can (by virtue of its greater size) coordinate several ligands at the same time. In addition metal ion catalysis can occur in pH ranges at which proton catalysis would not be effective (Hughes, 1981). I. METAL-BINDING SITESIN PROTEINS T h e major metal-binding amino acid side chains in proteins (Gurd and Wilcox, 1956; see Voet and Voet, 1990) (Table 11)are carboxyl (aspartic acid and glutamic acid), imidazole (histidine), indole (tryptophan), thiol (cysteine), thioether (methionine), hydroxyl (serine, threonine, and tyrosine), and possibly amide groups (asparagine and glutamine, although TABLE I1 Metal-Binding Croups in Proteins ~~

~~~

Group

Abbreviation

Carboxyl

ASP Glu

Imidazole

His

Formula CHZCOOCHpCH2COO-

Amide Amino”

0 0

N

Indole

Thiol Thioether Hydroxyl

Metal-binding atom

N

CYS Met

Ser Thr TYr Asn Glu =YS

CHpSH CHzCHpSCHs CHpOH CH(CH3)OH CHp-CeH4-OH CHpCONHp CH&H&ONHz (CH~).,NH; N

Main-chain carbonyl Main-chain amino”

” Questionable.

N-H

0 N

4

JENNY P. GLUSKER

generally via their side-chain carbonyl, rather than amino, groups). About 65% of the various types of amino acid side chains are potential metal-binding groups. In addition protein main-chain carbonyl and amino groups bind metal ions.

11. POLARIZABILITIES OF METALSAND LICANDS

T h e binding of a metal ion to a ligand can be considered in terms of Lewis acid-base theory (Lewis, 1923; Allred and Rochow, 1958; Brown and Skowron, 1990) because, in accepting an electronic pair, the metal ion acts as a Lewis acid. When a metal ion coordinates a ligand, it can affect the electron distribution of the ligand and therefore its reactivity. T h e interaction of an acid with a base is one of the best-known chemical reactions: Acid

+ base + salt + water

This reaction can be generalized by replacing the acid by an electron-pair acceptor [Lewis acid (A), e.g., metal ion] and the base by an electron-pair donor [Lewis base (B), e.g., ligand) to give: A Metal ion

+:B+A:B

+ ligand 4metal-ligand

complex

where a partial donation of electrons from electron-rich :B to electronpoor A results in the formation of a metal-ligand complex. T h e manner by which this partial electron transfer occurs depends on the natures of the atoms involved. Certain atoms or groups of atoms are polarizable; that is, when they are placed in an electric field, there tends to be a charge separation in the atom or group so that it acquires a dipole. This “deformability” or polarizability is measured by the ratio of the induced dipole to the applied field. In general atoms that are more polarizable hold on less firmly to their electrons. When two ions have the same inert gas structure, the negatively charged anion is more polarizable than the positively charged cation. This is because the net positive charge of a cation will cause electrons to bind more tightly to the nucleus, while for anions the electrons are bound more loosely. If the cation is small and has a high charge, it can polarize a large anion, perhaps even partially forming a covalent bond (Fajans, 1923). Results of measurements of polarizabilities lead to the concept of hard

5

STRUCTURAL ASPECTS OF METAL LIGANDING

TABLE 111 HardlSoft Classifications ~~

Classification Hard Soft Borderline

Cation H+, Li+, Na+, K + , Mgz+, Ca’+, Mnz+, Cr3+,Co3+ Cu+, Ag+, Au+, TI+,Pdz+,Ptz+, Cd2+, Hg+, Hg2+ Zn2+,Cuz+,Ni2+,Fez+,Fe3+, Coz+,Snz+,Pbz+, Rh3+, Ir3+, Ru3+

Ligand

H20, OH-, ROH, RO-, NH3, CO:-, RCOO- (mainly oxygen ligands) RSH, RS-, RzS, CN-, H-, I- (sulfur ligands) Pyridine, RNHz (mainly nitrogen ligands)

and soft acids and bases. The word “hard” means low polarizability, so that the atom or ion is deformed only with difficulty (i.e., like a hard sphere). By contrast the word “soft” means high polarizability, so that the electron cloud is readily deformed (i.e., easily “squished”). The donor atom of a hard base (a ligand) holds on tightly to its electrons, whereas a soft base contains electrons that are easily distorted or removed. A hard acid (a metal cation), has a high positive charge, a small size, and lacks easily excited unshared valence electrons, whereas a soft polarizable metal cation tends to have a large size, a small positive charge, and several unshared valence electrons that are readily distorted or removed. A list of classifications as hard, soft, or borderline is given in Table 111. The general principle put forward by Pearson (1966, 1968a,b, 1986), following the work of Ahrland et al. (1958), Schwarzenbach (1961), and others is that hard acids prefer to coordinate with hard bases and soft acids prefer to coordinate with soft bases. This has been rephrased by Mildvan (1970) as: “Cations that indulge in ionic binding prefer ligands that so indulge; cations that indulge in covalent binding prefer ligands that so indulge.” T h e reason that it is important to know how polarizable a metal ion is comes from the concept that the polarizability of a metal ion provides an indication of its ability to donate electrons for ‘TT bonding. The greater the polarizability, the greater the potential for ‘TT bonding and, on such metal binding the possibility exists for activation of the ligand. For example, if a metal ion binds to a carbonyl group, the latter is polarized and the carbon atom becomes, by virtue of its acquired partial positive charge, more susceptible to nucleophilic attack by a base. Thus, hard acids bind hard bases by ionic forces, whereas soft acids bind soft bases by partially forming covalent bonds (e.g., ‘TT bonds). As a result of this covalent bond formation, the effective charge on the metal ion is reduced. The electrons, loosely held in the outer d-orbitals of soft metal

6

JENNY P. GLUSKER

ions and able to expand as the effective charge is reduced, are particularly suitable for forming 7r bonds by donation to empty orbitals of suitable ligands, as shown in Fig. 1. This process can alternatively be thought of in terms of frontier orbitals (Klopman, 1968).The frontier orbitals are the highest occupied molecular orbitals (HOMOS) of the (a) Hard acid-hard base (M', L- ) hard base (ligand, e.g. 0, N)

hard acid (metal cation, e.g. Na, K, Mg, Ca)

no n-electron transfer

p -orbital of ligand (filled)

d -orbital

(solvated in solution) big difference in electronegativities

(b) Soft acid-soft base (M', :C *M

6-

a+

-L

6+

6-

+M = L)

a-bond

a-bond + *-"backbond"

soft base

soft acid

(ligand, e.g. thiol)

(metal cation, e.g. CU,AS, Hg)

p - (d- or n-') orbital of ligand (empty)

*-electron transfer

small difference in electronegativities

FIG. 1. Interaction of d-orbital electrons of metal ions with ligands. (a) In a hard acidhard base combination there is no electron transfer, and the two ions bind by ionic forces. (b) In a soft acid-soft base combination there may be m-bonding as a result of donation of electrons from the d-orbital of the metal to the ligand; the transfer of electrons from metal to ligand prevents the soft metal (usually in a low oxidation state) from becoming too negative.

7

STRUCTURAL ASPECTS OF METAL LIGANDING

donor atoms (in the Lewis base, the ligand) and the lowest unoccupied molecular orbitals (LUMOs) of the acceptor atom (in the Lewis acid, the metal ion). If these two orbitals have similar energy, there is electron transfer to the donor (ligand) from the acceptor (metal ion) and the resulting bond is primarily covalent (soft acid-soft base). If the energy difference between the HOMO and LUMO orbitals is large, no electron transfer occurs and the interaction between base and acid is primarily electrostatic (hard acid-hard base). Th e hard metal ions of interest in biochemistry (Table IV) are alkali metal ions (e.g., Na2+ and K2’), alkaline earth ions (e.g., Mg2+and Ca2+), Mn2+, Cr3+, and Co”. The soft metal ions are Cu+, Ag+, Hg+, Pd2+, Cd2+,Pt2+,and Hg2+.Metal ions with intermediate properties are Fe2+, Co2+, Ni2+, Cu2+, Zn2+, and Pb2+. Thus, most metals and ligands of biochemical interest are hard, except for cuprous ions and thiols and hydride ions, which are soft. For example, thiocyanate (SCN-) binds first transition-series metal ions (hard) by the nitrogen atom but second and third transition series (softer) by the sulfur atom (Hughes, 1981). Pretransition elements (e.g., Na+, K + , Mg2+,and Ca2+)are held to ligands by electrostatic forces. Note that most biological metal-ligand interactions are between hard metal ions and hard ligands. Some soft metal ions (e.g., Hg2+)and soft ligands (e.g., carbon monoxide, hydrogen sulfide, and cyanide) are poisons. They interact with soft species in the biological system and inhibit them. 111. REDOX BEHAVIOR OF METALIONS

T h e 3d orbitals of transition elements are not completely filled and they may have two or more oxidation states. Nature has taken advantage of this to use a change in the valence state of the metal to effect changes in

TABLE IV Compkx-Forming Properties of Metals in Biology

Metal ion Zn2+, Cd2+,Co2+,

Property

Na+, K+

Mg2+,Ca2+

Cu2+,Fe2+,Mo2+

Complex formation Preferred ligand atom

Weak 0

Moderate 0

Strong N and S

8

JENNY P. GLUSKER

the electron distribution in the substrate, thereby making it more reactive. Copper, iron, and cobalt are common examples of this. The valence changes involve copper [Cu(I) (d”) and Cu(I1) (81,iron [Fe(II) (8) and Fe(I1I) ( d 5 ) ] and cobalt [Co(I) (d’), Co(I1) (8,and Co(1II) (&)I. In addition other types of metal ions (e.g., molybdenum) may also take part in redox processes.

I N FIRSTCOORDINATION SPHERE IV. NUMBEROF ATOMSPACKED AROUND METALION

How many ligands can pack around a metal ion? The answer depends on the relative sizes of the metal ion and liganding atom. T h e early work of Alfred Werner (1893) led to the notion of coordination numbers and an understanding of the stereochemistries of arrangements of liganding atoms (Buckingham, 1973). X-Ray diffraction studies of the crystal structures of inorganic salts have shown that ions pack in such a way that there is a local neutralization of charge (Wyckoff and Posnjak, 1921; Goldschmidt, 1926, 1929). Each cation has as many anions as possible around it and each anion has as many cations as possible around it. In both cases the central ion is in contact with each of its neighbors. Allowance must also be made for repulsion between the ligands, because they are ions of like charge. This means that relative charges and sizes as well as the polarizabilities of ions are important. Measurements of metal ion-liganding atom distances in the crystal structures of many inorganic salts led to the derivations of atomic o r ionic radii for these atoms or ions. These “radii” are based on the idea that each atom or ion behaves as a solid sphere of defined size (Wasastjerna, 1923; Zachariasen, 1931). The shortest distance between adjacent ions of opposite sign is then presumed to be the sum of the radii of the respective ions: the cation and the anion. rexpt1

= T+

+ r-

However, since only values of rexptlare obtained, it is necessary to assume a value for the ionic radius of either r+ or r- in order to derive the ionic radius of the other. It is usual to assume a value of 1.40 A for the radius of the 0‘- and 1.94 A for the radius of C1- (Pauling, 1948) because these are half the minimum anion-anion distances found in crystal structures. Values for ionic radii (Shannon and Prewitt, 1969; Shannon, 1976; Brown, 1988) are listed in Table V for a coordination number of 6 around the metal atoms. Thus, values of radii are hypothetical, based on the idea of an additivity rule and a few initial assumptions on anion size.

9

STRUCTURAL ASPECTS OF METAL LIGANDING

TABLE V Effective Cation Radii of Some Metal Ions and Their Average Observed Coordination Numbers for Different Ligands in Structures of Small Molecules" Coordination number Element Group I Li Na K Rb Cs W1)

Cation radius (CN = 6)

0,s

F, CI, Br, I

0.60 0.95 1.33 1.48 1.69 1.49

4.9 6.4 7.9 8.0 8.8 6.9

5.3 6.7 9.0 9.8 10.4 8.3

Main group, highest oxidation state Be(I1) Mg(W Ca(I1) Sr(I1) Ba(I1) Al(111) Ga(I1I) In(I I I) TI(I I I) Main group, lowoxidation state Sn(I1) Pb(I1) Transition metals

CW) Ag(U Cr(I1) Mn(I1) Fe(I1) Co(I1) Ni(I1) CU(I1)

Zn(I1) Pd(I1) Cd(1I) Pt(I1) Hg(W Cr(II1) Mn(II1) Fe(I I I) Co(II1) La(II1) a

a w -

O,F

S, CI, Br, l

0.31 0.65 0.99 1.13 1.35 0.50 0.62 0.81 0.95

4.0 6.0 7.3 8.6 10.2 5.3 4.6 6.0 6.1

6.0 6.7 7.4 8.0 4.1 4.0 5.5

0.86 1.12

4.4 6.9

5.5 6.7

0.72 1.10 0.80 0.80 0.74 0.70 0.66 0.69 0.7 1 0.86 0.91 0.80 0.98 0.58 0.62 0.62 0.54 1.03

2.2 5.1 6.0 5.9 5.7 5.9 5.1 5.0 4.4 6.1

3.5 4.4 5.9 5.7 5.4 4.8

-

5.5 6.0 5.8 5.7 5.9 8.5

-

4.7 4.0 4.0 4.6 4.0 4.0 6.0 4.4 7.8

Radii are expressed in angstroms (A). CN, Coordination number. From Brown (1988).

10

JENNY P.GLUSKER

Octahedral complexes in d4 to d7 configurations may have more than one arrangement of electrons. Some are high spin, with a maximum number of unpaired electrons, while others are low spin, with as many electrons paired as possible. In practive tetrahedral complexes in d3 to d6 configurations should also show high and low spins, but generally only high-spin complexes are observed in these cases (Hughes, 1981). T h e radius of a metal ion is different if it is in a low-spin state than if it is in a high-spin state (Table VI); the high-spin ion has the larger radius. T h e coordination number of a cation depends on the number of anions or ligand atoms that can be fit around it in three dimensions (Fig. 2). In the hard-sphere model the coordination number is determined by the ratio of the radius of the cation to that of the anion Radius ratio = r + / r -

TABLE VI Radii and Electronic Configuration of Some High- and Low-Spin Transition Metal Cationsa (a) Radius (A); CN = 6’ Cation

Low spin

High spin

Ratio HS/LS

Cr(I1) Mn(I1) Fe(I I) Co(I1) Fe(II1) Co(1I I)

0.73 0.67 0.6 1 0.65 0.55 0.52

0.82 0.82 0.77 0.73 0.64 0.61

1.12 1.22 1.26 1.12 1.16 1.17

Unpaired electrons

V(W Ti(II), V(II1) V(II), Cr(II1) Cr(II), Mn(II1) Mn(II), Fe(II1) Fe(II), Co(II1) Co(I I I) Ni(II1) Cu(I1) Zn(I1) a

LS

HS

1

1 2

2 3 2 1 0 1 2 1 0

3

4 5 4 3 2 1 0

LS, Low spin; HS, high spin. From Shannon and Prewitt (1969).

(b) Configurations Low spin

t tt ttt r u t

t w t ? & t i t i t i t i t i t t w t w t i t i t i t i t t w t i t i t i

High spin

t tt

t t t

tttt ttttt

t i t t t t t i t i t t t t i t i t i t ? t i t i w i t tit it it it^

STRUCTURAL ASPECTS OF METAL LIGANDING

a

11

b

ratio 0.155

ratio 0.414

FIG.2. The two-dimensionalpackingof anions around a cation, where the ratio of cation to anion radii is (a) 0.155, so that the coordination number is 3, and (b) 0.414, so that the coordination number is 4. In three dimensions (b) is an octahedron if two or more anions bound above and below the cations. Note that in each case the anions are in contact and that the cation, if it were smaller, would not fill the hole.

The relationship between radius ratio and coordination number is shown in Fig. 2.Since cations are generally smaller than anions, the radius ratio is particularly important for the coordination of anions around cations. The coordination number is usually as high as possible, but the arrangement of surrounding ligand atoms should be such as to minimize repulsive energy between them. The observed coordination number is an integer and may be plotted for ranges of the radius ratio (Fig. 3 and 4). Overall, the most likely value of the coordination number is 6, but several metals have other preferences (see Table V). The radius of an ion increases as the coordination number increases, possibly because of repulsions between the coordinated anions. T h e radius ratio is considered important because the central ion must be prevented from “rattling around” in a cavity (see Orgel, 1966). However, the radius ratio is not a rigorous prognosticator, since the concept applies to hard spheres. It has already been noted that ions may be polarizable and deformable, sometimes with a tendency to directional covalent bond formation. These properties affect models based on hard spheres and the extent to which the radius ratio determines the coordination number of a particular ligand. Thus, “. . . we can accept the radius ratio rule as a useful, if imperfect, tool in our arsenal for predicting and understanding the behavior of ionic compounds.” (Huheey, 1983). Brown (1988) showed that the use of ionic radius ratios generally gives

12

JENNY P. GLUSKER

t

0.8

0.6

Radius ratio (cation/ anion)

0.4

0.2

0.0

0

2

4

6

8

10

Coordination number -w FIG.3. Plot of radius ratio (catiodanion) versus coordination number.

coordination numbers that are higher than those observed experimentally. Smaller coordination numbers, he found, are associated with strongly directed bonds such as are found for main-group elements in low-oxidation states and for transition elements with filled d shells, such as Cu(I), Hg(II), and Ga. Values of average experimental coordination numbers are listed in Table V and shown in Fig. 5. Since these are experimentally determined, they are fractional values, the maxima of histograms of coordination numbers in inorganic compounds. Obviously, in each specific case the coordination number is integral in value. What determines, in a given situation, what the coordination number (for which an approximate value has been derived) actually is? Brown (1988) and others (Brown and Faggiani, 1980; Dent-Glasser, 1981) suggested that one factor is the Lewis base strength of the anion. If the anion is a strong Lewis base, then the coordination number is lower than for a weaker Lewis base. Several shapes for coordination polyhedra (i.e., the spatial arrangements of anions around a cation) are possible. If there are six ligands and they are each as far apart as possible, the result is an octahedral coordination of ligands. If the coordination number is 4, the arrangement of ligands is tetrahedral or square planar. If the coordination number is 5, the arrangement of ligands is generally square pyramidal or trigonal

STRUCTURAL ASPECTS OF METAL LIGANDING

Radius ratio cation/anion

0.155 0.225

0.645 0.732

0.414

13

0.902

Coordination number

Polyhedron

tetrahedron equilateral triangle

Radiuscati n, oxygen 1 . 4 1

0.22

cube square antiprism

trigonal bipyramid square pyramid octahedron

0.32

0.91 1.02

0.58

BeAl

icosahedron

1.26

Li

Na

K

Mg Zn Mn(ll)

Ca

Ba

Fe(l1)

Cd La

FIG. 4. Radius ratios for various coordination numbers. Also shown are the types of coordination polyhedra, and cation radii that fit these criteria. Note that most metal ions of biological interest have coordination numbers of 6 or more.

bipyramidal. Some of these coordination polyhedra are shown in Fig. 6. A linear arrangement shows no limits imposed by the radius ratio, but a trigonal complex, AB3, is likely only if the ratio of radii of A to B is less than 0.225, but greater than 0.155, as shown in Fig. 4. If the ratio is greater than 0.225, a tetrahedral arrangement is more likely. As the coordination number increases, there are several possibilities for packing and these may have different radius ratios. For example, ABs may form either a square pyramid or a trigonal pyramid; there are several B-A-B angles of 90" in each. Therefore, the radius ratios are the same and, more importantly, the same as for an octahedron; if five ligands can fit around a metal ion, so can six, with the result that a coordination number of five is not as common as one of six. The shapes of coordination polyhedra, that is, of the solid figures obtained by joining all ligand atoms directly bonded to the metal cation, have been analyzed by inorganic chemists. Some results are diagrammed in Fig. 6 and listed in Tables VII and VIII. Models can be constructed readily for clarification (He et al., 1990).

14

JENNY P. GLUSKER

30-

1

loo

p

20-

$8

'6

3E,

10-

2

n "

3

4

5

6

7

8

9

1

0

"

3

4

5

6

1

8

9

1

0

Coordinationnumber

Coordination number

20 -

K

Ca

10

0 Coordination number

I 10

3

4

5

8

7

8

9

Coordinationnumber

FIG. 5. Coordination numbers of some metal ions from data on small molecules contained in the Cambridge Structural Database (Liao el al., 1992).Shown are data for Mg, Na, Ca, K, Zn, Cd, Fe, Co, Cu, and Mo.

30C

I

Zn

9 200

6

B

E, 100

0 3

4

5

8

7

B

0

0

10

4

3

5

Coordinationnumber

8

7

8

0

10

Coordination number

800

1500

500

g

rl E

g 1000

400

Fe

co

1

300

6

?

{

500

z'

3

4

5

8

7

8

0

1

"

0

5

8

7

8

0

500 1

-

cu 800'

Ei

-E?

4

Coordination number

Coordinationnumber

1200 -

1000

3

400-

g

Mo

300'

5

600-

j c

400-

200'

1

z

100'

Coordinationnumber

Coordination number

10

16

JENNY P.GLUSKER

L-M-L

I linear

I I trigona~planar

I v tetrahedron

I I I trigonal pyramid

V square planar

v Itrigonal bipyramid

L

L

v I Isquare pyramid I x trigonai

x

i pentagonal bipyramid

xI

L capped octahedron

biprism

x I Icapped

trigonal biprism

X I I I Quare

antiprism

x Iv dodecahedron

FIG.6. Shapes of commonly encountered coordination polyhedra representing ligands (L) around metal ions (M). These polyhedra are listed in Table VII.

17

STRUCTURAL ASPECTS OF METAL LICANDING

TABLE VII Coordination Polyhedra for Various Coordination Numbers Coordination number

Ligand arrangement

2

Linear Trigonal planar Trigonal pyramidal Tetrahedral Square planar Trigonal bipyramidal Square pyramidal Octahedral Trigonal biprismatic Pentagonal bipyramidal Capped octahedral Capped trigonal biprismatic Square antiprismatic Dodecahedra1

3 4

5

6

7 8 a

Polyhedron"

I I1 111 IV V VI VII VIII IX X XI XI1 XI11 XIV

Illustrated in Fig. 6.

V. METAL-LIGAND BONDDISTANCES T h e crystal structures of many metal-containing enzymes are now known, and the coordination around the metal can often be analyzed. However, useful information on the tendency to coordination also comes from X-ray crystallographic studies of small molecules. There are now approximately 90,000 crystal structures in the Cambridge Structural Database (Allen et al., 1979), and a large number of these structures contain metals. Information on the geometry of how metal ions bring other groups around them when a metal-containing compound forms crystals is readily available from the Cambridge Structural Database. Some minimum metal ion-oxygen, metal-nitrogen, and metal-sulfur distances are listed in Table IX, together with data for magnesium ions on the variation of bond distance with coordination number (Carrel1 et al., 1988; Vedani and Huhta, 1990; Baur, 1970). In addition analyses of stereochemical data in the Cambridge Structural Database give some information on dynamic stereochemistry during chemical reactions (Fukui, 1971; Murray-Rust et al., 1975; Biirgi, 1975; Chandrasekhar and Biirgi, 1984; Auf der Heyde and Nassimbeni, 1984).

18

JENNY P. GLUSKER

TABLE VIII Geometries of Coordination Polyhedra around Some Transition Metal Cations" Electronic configuration

Coordination number

Mn(I1)

d5

Cr(I1) Cr(II1) Fe(1I)

d4

4 6 6 6 4 5 6

Fe(111)

d5

Cation

d3

d6

4

6

d8

Co(1I)

d7

Co(II1)

d6

Ni(I1)

d8

cum

d'O

Cu(I1)

d9

7 4 5 6 4 5 6 4 5 6 4 5

5 6 4 5

Zn(I1)

d"'

Mo(IV), W(IV)

d2

6 8

Mo(V), W(V)

d'

5 6 8

Mo(VI), W(V1)

do

4 6

6

a

Polyhedron geometry Tetrahedral, square planar Octahedral Octahedral (distorted) Octahedral Tetrahedral Trigonal bipyramidal Octahedral Tetrahedral Octahedral Pentagonal bipyramidal Square planar Trigonal bipyramidal Octahedral Tetrahedral, square planar Trigonal bipyramidal, square pyramidal Octahedral Tetrahedral Square pyramidal Octahedral Square planar, tetrahedral Square pyramidal, trigonal bipyramidal Octahedral Linear Planar Tetrahedral Tetrahedral (distorted), square planar Trigonal bipyramidal, square pyramidal Octahedral (distorted) Tetrahedral Distorted trigonal bipyramidal, square pyramidal Octahedral Octahedral Dodecahedra1 Trigonal bipyramidal Octahedral Decahedral Tetrahedral Octahedral

Including Mo and W. The most common motifs are italicized.

19

STRUCTURAL ASPECTS OF METAL LIGANDING

TABLE IX Metal-Ligand Distances Measured in Crystal Structures of Small Molecules (a) Minimum metal-oxygen (M...O) distances (A) in metal carboxylates Cation

1.93 1.95 1.96 2.08 2.23 2.25 2.46 2.61 1.82 1.83 1.83 1.96 1.96 1.87

Ni(I1) MgW Co(I1) Mn(I1) Ca(I1) Na(I)

KO) Ba(I1) CU(I1) CU(I) Co(I I I) CO(I1) Fe(II1) Fe(I1)

Reference

Carrel1 et al. (1988)

(b) Minimum metal-ligand (M...L) distances (A). Complex Zn(I1)

Co(I1)

Ligand atom

CN-4

CN = 5

CN=6

Reference

0 N S 0 N S

1.91 1.92 2.25 1.84 1.79 2.12

1.93 2.00 2.36 1.86 1.80 2.30

1.97 2.05 1.94 1.87 2.25

Vedani and Huhta (1990)

Bond

Coordination number

(c) Average bond length (A)b

Reference

Mg...O

3 4 5 6 7

2.02 2.07 2.05 2.08 2.1 1 2.12 2.13

Baur (1970)

8

9 a

As a function of ligand atom identity and coordination number (CN). As a function of coordination number: in minerals.

20

JENNY P. GLUSKER

VI. ASYMMETRY IN IONICSHAPE Several types of ions exhibit asymmetry in their apparent shapes. This is evident in octahedral complexes of Cu(I1) in which the Jahn-Teller effect Uahn and Teller, 1937) is observed; the coordination octahedron of Cu(I1) is found not to contain six bonds of equal lengths, but has four short bonds and two long bonds. This effect occurs because the Cu(I1) cation, with a 8 configuration, contains an unpaired electron; this unpaired electron may lie either in the d x n - y 2 o r dL2orbital. T h e Jahn-Teller theorem states that any nonlinear molecule with a degenerate electronic state distorts and so lowers its symmetry and reduce the degeneracy. In so doing the system is stabilized. For example, if, in an environment of octahedrally disposed ligands, the unpaired electron is in the dXn-p orbital, then the nucleus of the central cation is more shielded in the z direction and less shielded in the xy plane. Therefore, ligands in the xy plane are attracted to the greater apparent nuclear charge in this (equatorial) plane than are ligands in the z-axial direction. As a result metalligand bonds are shorter in the xy plane than in the z direction. The result is shown in Fig. 7 for two hydrates of copper sulfate (Beevers and Lipson, 1934; Bacon and Curry, 1962; Zahrobsky and Baur, 1968). Here, the difference between the lengths of equatorial and axial ligands is on the order of 0.44 A. If, for a cation, this difference is considerably smaller than that observed for Cu(II), the coordination polyhedron is described as pseudooctahedral. On the other hand, if the difference is much larger than for Cu(II), then square-planar or two-coordinated structures result (depending on where the unpaired electron went). Other ions, besides Cu(I1) (dg),showing this effect are high-spin Cr(I1) (d4) and Mn(II1) (d4) and, to a smaller extent, low-spin Co(1I) (d7) and Ni(II1) (d7). Another factor that modifies metal-ligand bond lengths is the trans effect. This is the labilization of ligands trans to other ligands in octahedral complexes and is a smaller effect, observed in octahedral Co(II1) complexes, in which one ligand may affect another ligand 180" away (trans).For example, while the Co(NH3);' ion has six equal Co-N bonds (1.97 A) arranged octahedrally (Kruger and Reynhardt, 1978), if one NH3 group is replaced by another group, a change is observed in the Co-N axial to the substituted ligand (Elder and Trkula, 1974; Elder et al., 1978) (see Fig. 8). When one ammonia is replaced by sulfite, the trans Co-NH3 bond is increased 0.09 A, while the other four Co-NH3 bonds remain essentially unchanged in length. Soft ligands (e.g., sulfur) can also show asymmetry of shape. T h e directions of approach of atoms to sulfur in a C-S-C group have been analyzed by Parthasarathy and co-workers (Rosenfield et al., 1977). They

STRUCTURAL ASPECTS OF METAL LIGANDING

21

03S0

H20

r3 I

2.41

1.98%$ H 2 ° 1 . 9 6

I

1.96

cu

2.41

H20

k . 9 8

03S0 FIG. 7. The influence of the Jahn-Teller effect on bond lengths (A) of copper sulfate trihydrate (top) and pentahydrate (bottom). Note that in the trihydrate one of the Cu(II)...OSOsbonds is short so that all equatorial Cu(II)...Obonds are 1.94-1.98 A, while the axial bonds are 2.40-2.45 A.

studied the orientations of groups near the C-S-C group in a variety of crystal structures containing it: the Cambridge Structural Database (Allen et al., 1979) was searched for this information. Not only did these investigators find that the sulfur atom appeared to have a different radius in different directions but they found that nucleophiles and electrophiles approach it from different directions (Fig. 9).

VII. STRENGTHS OF BONDSFROM METALIONSTO LICANDS In analyzing inorganic structures, Brown (1978) extended the “balland-stick” valence models of organic structures to inorganic structures,

22

JENNY P. GLUSKER

1.97

r3

Co(NH3): +

NH3

2.22 1.96

To3 I ,,J 1.97

NH3

H7N

NH3

FIG. 8. tram-Effects on bond distances (A) in octahedral Co3+ complexes. Here, the length of a bond is affected by the bond trans to it. The Co-NH3 bond of 1.96-1.97 A is elongated to 2.06 A when it is tram to an SO3 group.

particularly metal complexes. Each atom is surrounded by a number of bonds, its coordination number. The valence of an atom is the number of electrons used o r required for bonding. It is convenient to define the bond valence of a metal-ligand interaction in the following way (Pauling, 1948; Brown, 1978, 1980). Pauling (1948) wrote, “in a stable ionic structure the valence of each anion, with changed sign, is exactly or nearly equal to the sum of the strengths of the electrostatic bonds to it from the adjacent cations.” In metal complexes the valence is shared among the ligands. As a result if a metal ion has a charge n, the bond valence of each of the individual metal-ligand bonds is Bond valence =

total charge of cation, n coordination number of cation

STRUCTURAL ASPECTS OF METAL LIGANDING

23

a

elecrophiles interact with HOMO (lone pair of S) H+ , Na+ , Cu2+

nucleophiles interact with

300

LUMO [a*(S-C) orbital]

plane of C-S-C b

C

electrophiles

FIG.9. Asymmetry of interactions of C-S-C groups. Nucleophiles approach the C-S-C bond in its plane, while electrophiles approach nearly perpendicular to this plane. (a) General view, (b) view along C-S-C plane, and (c) view onto C-S-C plane.

Thus, if a magnesium ion binds six oxygen atoms, the strength of each metal-ligand interaction is 2/6 = 0.33. If the cation is sodium, with the same coordination number, the bond strength is lower, 0.17. The above equation can be rewritten as Valence (charge) of metal ion = Call atoms (bond valence) and the bond valence is effectively the extent to which the charge on each liganding atom is contributed to the metal-ligand bond (valence sum rule). Analyses of metal coordination and the bond strengths started with the publication by Pauling (1929)of an article entitled “The Principles Determining the Structure of Complex Ionic Crystals.” In this article Pauling defined the bond strength as listed above. Later, it became clear from measurements of bond lengths by X-ray crystallographic analyses that

24

JENNY P. GLUSKER

the shorter bonds are the stronger ones. T h e simplest example of this is found in the measurement of the C-C bond length: 1.54 8, for a single bond, 1.34 8, for a (stronger) double bond, and 1.20 8, for a triple bond. Such information led Zachariasen (1954) and others to fit them to formulas relating bond length to bond order. Similar formulas have been derived for inorganic compounds (see Brown, 1978). Values of ionic radii for metals (Shannon, 1976; Brown, 1988) are listed in Table V. These radii, however, vary with coordination number (Baur, 1970). An ion with tetrahedral coordination has 93-95% of the radius for the same ion with coordination number 6. If the coordination number is 8, the ionic radius for coordination number 6 is increased by a factor of 1.03. Anions also appear to have different ionic radii for different coordination numbers. Values for oxide anions vary from 1.35 A (coordination number 2) to 1.40 (coordination number 6) to 1.42 8, (coordination number 8). Brown has studied rules for cation coordination in inorganic structures, including minerals (Brown, 1987). T h e Inorganic Crystal Structure Database (Bergerhoff et al., 1983) formed the basis from which the “bond valence model” was derived. Such analyses require that the sum of the partial negative charges contributed by each ligand around a cation must be the total cation charge. Thus, if a sodium ion, Na+, has six oxygen ligands, each of these ligands contributes a charge of -&. However, ligands with shorter Na+... 0 distances contribute a negative charge greater in value than Q, while those with longer Na+... 0 distances contribute less than the average. This is diagrammed in Fig. 10. Anionic ligands distribute themselves around a metal cation, so that the total of the bond valence contributions is equal but opposite in value to the charge on the cation. For example, in a study of compounds containing C-F bonds (Murray-Rust et al., 1983), it is shown that the bond valences can be calculated by use of an equation such as Bond valence = S = (R/Ro)-N where R is the metal-oxygen or metal-fluorine distance, Ro = 1.622 and N = 4.290 for Na+ for oxygen coordination, and Ro = 1.538 and N = 4.290 for Na+ for fluorine coordination (Brown and Shannon, 1973). These are empirically derived constants derived from experimental data. Values for each bond are given in Table X. Note in Table X that the shorter the metal-ligand distance, the greater the bond valence and that the fluorine atom contributes to the coordination sphere so that the sum of bond valences becomes approximately 1 .O. Many metal-binding sites involve carboxylate groups. Each carboxylate

STRUCTURAL ASPECTS OF METAL LIGANDING

25

-0.17

Q I

I I

-0.19

i2.47

-0.19

-0.17

FIG. 10. Valence sum around a cation. If the oxygen atoms (from Table X) are closer to the sodium ion, they have a higher bond valence. The bond length (A) is drawn for each Na...O bond along it, and the bond valence is shown on the oxygen atom. Thus, the total negative charge of - 1.0 that must surround the Na+ for local electroneutrality is divided in accord with the distances.

group has a total charge of - 1, divided between its two oxygen atoms. There may be more carboxyl groups than expected from the positive charge of the metal ion. This excess of negative charge can be offset in several ways. First, carboxyl groups may bind only one oxygen to the metal, neutralizing the anion charge by -1, rather than -1. Second, carboxyl groups in proteins are held in place in the active site by hydrogen bonds from main-chain (or sometimes side-chain) NH groups. This also reduces their apparent charge (Baker and Hubbard, 1984). Since carboxyl groups may bind metal ions via one o r both of their oxygen atoms, some flexibility in metal ion requirements with respect to coordination number results. For example, calcium in its complexes may have seven ligands and, by swinging one oxygen of a shared carboxyl group

26

JENNY P. GLUSKER

TABLE X Metal-Ligand Distances and Bond Valences in Sodium Compounds

Metal-ligand distance (A) Compound

Na+*...O Na+....F

Sodium fluoroacetate

2.430

0.18

2.503 2.409 2.607 2.351 2.988

0.16 0.18 0.13 0.20 0.07 0.11

2.562 Sodium acetate

Bond valence S(M...L)

Sum of charge around Na'

Reference Vedavathi and Vijayan (1977)

- 1.03 Hsu and Nordman ( 1983)

2.469 2.375 2.668 2.470 2.420 2.374

0.17 0.19 0.11 0.17 0.18 0.19

-1.01

2.346 2.346 2.371 2.371 2.635 2.635

0.20 0.20 0.19 0.19 0.12 0.12

- 1.02

out of the metal-binding site, the active site can accommodate magnesium, which binds only six ligands (Strynadka and James, 1989).

VIII. SELECTION OF METALIONSFOR COMPLEXATION Selective metal-binding compounds (e.g., proteins) must use some feature of the coordination specificity of metals in order that they bind the one appropriate metal ion much more tightly than others. How this is done is not completely clear. Among requirements are a binding site of the correct size and shape (i.e., directionality of ligands). T h e appropriate metal ion forms a more stable complex than do others. A ranking of the stability of complexes for doubly charged metal ions of the first long period is provided by the Irving-Williams order (Irving and Williams,

STRUCTURAL ASPECTS OF METAL LIGANDING

27

1948, 1953) (zinc is less than copper, but its location is not clear in this list): Ba2+ < Sr2+ < Ca2+ < Mgz+ < Mnz+ < Fez+ < Co2+ < Ni2+ < CuP+> Znzi Ionic radius decreasesIonization potential increases ---+

This is the order of reactivity of model compounds involving Lewis acid catalysis by metal ions; the metal ions to the right are the most active. Both ionic radii and ionization potentials show these trends, radii decreasing and ionization potentials increasing as one proceeds to the right in the series. As the size of the metal ion decreases, its inherent acidity increases. In addition, however, metal ions to the right of the series have more d electrons and therefore are softer (and bind sulfur in preference to nitrogen or, particularly, oxygen). Although metals in enzymes d o not exactly follow the rules found for compounds, they do show some general activities in line with the series just given. For example (Hughes, 1981), zinc and cobalt are involved in the hydrolysis of phosphates (by phosphatases) and esters (by esterases). On the other hand, magnesium, manganese, and calcium catalyze reactions of substrates that are weaker bases (e.g., polyphosphates). Magnesium is very active in this way, particularly in phosphate group transfer, which nearly always requires a metal. In proteins the folding of the polypeptide backbone is such as to have several functional groups, mostly in side chains, congregate to form a metal-binding site. For such side-chain functional groups the lower the pK,, the lower the pH at which hydrogen ions are lost, so that a donor atom is available to form a metal-ligand bond. This may be viewed as a competition between hydrogen ions and metal ions for the ligand. As a result metal-binding tendencies follow the order carboxyl (pK 4.5) > imidazole (pK 6.5) > amino (pK 9.0). Carboxyl groups are the strongest, mainly because they are charged. However pK values may vary in a protein, depending on the local environment, and may further differ in the crystalline state from those in solution (Smith et al., 1989). There is also a tendency for functional groups (e.g., carboxylates) to form, in cooperation with a second donor atom in a suitable site, a chelate five- or six-membered ring. While these five- and six-membered chelate rings show extra stability in small structures, they are generally not possible in simple proteins. Some additional chemistry must have taken place on a protein to make this possible (e.g., the presence of a heme group or some posttranslational modification).

-

-

-

28

JENNY P. GLUSKER

On the other hand, many substrates and inhibitors bind to protein-bound metals by the formation of such rings. T h e actual binding of a metal ion to a ligand is governed, in aqueous solution, by its competition with protons. Breslow (1973) considered a ligand-binding site, L-, a protein side chain, for example, that can interact with either a hydrogen ion, H + , or a metal ion, M2+:

Breslow defined an apparent pH-dependent constant, K A , which relates the number of binding sites carrying metal [LM+] to those not carrying metal, as KA = [LM+]/([L-

+ LH][M'+])

= Km/(I

+ KH[H+])

As a result the apparent affinity of the ligand for the metal ion is less than the intrinsic affinity unless the pH is approximately 2 or more units above the pKa of the ligand. Thus, at neutral pH an arginine side chain, which has a very high pK, value (>12; see Table XI), will not bind as a single participant. If a ligand has a single ionizable group, there is an apparent change in the metal ion affinity at different pH values, as a result of the extent to which it can successfully compete with protons for the ligandbinding site. For example, the intrinsic affinities of carboxyl groups for H + ion and Cu(I1) are 104.5and respectively (from pK, and log K , values; see Table XI), while those for imidazole side chains are lo7 and lo4, respectively, for H+ and Cu(I1). Thus, at pH 4.0, Cu(I1) binds carboxyl, rather than imidazole (KA carboxyl > KA imidazole), but the reverse is true at pH 7.0. Of course, the latter situation, pH 7.0, is more nearly that found in vivo. TABLE XI Relative Affinities of Protons, Cu(11). and Zn(l1) for Protein Szde Chains"

Side chain Carboxyl (Asp, Glu) Irniclazole (His) E - N H(LYs) ~ Phenolic OH (Tyr) Sulfhydryl (Lys) Guanidiniurn (Arg) a

4.4-4.7 6.4-7.2 9.6-10.5 9.6-9.8 9

1.8 (Acetate) 3.1-4.4 4.3 (NH3)

>12

K, = [H+I[L-]/[HLl; pKa = -log (Ka);K, = [LM+]/([L-][M2+]).

1.0 (Acetate) 2.5 (Irnidazole) 2.6 (NHs) 7

STRUCTURAL ASPECTS OF METAL LIGANDING.

29

T h e ligands around a metal ion generally form additional interactions. However, these may be sterically limited. For example, in crystal structures hydrogen bonds are generally not formed between two oxygen atoms coordinated to the same metal ion. This is not universally true, but has been observed in most small crystal structures. In hydrated ions the oxygen atoms of water molecules pack around the metal ion with the expected coordination number. Magnesium is particularly prone to form structures with a fully hydrated cation. The structure of a hydrated magnesium ion, found in the crystal structure of magnesium citrate decahydrate Uohnson, 1965), is shown in Fig. 1 1 . Manganese (Carrel1 and Glusker, 1973) behaves in the same way, but less frequently than magnesium. The hydrogen atoms of the water molecules generally lie as far as possible from the positively charged metal ion, as shown in Fig. 1 1 , and therefore do not point to other oxygen atoms in the same polyhedron unless there is no other hydrogen-bond acceptor available. Similarly, in other coordination complexes involving oxygen, it is generally not likely (although not impossible) that hydrogen bonds are formed from the coordinated oxygen to another in the same coordination polyhedron. Neutron diffraction studies, in which the hydrogen atom is well located, show many examples of this. In nickel sulfate hexadeuterate the deuterium atoms of water molecules around the nickel ion are hydrogen bonded to sulfate oxygen atoms, except for one that is hydrogen bonded to the oxygen of another hydrated cation (O’Connor and Dale, 1966); no hydrogen atoms lie between two oxygen atoms in the same coordination polyhedron.

FIG. 1 1 . Stereo of the Mg2f(H20)6ion in the crystal structure of magnesium citrate decahydrate (coordinationnumber 6) (Johnson, 1965). Oxygen atoms are stippled; hydrogen atoms are small and white. Note that none of the hydrogen atoms points to the oxygen atom of another water molecule coordinated to the same Mg2+ion.

30

JENNY P.GLUSKER

IX. METALBINDING TO ISOLATED CARBOXYLATE GROUPS T h e carboxylate ion has one negative charge delocalized within it, and each oxygen atom has two lone pairs disposed at 120" to its C-0 bond and in the plane of the carboxyl group. It is evident from a variety of crystal structures that a given carboxylate group may bind several cations, and, in certain circumstances, the carboxyl group may share the metal cation between both of its oxygen atoms, as has been found for calcium carboxylates. This leads to a study of the importance of the directionalities of the oxygen lone pairs and also which lone pair of each oxygen atom of a carboxylate group is preferred for metal ion binding. T h e geometry of the position of the hydrogen atom in a carboxyl group has been investigated by Rebek et al. (1985, 1986). They designed, prepared, and studied some compounds in which the carboxylic acid groups, from steric effects caused by the rest of the molecule, are obliged to approach each other in a controlled way. Two locations for a proton to lie, when attached to a carboxylate group in the directions of lone pair electrons, are designated syn and anti; these are illustrated in Fig. 12. In the syn conformation (2 form) the proton is on the same side of the C - 0 bond as the other C-0 bond; this is the conformation found when carboxyl groups dimerize by forming two hydrogen bonds. On the other hand, hydrogen bonding in the anti conformation (E form) cannot result in dimerization. Ab initio studies of formic acid indicate that the syn (2) conformation is more stable than the anti (E) conformation by 4.5 kcal/mol, implying that the syn lone pairs are more basic than the anti lone pairs (Peterson and Csizmadia, 1979). Gandour (1981) noted that the carboxylates in active sites of enzymes generally employ the more basic syn lone pairs for metal chelation, rather than the less basic anti lone

anti FIG.12. syn and anti lone pairs in carboxyl and carboxylate groups. Shown are q n ,anti, and direct directions used to describe results of the analysis (see text).

STRUCTURAL ASPECTS OF METAL LIGANDING

31

1. Search the datafile for crystal structures containing metal ions and carboxyl

groups. Select data for a metal ion of interest. 2. Eliminate entries with m r e than one interactionwith the metal ion or other undesirable features.

3. Lay each carboxyl group in a constant defined orientation and mark where the metal ion lies. This gives a scatterplot.

..

* ’ 4.

*.

“ Y O

Put a Gaussian function on each point of the scatterplot.

5. Contour the result.

6. This gives directional preferences of binding. Contours indicate the likelihoodof finding a metal ion in a given region. The higher the peak, the greater the number of points in that area (see step 3) and the higher the probability of finding the selected metal ion in that region.

(a) Onto plane of carboxyl group

(b) Perpendicular to plane of carboxyl group (looking along plane of paper from left to right)

FIG. 13. The strategy used for the construction of contoured scatterplots. Positions of metal ions near a carboxyl group are plotted for different crystal structures. The result is a scatterplot. Gaussian peaks are put on each scatterplot point. The resulting “density” is contoured as for an electron density. The result is a plot of the orientational preferences for binding.

32

JENNY P. GLUSKER

pairs; he estimated that syn protonation is 104-fold more favorable than anti protonation. This means that the carboxylate is a weaker base when constrained to accept a proton in the anti ( E ) direction. An analysis was made from crystal structures of small compounds of the directions in which metal ions approach a carboxyl group. The positions of metal binding to a carboxyl group were determined from each appropriate structure by use of the Cambridge Structural Database (Allen et al., 1979). All available data on carboxylate structures were extracted from this database and divided into groups, depending on the identity of metal cation. Structures in which there was a second intramolecular contact in the ligand that interacted with the metal ion were eliminated from this study because the aim was to analyze contacts of metal ions with an isolated carboxylate group. T h e analysis was carried out (see Fig. 13) by constructing scatterplots of the locations of metal ions around a carboxyl group which was fixed in place as the scatterplot was drawn. The result was, for each metal cation, a series of points illustrating the location of the metal ion in a crystal structure with respect to a fixed carboxylate group. Gaussian-shaped peaks were placed on each scatterplot point, and the resulting density was contoured to give a probability plot (Rosenfield et al., 1984). The metalcarboxylate geometry used in the analysis is shown in Fig. 14. Some examples of the results of this analysis are given in Fig. 15. T h e contoured scatterplots showed that the most likely arrangements of metal cations are those designated syn, anti, and direct. The percentages of directionalities of metal liganding for a total of 1558 metalcarboxylate interactions are 62.9% syn, 22.7% anti, and 14.4% direct. Minimum metal ion-ligand distances are given in Table IX(a). ~ y nis generally preferred, except when the Mn+..*O distances are short (i.e., <1.95 A). T h e elements potassium, sodium, and Cu(I1) have a high number of entries in Table X, and of these, sodium and potassium ions show extensive out-of-plane interactions. T h e conclusions from this analysis of carboxylate-metal binding in small structures studied to high resolution are (1) The metal cation generally lies in the plane of the carboxyl group. There are some notable exceptions. Alkali metals and some alkaline earth metals, which ionize readily and form strong bases, appear to have less specific locations of binding. (2) If the Mn+...O distance lies in the range 2.3-2.6 A, it is common to have “direct bonding,” in which the metal lies equidistant from the two oxygen atoms of the carboxylate group (Figs. 15 and 16). At other Mn+.*.O distances direct bonding is rare; it is a function of the carboxylate “bite” size (2.2 A) and of a need to keep O...Mn+*.*O angles reasonably large (i.e., larger than approximately 60”). Elements with a

STRUCTURAL ASPECTS OF METAL LIGANDINC

a

33

direct

b metal out-of-

FIG. 14. Views of the C-O...M"+ plane and the deviation of the metal from this plane. The metal ion may lie at any point along the arc and may, or may not, be in the plane of the diagram. (a) View directly onto the plane of the carboxyl group; (b) general view of the metal-carboxyl group system showing the deviation of the metal ion from the carboxyl group planar.

reasonably high percentage of direct bonding are Ca(II),Cd(II), Hg(II), Sm(III), Tl(III), Ce(IV), and U(V1). (3) The arrangement with the metal in the syn position is much more common than that for which it is anti. Exceptions are found at very short and very long M"+...O distances (i.e., for very small and very large cations). The information that there is a high probability that the metal lies in the carboxyl plane, but with possible deviations for specific metals, provides a mechanism for searching for metal-binding positions in proteins. This method was used in a study of the enzyme xylose isomerase from Streptomyces rubiginosus (Carrel1 et al., 1989).T w o metal sites were located. One metal-binding site involves three carboxylates (aspartate and glutamate), histidine, and water, and the other involves four carboxylate

34

JENNY P.GLUSKER

(d) Ca2+

(e) cu2+

(9 Fe2+

8

Q

FIG.15. (a-j) Examples of contoured scatterplots of metal-carboxylate binding, viewed onto the plane of the carboxyl group, for Na+, Mg2+, K+, Ca“, Cu’+, Fez+,FeS+, Zn2+, Co2+,and Ni2+.

groups and water. The method is complementary to one in which sites of high hydrophilicity (0,N , and S), embedded within a larger shell of hydrophobic groups (C), are sought; thus, sites of high “hydrophobicity contrast” are likely metal-binding sites in proteins (Yamashita et al., 1990). Such hydrophobicity contrast, Yamashita et al., note, may be an

STRUCTURAL ASPECTS OF METAL LIGANDING

(I)

35

(j) Ni2+

co2+

@Ye @yo

FIG. 15.

indication of an environment that is honed to be ideal for binding the required metal ion, excluding others. These two methods provide good criteria for searching for sites of aspartic and glutamic acid residues in proteins at which metals can bind with a reasonably high affinity (Schmidbaur et al., 1990). Mutagenesis studies of staphylococcal nuclease show less than an order of magnitude decrease in affinity for metal for each removal of a carboxyl ligand (Mildvan and Serpersu, 1989).

36

JENNY P. GLUSKER

O-M DISTANCES

FIG. 16. Percentages of the three types of binding-xyn, of 0 - ~ 7 1 + distance.

anti, and direct-as

a function

X. METALBINDING TO IMIDAZOLE GROUPS Imidazole groups in histidyl side chains bind metal ions in a variety of enzymes. One imidazole can bind one or two metals, depending on its ionization state and the suitabilities of the metal. Freeman (1973) found that the metal-N (imidazole) bond can be up to 30” from the imidazole plane, as is found in the cadmium complex [Cd(~-His)2*5H~O]. The M*.*N(imidazo1e)-C angles at the donor atoms range from 121”to 131” (Candlen and Harding, 1967). With copper, Freeman found, sterically unhindered imidazole rings tend to be coplanar with the M-N (imidazole) bond. Some scatterplots are shown in Fig. 17. Rotation about the M.-.N (imidazole) bond appears to be governed by the steric requirements of the rest of the histidyl residue, by contacts between the imidazole group and neighboring ligands, and by hydrogen bonding of the free (“pyrrole”) N (imidazole) atoms, rather than by electronic factors. The flexibility of imidazole-metal bonds may be one reason for the effectiveness of histidine side chains as metal-binding sites in proteins. The imidazole group is an excellent electron donor, available for action at physiological pH. Imidazole liganding of Zn(I1) and Co(I1) has been studied (Vedani et al., 1986; Vedani and Huhta, 1990). Vedani and co-workers found that “there is a clear preference for metal-bound li-

STRUCTURAL ASPECTS OF METAL LIGANDING

N

37

N

FIG. 17. Scatterplot for metal ion binding to an imidazole group.

gand atoms to orient their lone pair towards the metal center like a H-bond acceptor atom prefers to orient its lone pair towards the donor-H atom.” As a result these authors developed a new force field that can be used for modeling metalloproteins which takes into account directional preferences of bonding, metal charge transfer, and ligand field stabilization. Some minimum metal-ligand distances are given in Table IX(b). In metalloporphyrins axially bound imidazole groups generally bind in such a way that they eclipse Mn+..*N (porphyrin) bonds (Scheidt and Chipman, 1986). Use can be made of the affinities of metals to imidazoles to modify enzyme activity. For example, site-directed mutagenesis was used to add a second histidine to a serine protease in order to enhance the interaction between a transition metal ion and the side chain of His-57 (Higaki et al., 1990). T h e strengths of association of metals were found to be Cu2+ (21 p M ) > Ni2+ (49 p M ) > Zn2+ (128 p M ) . This is the order of association constants, K,, for metals binding to imidazole. In this way it was shown that it is possible to regulate the catalytic activity of trypsin, since, if histidine binds a metal, it can no longer act as a general base in catalysis.

XI. METALBINDING TO SULFUR-CONTAINING GROUPS Many metals bind to the sulfur of sulfhydryl groups in proteins. Metals that bind sulfur in preference to oxygen not only form strong CT bonds with the readily polarizable ligands, but also v bonds by back-donation of electrons from metal dv to ligand dv or pv orbitals. The electronegativity

38

JENNY P. GLUSKER

of sulfur is low and its polarizability is high and it becomes highly polarized in the field of a small metal ion [even Cu(I),Ag(I), or Hg(II)]. T h e fact that Ag(I), Au(I), and Hg(I1) bind readily to sulfhydryl groups (cysteinyl side chains) is used in the preparation of heavy-atom derivatives in protein crystallography. It is found from crystal structure analyses than Zn(I1) binds N and S of cysteine at high pH (NHp), but binds 0 and S at low pH (NH;), see Table IX(b) for some metal-ligand distances. Vallee and Auld (1990a) wrote, “The realization that the affinity of zinc for nitrogen and oxygen is nearly equal to that for sulfur ligands has brought about a major change of viewpoint regarding zinc coordination chemistry and its manifestation in biological systems.” Thioether sulfur atoms have smaller polarizabilities and are weaker donors than S (sulfhydryl) atoms, but they have fewer base-pair electrons and therefore should be better electron acceptors. Methionine generally binds ds and d’’ configuration atoms Pd(II), Pt(II), Pb(II), Ag(I), Cu(I), and Hg(I1) (Dickerson et al., 1969; Freeman and Golomb, 1970; Blundell and Johnson, 1976; Sheriff et al., 1987).

TO MAIN-CHAIN CARBONYL GROUPS XII. METALBINDING

An analysis of metal binding to peptide carbonyl groups (Chakrabarti, 1990), mainly calcium ions in protein crystal structures, shows that the cations tend to lie in the peptide plane near the C=O bond direction. Generally, this binding occurs in turns in proteins or in regions with no regular secondary structures. Ca..-0 distances range from 2.2 to 2.5 A, and metal ions do not deviate by more than 35” from the peptide plane. Thus, metal ions in proteins do not, Chakrabarti observed, bind in lonepair directions.

XIII. METALBINDINGTO T w o GROUPS Chelating groups that bind metals are found in some iron-binding ferrichromes o r siderophores that bind Fe(111) via an octahedral arrangement of catecholate or hydroxamate groups (Nielands, 1973). Several crystal structures of such complexes have been determined (Hossain et al., 1987; van der Helm et al., 1987;Jalal et al., 1988).T h e ferrichromes, which have three hydroxamate groups, bind Fe(1II) very tightly and have log K values in the range of 30-32. An a-hydroxycarboxylate group, found in citrate and isocitrate (Glusker, 1980), which are substrates of many enzymes, can also bind metal ions. A scatterplot is shown in Fig. 18.

STRUCTURAL ASPECTS OF METAL LIGANDING

39

b

FIG. 18. a-Hydroxycarboxylate binding of metal ions. In this case there is a strong tendency for chelation of the metal ion, although it may lie well out of the plane of the liganding bidentate group. (a) View onto OOC-C-OH plane and (b) view along OOC-COH plane.

Based on affinity and on distances from Mn(I1) to 13Cand from Mn(I1) to protons, measured in solution by nuclear magnetic resonance (NMR), Mn(I1) is shown to be chelated by the a-hydroxycarboxylate group of lactate, but to coordinate only the carboxylate (not the a-carbonyl group) of pyruvate (Fung et al., 1973; A. S. Mildvan, unpublished observations, 1991).

XIV. METALSIN PROTEIN CRYSTAL STRUCTURES The stereochemistry of liganding of metal ions in proteins is now known for several proteins (see Armstrong, 1988). Some selected examples follow with data derived from the Protein Data Bank (Bernstein et al., 1977). I n the cases in which two different metals are bound, information can be obtained on preferential sites for each metal in the presence of the other. A . Copper The “blue,” or type 1, copper proteins, azurin from Pseudomonas aerugznosa (Adman et al., 1978; Adman and Jensen, 1981) and from Alcaligenes denitrificans (Norris et al., 1983, 1986) and poplar plastocyanin (Guss and Freeman, 1983; Guss et al., 1986), have been studied by X-ray diffraction. These involve a Cu(I)/Cu(II) redox system. Cu(1) (d”) is

JENNY P. GLUSKER

cys112

\

\

Me1121

SI

FIG. 19. (a) Atomic designationsand (b) stereo view of azurin. Cu(I1) [2N, 2S, 01.In this and Figs. 20-35 oxygen atoms are stippled, nitrogen atoms are black, and carbon atoms are white. Generally, hydrogen atoms are not included, hut if they are, they are represented by smaller circles.

STRUCTURAL ASPECTS OF METAL LIGANDING

41

His87

cyst34 His37

N NH

Cys92

FIG. 20. (a) Atomic designations and (b) stereo view of plastocyanin.Cu(I1) [2N, 2S].

usually found to be tetrahedrally ligated, while Cu(I1) (8) is octahedrally ligated with Jahn-Teller distortion (sometimes giving a square-planar complex). In oxidized azurin from Alcaligenes (Norris et al., 1986) (pH 6.0, refined to 1.8 8, resolution), the copper ion [Cu(II)]is surrounded by five atoms (2N, 0, and 2s).An adequate description of the coordination geometry is hard to make because there are two very long bonds, the Cu-0 (peptide) and Cu-S (methionine) distances (3.11 and 3.13 A, respectively). If these are both considered part of the coordination sphere, this is a distorted trigonal bipyramid with the oxygen and sulfur atoms in axial positions (Fig. 19). In oxidized poplar plastocyanin (Guss and Freeman, 1983) the copper ion is surrounded by four atoms (2N and 2s) in a distorted tetrahedral geometry, one bond (to methionine) being long (2.90 A). This coordination, intermediate between tetrahedral and square planar, is diagrammed in Fig. 20. This intermediate character

42

JENNY P. GLUSKER

probably facilitates electron transfer. There is no change in spin state for the copper, and the rate of electron exchange is related to the depth of the active site within the protein (Mauk et al., 1980). Near physiological pH there does not seem to be much difference between the Cu(1) and Cu(I1) environments (Cuss et al., 1986; Shepard et al., 1990). Hemocyanin is a copper-containing oxygen storage and transport proteins. T h e crystal structure (Gaykema et al., 1984, 1985) shows a Cu/Cu distance of 3.7 A, although spectroscopic data suggest that there may be a tyrosine bridge involved. Each copper atom is bonded to three histidines.

B . Iron The iron-sulfur proteins (Lovenberg, 1973a,b, 1977; Carter, 1977) are electron carriers. Their iron-sulfur centers may contain one, two, o r four iron atoms. Rubredoxin from Clostridium pusteurianum (Herriott et al., 1970; Adman, 1979; Watenpaugh et al., 1980), Desulfovibrio vulgaris (Adman et al., 1977), and Desulfovibriogigas (Frey et al., 1987) contain such a center, with one iron surrounded by four cysteinyl sulfur atoms in a slightly distorted tetrahedral arran ement. T h e structure of the oxidized form of the D . @gasenzyme at 1.4 resolution is shown in Fig. 21. Fe...S distances are lengthened by about 0.08 A. There is an extensive network of N H . * - Shydrogen bonds, which may help to form the specific ironbinding region (Adman et al., 1975). T h e 2Fe-2S ferredoxins from Spirulina plantensis (Fukuyama et al., 1980) and Aphanothece sacrum (Tsukihara et al., 1981) show two iron atoms, each surrounded by two cysteinyl sulfur atoms and each iron sharing two additional sulfur atoms. Model studies (Holm, 1975) had already indicated a shape for this cluster. T h e 4Fe-4S cluster resembles a cube (Fig. 22), and many small structures containing this group have been studied (see, e.g., Carrel1 et al., 1977). The dimensions of the cubane-like structure reflect the oxidation state. Enzymes containing them include the high-potential iron proteins from Chromatiurn (Freer et al., 1975) and from Peptococcus aerogenes (Adman et al., 1976) and the sulfate reductase from Escherichia coli (McRee et al., 1986). Clusters lacking one or more atoms are known (Armstrong, 1988). Hemoglobin and myoglobin are proteins that transport molecular oxygen (Kendrew et al., 1960; Perutz et al., 1968, 1987). These proteins contain iron surrounded by four equatorial ligands from protoporphyrin IX and two axial groups, one of which is a proximal coordinated histidine nitrogen atom and the other the oxygen molecule (Fig. 23). The “trigger” mechanism (Perutz, 1970) involves a high-spin Fe(I1) atom (radius of 0.92 A) in a heme lacking oxygen. There is not enough space for such a

1

STRUCTURAL ASPECTS OF METAL LIGANDING

43

CyS42

FIG.21. (a) Atomic designations and (b) stereo view of rubredoxin. Fe(I1) [4S].

large cation, and therefore the Fe(I1) atom lies about 0.8 8, above the plane of the heme. When oxygen binds to this iron atom, the electrons become spin paired and the smaller low-spin Fe(1I) (radius of 0.75 h;) can then fit in the heme plane (Hoard, 1966). This small radius change pulls the histidine attached to the iron, thereby disrupting other parts of the protein, converting it from the deoxy T state to the oxy R state (where T and R originally indicated “tense” and “relaxed” states, but now are symbols for deoxy and oxy states) (Gelin et al., 1983). A distal uncoordinated histidine serves to control the oxygen-binding site, inhibiting the binding of carbon monoxide; this occurs because carbon monoxide binds iron in a linear fashion, while oxygen binds in a bent manner (Scheidt and Lee, 1987). Several enzymes, such as catalase (Vainshtein et al., 1986), cytochrome c peroxidase (Edwards et al., 1987), and cytochrome P-450

JENNY P.GLUSKER

\

cys41

Cysl8 cys35

FIG. 22. (a) Atomic designations and (b) stereo view of ferredoxin. Fe(I1) [4S]. FE-S distances range from 2.0 to 2.4 A.

(Poulos et al., 1985,1986),also have a porphyrin-liganding group, but the axial ligands may vary. In catalase the axial groups are water and tyrosine, while in cytochrome P-450 (Fig. 24) they are cysteine and water o r oxygen. Non-heme iron proteins, such as hemerythrin (Stenkamp et al., 1981, 1984; Ward et al., 1975; Smith et al., 1983),have two iron atoms bridged by an 0x0 group. As a result [in metazido myohemerythrin from sipunculan worms, refined to 1.3 A resolution (Sheriff et al., 1987)] each iron, as shown in Fig. 25, is surrounded by three nitrogen atoms and three oxygen atoms. T h e two iron atoms are bridged by a glutamate and an aspartate. In each case one carboxyl oxygen atom is bound to one iron atom, and the other carboxyl oxygen atom is bound to the other iron

STRUCTURAL ASPECTS OF METAL LIGANDING

deoxyhemoglobin

oxy hemoglobin

T (tense)

R (relaxed)

High-spin 5-coordinate

Low-spin 6-coordinate Fe(ll)

Fe(ll)

45

FIG. 23. Diagram of hemoglobin, looking along the porphyrin plane. Fe(I1) [5N, 01.

atom. In addition an oxide anion, O", is bound to each iron. One of the iron atoms also binds azide. The Fe.-.Fe distance is 3.23 A. Note that each iron atom would be coordinated by only five atoms until azide binds. C . Manganese

Manganese can also be a catalyst. Manganese [as Mn(III)] in superoxide dismutase from Thermus thermophilus (Stalling et al., 1984, 1985) is surrounded by three histidines, one aspartate oxygen, and water in a trigonal bipyramidal arrangement. The fifth coordination site is occupied by a water molecule. In copper, zinc-superoxide dismutase (Cu,Zn = SOD), as described later, there are two metals (copper and zinc). Each bonds to and are separated by this same histidine group. D . Zinc

The stereochemistry of reactions at zinc atoms has been studied in small molecules (Auf der Heyde and Nassimbeni, 1984) and in proteins (Holmes and Matthews, 1981; Vallee and Auld, 1990a,b). Zinc enzymes include carboxypeptidase A (Quiocho and Lipscomb, 1971; Rees et al., 1983), in which the zinc is coordinated to two histidine nitrogen atoms, two glutamate oxygen atoms, and water (involved in hydrolysis) (Fig. 26).

JENNY P. GLUSKER

kcH3

Leu245

C H 3,@ ,

CH3

Camphor

CH3

HO cys357

FIG.24. (a)Atomic designations and (b) stereo view of cytochromeP-450. Fe(I1)[4N, S].

FIG. 25. (a) Atomic designations and (b) stereo view of myohemerythrin. Fe(I1) [3N, 301.

STRUCTURAL ASPECTS OF METAL LIGANDING

47

JENNY P. GLUSKER

f

Glu72

FIG. 26. (a) Atomic designations and (b) stereo view of carboxypeptidase. Zn(l1) [2N, 301.

'This is an enzyme that cleaves peptides and ester substrates, but the mechanism is still not clear. The enzyme has been refined to 1.54 8, resolution (Rees et al., 1983). Note in Fig. 26 that the zinc is coordinated to carboxylate in a bidentate manner. When zinc is substituted by Co(I1) the enzyme still works.

STRUCTURAL ASPECTS OF METAL LlGANDlNG

49

Another zinc-utilizing enzyme is carbonateldehydratase C (Kannan et al., 1972). Here, the zinc is firmly bound by three histidyl side chains and a water molecule or a hydroxyl ion (Fig. 27). The coordination is that of a distorted tetrahedron. Metals such as Cu(II), Co(II), and Mn(I1) bind at the same site as zinc. Hg(I1) also binds near, but not precisely at, this site (Kannan et al., 1972). Horse liver alcohol dehydrogenase (Schneider et al., 1983)contains two zinc sites, one catalytic and one noncatalytic. X-Ray studies showed that the catalytic Zn(II), bound tetrahedrally to two cysteines, one histidine, and water (or hydroxyl), can be replaced by Co(I1) and that the tetrahedral geometry is maintained. This is also true with Ni(I1). Insulin also binds zinc (Adams et al., 1969; Bordas et al., 1983)and forms rhombohedra1 2Zn insulin crystals. The coordination of the zinc consists of three symmetry-related histidines (from B 10) and three symmetry-related water molecules. These give an octahedral complex

po I I I

12.1 I I

I I

His94

FIG. 27. (a) Atomic designations and (b) stereo view of carbonate dehydratase. Zn(I1) [O, 3Nl.

50

JENNY P. GLUSKER

with Zn...O distances of 2.2-2.3 8, and Zn-..N distances of 2.0-2.1 A, in agreement with extended X-ray absorption fine structure results (Bordas el al., 1983). A novel property of zinc is its putative formation of “zinc fingers” (Klug and Rhodes, 1987; Berg, 1986). These are regions of protein containing four residues of histidine andlor cysteine that are considered to bind zinc and form a loop that can take part in protein-nucleic acid interactions. A tetrahedral Zn(I1)-binding site has been engineered in a protein (Regan and Clarke, 1990). The standard zinc finger contains two cysteines at a turn connecting p strands, a histidine in an a helix, and a histidine between the end of an a helix and a turn. Crystallographic examples of such metal-binding sites have been seen for copper and iron. Most such zinc fingers have been analyzed by NMR methods (Omichinski et al., 1990), but have the caveat “. . . the so-called zinc finger motif is not unique for a class of DNA binding proteins but may represent a general folding motif found in a variety of proteins irrespective of their function.” Vallee and Auld ( 1990b) noted, “Considering the vast number of articles whose titles refer to ‘zinc fingers,’ it is important to realize that the presence of zinc has been confirmed analytically in only four instances . . .” However, the crystal structure of a zinc finger has just appeared in the literature (Pavletich and Pabo, 1991). E . Magnesium

Magnesium is bound in the active site of D-xylose isomerase (Carrel1 et al., 1984, 1989; Farber et al., 1987; Rey et al., 1988; Henrick et al., 1989). Here, the site at which two metals [from among Mg(II), Mn(II), and Co(II)] bind is similar to that found for Fe(I1) in ribonucleotide reductase (Nordlund et al., 1990). T h e active site of xylose isomerase is shown in Fig. 28. Magnesium ions are preferred in ATP-utilizing enzyme reactions (Mildvan, 1987). Magnesium is also of interest as a replacement for Ca(1I) in calciumrequiring enzymes. In some of these, the replacement is simple (Lewinski and Lebioda, 1986), and in others it cannot occur. NMR studies show that magnesium can bind in the calcium sites of troponin C (Tsuda et al., 1990). The structure of turkey skeletal muscle troponin C has recently been reported (Herzberg and James, 1985). In one domain the replacement of Ca(I1) by Mg(I1) causes a conformational change, but in the other domain it does not. FIG.28. (a) Atomic designations and (b) stereo view of o-xylose isomerase. Mn(I1) [ 6 0 and 5 0 , N].

51

STRUCTURAL ASPECTS OF METAL LICANDING

'--o

'0 Asp287

Asp257

I

His220

52

JENNY P. GLUSKER

F. Calcium

In small organic crystal structures the normal coordination number of calcium varies from 6 to 10 (Einspahr and Bugg, 1980, 1981). The geometry varies from octahedra to square antiprism to the latter with additional capping ligand atoms. The binding of calcium ions to proteins has been reviewed by Strynadka and James (1989), and the reader is referred to this article for details. In general in protein calcium ions have seven oxygen ligands donated by protein backbone or side chains. Only in a few instances are there six or eight ligands. Nine oxygen atoms have not, so far, been observed around a calcium ion in a protein structure. When there are seven ligands (which may include water molecules), they may be described as lying approximately at the vertices of a pentagonal bipyramid, approximately 2.4 8, from the calcium ion in the center. Some of the ligands may be water molecules. T h e stereochemistry of the mechanism of action of staphylococcal nuclease, a calcium-utilizing enzyme, has been studied to 1.5 8, resolution (Cotton et al., 1979) but not refined. It was independently solved to 1.7 8, resolution and refined by Loll and Lattman (1989). The refined structure revealed an additional water ligand, leading to an overall coordination number of 7. The effects of mutating ligands and catalytic residues on the binding of metal ions and substrate molecules and on activity have been studied by Mildvan and Serpersu (1989). Two types of calcium-binding proteins have been identified. The first group includes enzymes stabilized by calcium ions, some of these using the calcium ions in catalysis. In these cases the calcium ligands are oxygen-containing groups from different portions of the polypeptide chain. The second group comprise proteins that bind calcium ions reversibly and, as a result, modulate the action of other proteins or enzymes. The calcium-binding site in the second group consists of a helixloop-helix motif, the loop consisting of 12 contiguous residues that provide oxygen atoms for metal liganding. In the helix-loop-helix motif (EF hand), one of the ligands deviates from the pentagonal plane. Five of the 12 residues in the loop of a helix-loop-helix motif provide oxygen ligands to the calcium ions. T h e other residues stabilize the loop by means of main-chain NH groups. T o aid in describing the helix-loop-helix motif, the structure has been compared to a right hand with an index finger (E helix), a curled second finger representing the loop and a thumb (F helix). This helix-loop-helix is therefore often referred to as an EF hand (Kretsinger and Nockolds, 1973). When such a calciumbinding motif is found, there are usually two of them, linked as pairs,

STRUCTURAL ASPECTS OF METAL LIGANDING

53

suggesting that gene duplication may have occurred (Kretsinger, 1972). EF hands, shown in Fig. 29, have a well-conserved calcium-binding site. This site is essentially octahedral, with one site shared by the two carboxyl oxygen atoms of a glutamic acid side chain. The vertices of this octahedron are designated X , Y , 2, -X, -Y, and -2 in a right-handed system, with -Y as a conserved backbone carbonyl group (Ox) and -2 as the position of the shared carboxyl group of glutamate. Some of these calcium-binding side chains are conserved. There is a strong preference for aspartic acid (D) at the X position, as it helps to maintain the required localized protein folding. -Y is usually a main-chain oxygen atom and -Xis generally a water molecule. The -2 ligand is always a glutamate (E) that coordinates both carboxylate oxygen atoms to the metal to account fully for seven groups, while - X has a strong tendency to bind water. The ordered sequence of protein folding around the calcium requires the positions -X and -Y to be inverted, as shown in Table XII. There seems to be two types of EF hands, one type structural, which can also bind magnesium, and the other type regulatory and calcium specific (Szebenyi and Moffat, 1986). Most of the EF-hand motifs have one water (-X). On the other hand, in Rhizopus chinensis aspartic proteinase (Suguna et al., 1987) there is one main-chain carbonyl oxygen bound to calcium and six water molecules to complete the pentagonal bipyramidal coordination. Calcium coordination has been measured in several viruses, such as Southern bean mosaic virus (Silva and Rossmann, 1985), satellite tobacco necrosis virus Uones and Liljas, 1984), and tomato bushy stunt virus IV (Olson et al., 1983). Calcium binding is typified in crystal structures of vitamin Ddependent calcium-binding protein from bovine intestine (Szebenyi and Moffat, 1986), which contains two EF hands; troponin C (Herzberg and James, 1985), which contains four EF hands (Fig. 30); parvalbumin (Moews and Kretsinger, 1975; Kumar et al., 1990), which contains two EF hands (Fig. 31); a-amylase (Boel et al., 1990); and calmodulin (Babu et al., 1985) (Fig. 32). Calcium-binding proteins that do not contain EF hands include bovine trypsoinogen (Fehlhammer et al., 1977; Kossiakoff et al., 1977), bovine pancreatic phospholipase A2 (Dijkstra et al., 1981), and concanavalin A (Hardman et al., 1982) (Fig. 33). In concanavalin A there are two metal sites. One is normally filled with calcium; the other, with a transition metal (Hardman et al., 1982). X-Ray absorption fine-structure spectroscopy (Lin et al., 19YO), with Zn(I1) or Cd(I1) replacing Ca(I1) and with Ni in the transition metal site, shows that, while in the crystal structure the transition metal has five ligands, it has six ligands in solution.

54

JENNY P. GLUSKER

l

D, N,s, ox, Wa

e

P

FIG. 29. EF hand showing the folding of the protein around sites X, Y, Z, -Y, -X, -Z. (a) The basic diagram of hands, (b) the identification of six locations of ligands (see Table XI1 for details of the symbols), (c) the octahedron built from (b), (d) the types of atoms found in each location (see Table XII), and (e) the direction of folding of the polypeptide chain around the calcium ion.

55

STRUCTURAL ASPECTS OF METAL LIGANDING

TABLE XI1 Calcium-Binding Ligands in Various Proteinsa Amino acid EF hands Troponin C

Calmodulin

Parvalbumin Intestinal calcium-binding protein

s-1 ooa Other Trypsin Thermolysin

Concanavalin A Phospholipase A2 Staphylococcus nuclease ~~

X

Y

Z

-Y

-

D D D D D D D D D D

-

D D N N D D D D D D

-

Wa

-

N D

-

D

-

0 D ox D

---

0 N ox D

-

0 D Ox D

-Ox - O x -Ox - O x

Wa Wa Wa D Wa D Wa Wa

-

E E E Wa D Wa D Ph

Ox D Wa D ox Wa ox D

--

-

-

-

--

-

s

D N N

D

s

Ox Ox O x O x O x O x O x O x O X O x

Ox ox ox ox Ox ox ox ox

-X -

Wa Wa Wa W a W a W a W a W a - E - W a

-

-

-

-

Wa W a Wa W a Wa E D Wa T N ox D

-2

--- - - -

E E E E E E E E E E

--

E

-

-

--

-

-

E E - E

--

E D E Wa ox D Wa Wa

~~

Amino acid side chains at the positionsx, Y , Z, -Y, -X, and -Z (illustrated in Fig. 29) of EF hands and other calcium-binding regions are listed. D, Aspartic acid; E, glutamic acid; N, asparagine; S, serine; T, threonine; Ox, main-chain carbonyl; Wa, water; Ph, phosphate 0;-, a number of intervening amino acids.

G . Copper and Zinc In Cu,Zn-SOD the copper atom is bound to three histidine groups and the zinc is bound to two histidines and an aspartate oxygen atom (Tainer et al., 1982, 1983) (Fig. 34). The Cu.-.Zn distance is 6.3 A. The zincligand geometry is tetrahedral, with a strong distortion toward a trigonal pyramid with aspartic acid at the apex. The coordination of the Cu(I1) is tetrahedrally distorted square planar. The axial position of copper is more open on the solvent side than on the protein side; probably, water is bound there. The Zn(I1) is buried, while the Cu(I1) site is solvent accessi-

JENNY P. GLUSKER

11

Asp1 10

0

-x \\,2.9

8

AsnlO8

Y

88

,

\ \

p

888

2.4

Glu117

FIG. 30. (a and c) Atomic designations and (band d) stereo view of troponin C. Ca(I1) [70].

ble. In spite of this, copper from one subunit has been found to exchange into an empty Zn(I1) site in another subunit, giving Cu,Cu-SOD (Valentine et al., 1979).

H . Copper, Zinc, and Cadmium Metallothioneins are small proteins of about 60 amino acids (20 of which are conserved cysteine residues) that bind copper, zinc, and cadmium (Melis et al., 1983; Furey et al., 1986). Solution structures have been elucidated by NMR, showing the coordination schemes (Schultze et al., 1988).

STRUCTURAL ASPECTS OF METAL LIGANDING

57

Asp146

Z 0

;2.5 Asn144

2.3 I I

0.

Asp142

Gln153

-Z

FIG. 30. (continued)

I . Zinc and Calcium In thermolysin (Matthews et al., 1974; Holmes and Matthews, 1982) zinc is bound approximately tetrahedrally to glutamate (monodentate), two histidines, and water. While zinc in the native enzyme is tetracoordinate, in some inhibitor complexes it is pentacoordinate. T h e four calcium ions are bound by six to eight oxygen ligands, as shown in Fig. 35, with Ca...O distances of 2.23-2.7 1 A. The RNA polymerase from E. cola contains Zn(I1) and Mg(II), which may be substituted by Mn(I1) (Chuknyisky et al., 1990).

JENNY P. GLUSKER

GlulOl

-2

i

f

XV. ELECTRON TRANSFER IN METALLOPROTEINS

The general properties of simple electron transfer proteins (e.g., the ferredoxins, the blue, or type 1 , copper proteins, cytochrome c, and

STRUCTURAL ASPECTS OF METAL LIGANDING

\ \

0

'\

.'

59

,'2.3

-Z L.."

backbone 51

-Y

; I

2.0 '\\

Asp51

FIG. 3 1. (continued)

cytochrome b5) have been reviewed by Williams (1990). The ligands in such proteins assist electron transfer either by reducing the charge on the metal ion or by inducing a low-spin, rather than high-spin, form of the metal. Such obliging ligands include sulfur, imidazole, and porphyrins. An essential feature of such electron transfer metalloproteins is a strong firm protein fold that can accommodate a change in charge on the metal ion without much change in the ligand geometry around that metal. In such metalloproteins the metal ions are buried inside the protein and the coordination sphere is complete; as a result of this protective shield, small ions and water are unable to reach the metal ion and interact with it.

JENNY P. GLUSKER

Asp56

X

n

\

fiyo

\

n

b a c b n e 62

-X

FIG.32. (a and c) Atomic designations and (b and d) stereo view of calmodulin Ca(I1). [ 7 0 , 701.

Changes in valence states of metals may cause large changes in metalligand distances. For example, Co(I1) complexes are high spin, while Co(II1) complexes generally are low spin (Hughes, 1981).An examination of Table VI(a) shows that this involves a radial change from 0.73 to 0.52 A, over 0.2 8,. Several factors make the electron transfer between Co(I1) and Co(II1) slow. Because of the Franck-Condon principle, which requires no movement of nuclei during electronic transitions, there are several intermediates in this electron transfer, explaining the slow rate. In order for a reaction to be fast, the geometries of the species before and after reaction must be identical. T h e protein, of course, must provide a very selective route by which electrons can enter and leave it. This is done by careful location of the metal site in the protein. First, the metal is usually located about 10 8,

STRUCTURAL ASPECTS OF METAL LIGANDING

61

Backbone 26

-Y

A

FIG. 32. (continued)

from the protein surface, below a recognition site, so that the electrons are transferred over a distance of about 10 A. In such a location the metal is greater than, say, 15 A, from any other surface, so that specificity results. Williams (1990) pointed out that, ideally, if metal coordination spheres could come in contact with each other, the rate of electron transfer could reach 10" per second (in the absence of thermodynamic

JENNY P. GLUSKER

FIG.33. (a) Atomic designations and (b) stereo view of concanavalin A. Mn(I1) [ 5 0 , N]; Ca(I1) [60].

STRUCTURAL ASPECTS OF METAL LIGANDING

63

His118

FIG. 34. (a) Atomic designations and (b) stereo view of Cu/Zn-superoxide dismutase. Cu(I1) [4N];Zn(I1) [3N, 01.

JENNY P.GLUSKER

Asp1 38 backbone 187

HN

I \

2.5

t I

1

'.

/2.5

H20'

I

0.

' c

backbone 183

I

HN

FIG. 35. (a) Atomic designations and (b) stereo view of the calcium surroundings in thermolysin. Ca(I1) [SO and 601.

STRUCTURAL ASPECTS OF METAL LIGANDING

65

barriers). However, burying the metal in order to protect it reduces the rate to a value more like lo5 per second; a compromise between rate of reaction and protection of the metal must be reached. Calcium ions are plentiful outside the cell, while low inside the cell. Mg2+,Fe2+,and Zn2+ are available intracellularly.

XVI. METALCOMPETITION AND REPLACEMENT How does a protein select out, from the sea of metal ions around it, precisely the metal it requires? T h e answer is not yet clear, but several attempts have been made to resolve this question. For example, Ca(I1) site specificity in the E. coli receptor for D-galactose and D-glucose has been examined (Snyder et al., 1990). The authors state, “Ca(I1) binding sites are faced with a considerable specificity problem: they must selectively bind a relatively rare substrate metal ion and exclude competing metal ions present in much higher concentrations.” The binding site is like an EF-hand site. The Ca(I1) site was probed with Tb(III),which has a similar size. A Tb(II1) phosphorescence assay revealed the extent of displacement of Tb(1II) by other ions. The protein, it appears, selects metal ions on the basis of both charge and size; the higher the charge, the better the binding, so that alkali metal ions are effectively excluded. Second, the metal-binding site is of the correct size to bind Ca(II), but is large for Mg(I1); however, the cavity size is flexible. Interestingly, other metals not generally available in biological systems, such as Yb(II1) and Lu(III), bind much better. Nature has only protected its systems against readily available ions. Selectivity for metals is also under active study in many laboratories. In pyruvate kinase (Buchbinder and Reed, 1990), which binds two divalent and one monovalent metal cation, the relative sizes of the divalent cations are major factors controlling selectivity. Ions smaller than Mn(I1) [Mg(II), Ni(II), Co(II), and Zn(II)] bind preferentially at the ATPbinding site, while the larger ions [Cd(Il) and Ca(II)] bind at the oxalatebinding site. A metapotyrosinase (Yong et al., 1990)binds two copper ions to give the met form [Cu(II)Cu(II)], the deoxy or reduced form [Cu(I) &(I)], or the oxy form [Cu(II) Cu(I1) OZ-1. Copper may be removed from one site by oxalate (which inactivates the enzyme), but, on storage [as for Cu,Zn-SOD (Valentine et al., 1979)], the copper migrates so that equal proportions of reconstituted holoenzyme and apoenzyme result. It is not clear why only one Cu(I1) is lost. It may be that only one is accessible, or that removal of one makes the other inaccessible. When Cu(I1) is removed in this way, it may be replaced by other ions, with

66

JENNY P. GLUSKER

affinities Cu(I1) > Co(I1) > Zn(I1). Both Cu(I1) ions are in distorted tetragonal environments, while Co(II), Zn(II), and Cu(1) prefer tetrahedral complexes. Thus, Co(I1) and Zn(I1) are more readily lost than Cu(II), the required metal. T h e active sites of enzymes are uniquely designed to result from polypeptide folding in such a way that a reaction can be catalyzed and the components of the reaction controlled. In many cases metal ions, bound to active-site groups, assist in providing the correct environment for this to occur. The suggestion has been made (Vallee and Williams, 1968)that the metal is bound in a geometry that approaches that of the transition state of the rection being catalyzed. This was termed the entatic state and highlights the importance of active-site three-dimensional structure for metals directly involved in catalysis. ACKNOWLEDGMENTS I wish to thank Andrew B. Carrell, who assisted with many of the calculations and figure preparations necessary for the production of this chapter. I also thank H. L. Carrell, G. D. Markham, A. S. Mildvan, and E. K. Patterson for many helpful discussions. Ball-and-stick drawings were drawn with the computer program VIEW (Carrell, 1976). Use of the Cambridge Structural Database (Allen et al., 1979) and the Protein Data Bank (Bernstein et al., 1977) is acknowledged. This work was supported by National Institutes of Health grants GM 44360, CA 10925, and CA 06927 and by an appropriation from the Commonwealth of Pennsylvania.

REFERENCES Adams, M. J., Blundell, T. L., Dodson, G. G., Vijayan, M., Baker, E. N., Harding, M. M., Hodgkin, D. C., Rimmer, B., and Sheet, S. (1969). Structure of rhombohedra1 2Zn insulin crystals. Nature (London) 224,49 1-495. Adman, E. T . (1979). A comparison of the structures of electron transfer proteins. Biochim. Biophys. Acta 549, 107-144. Adman, E. T., and Jensen, L. H. (1981). Structural features of azurin at 2.7 A resolution. Isr. J. Chem. 21,8-12. Adman, E. T., Watenpaugh, K. D., and Jensen, L. H. (1975). NH...S hydrogen bonds in Peptococcw aerogenes ferrodoxin, Clostridium pasteurianum rubredoxin and Chromutium high potential iron protein. Proc. Natl. Acad. Sci. U.S.A. 72,4854-4858. Adman, E. T., Sieker, L. C., and Jensen, L. H. (1976). Structure of Peptococcw aerogenes ferredoxin. Refinement at 2 A reso1ution.J. Biol. Chem. 251,3801-3806. Adman, E. T., Sieker, L. C., Jensen, L. H., Bruschi, M., and LeGall, J. (1977). A structural model of rubredoxin from Desulfouibrio vulgaris at 2 ,h resolution.]. Mol. Biol. 112, 113-120.

Adman, E. T., Stenkamp, R. E., Sieker, L. C., and Jensen, L. H. (1978). A crystallographic model for azurin at 3 A resolution. J. Mol. B i d . 123, 35-47.

STRUCTURAL ASPECTS OF METAL LIGANDING

67

Ahrland, S., Chatt, J., and Davies, N. R. (1958).The relative affinities of ligand atoms for acceptor molecules and ions. Rev. Chem. SOC. 11,265-271. Allen, F. H., Bellard, S., Brice, M. D., Cartwright, B. A., Doubleday, A., Higgs, H., Hummelink, T., Hummelink-Peters, B. G., Kennard, O., Motherwell, W. D. S., Rodgers, J. R., and Watson, D. G. (1979). The Cambridge Crystallographic Data Centre: Computerbased search, retrieval, analysis and display of information. Actu Crystallogr. Sect. B 35, 2331-2339. Allred, A. L., and Rochow, E. G. (1958). A scale of electronegativity based on electrostatic force. J. Inorg. Nucl. Chem. 5,264-268. Armstrong, W. H. (1988). Metalloprotein crystallography. Survey of recent results and relationships to model studies. ACS Symp. Ser. 372, 1-27. Auf der Heyde, T. P. E., and Nassimbeni, L. R. (1984). Reaction pathways from structural data: Dynamic stereochemistry of zinc(I1) compounds. Acta Crystallogr. Sect. B 40, 582-590. Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means, A. R., and Cook, W. J. (1985).Three-dimensional structure of calmodulin. Nature (London) 315,37-40. Bacon, G. E., and Curry, N. A. (1962). The water molecules in CuS04.5H20. Proc. R . SOC. London, Ser. A 266,95-108. Baker, E. N., and Hubbard, R. E. (1984). Hydrogen bonding in globular proteins. Prog. Biophys. Mol. Biol. 44,97-179. Baur, W. H. (1970). Bond length variation and distorted coordination polyhedra in inorganic crystals. Trans. Am. Crystallogr. Assoc. 6, 129-155. Beevers, C. A., and Lipson, H. (1934). Crystal structure of CuS04.5H20. Proc. R . SOC. London, Ser. A 146,570-582. Berg, J. M. (1986). Potential metal-binding domains in nucleic acid binding proteins. Science 232,485-487. Bergerhoff, G., Hundt, R., Severs, R., and Brown, I. D. (1983). The inorganic crystal structure database. J. Chem. Inf. Comput. Sci. 23,66-69. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr., Brice, M.D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977). The Protein Data Bank: A computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535-542. Blundell, T . L., and Johnson, L. N. (1976).“Protein Crystallography.” Academic Press, New York. Boel, E., Brady, L., Brzozowski, A. M., Derewenda, Z., Dodson, G. G., Jensen, V. J., Petersen, S. B., Swift, H., Thim, L., and Woldike, H. F. (1990). Calcium binding in a-amylases: An X-ray diffraction study at 2.1-A resolution for two enzymes from Aspergillus. Biochemistry 29, 6244-6249. Bordas, J., Dodson, G. G., Grewe, H., Koch, M. H. J., Krebs, B., and Randall, J. (1983). A comparative assessment of the zinc-protein coordination in 2Zn-insulin as determined by X-ray absorption fine structure (EXAFS)and X-ray crystallography. Proc. R . SOC.London, Ser. B 219,21-39. Breslow, E. (1973). Metal-protein complexes. In “Inorganic Biochemistry” (G.L. Eichhorn, ed.), pp. 227-249. Elsevier, Amsterdam. Brown, I. D. (1978). Bond valences-A simple structural model for inorganic chemistry. Chem. SOC.Rev. 7, 359-376. Brown, I. D. (1980). A structural model for Lewis acids and bases. An analysis of the structural chemistry of acetate and trifluoroacetate ions. J. Chem. SOC.,Dalton Trans. pp. 1118-1123. Brown, I. D. (1987). Recent developments in the bond valence model of inorganic bonding. Phys. Chem. Miner. 15,30-34.

68

JENNY P. GLUSKER

Brown, I. D. (1988). What factors determine cation coordination numbers? Actu Crystallogr. Sect. B 44,545-553. Brown, I. D., and Faggiani, R. (1980). The structure of thallium(1) tetraacetatothallate(II1): When is the lone pair of electrons on TI’ stereoactive? Actu Ctystullogr. Sect. B . 3 6 , 1802- 1806. Brown, I. D., and Shannon, R. D. (1973). Empirical bond-strength-bond-length curves for oxides. Acta Crystullogr. Sect. A 29, 266-282. Brown, I. D., and Skowron, A. (1990). Electronegativity and Lewis acid strength. J . Am. Chem. Sac. 112,3401- 3403. Buchbinder, J. L., and Reed, G. H. (1990). Electron paramagnetic resonance studies of the coordination schemes and site selectivities for divalent metal ions in complexes of pyruvate kinase. Biochemistry 29, 1799- 1806. Buckingham, D. A. (1973). Structure and stereochemistry of coordination compounds. In “Inorganic Biochemistry” (G. L. Eichhorn, ed.), pp. 3-6 1 . Elsevier, Amsterdam. Burgi, H.-B. (1975). Stereochemistry of reaction paths as determined from crystal structure data-A relationship between structure and energy. Angew Chem., Int. Ed. Engl. 14, 460-473. Candlen, R., and Harding, M. M. (1967). The crystal structure of bis(histidin0) cadmium dihydrate. J. Chem. Sac. A 42 1-423. Carrell, H. L. (1976). Computer program VIEW. Inst. Cancer Res. Fox Chase Cancer Cent., Philadelphia, Pennsylvania. Carrell, H. L., and Glusker, J. P. (1973). Manganous citrate decahydrate. Actu Crystullogr. Sect. B 29,638-640. Carrell, C. J., Carrell, H. L., Erlebacher, J., and Glusker, J. P. (1988). Structural aspects of metal ion-carboxylate interactions. J . Am. Chem. Sac. 110,865 1-8656. Carrell, H. L., Glusker, J. P., Job, R., and Bruice, T. C. (1977). A synthetic tetranuclear iron-sulfur complex with ionized side chains: The crystal structure of (Fe4S4(S(CH2)2C00)4)6-.(Na5.N(C4H9)4)6+.5CsHgN0. J . Am. Chem. Soc. 99,36833690. Carrell, H. L., Rubin, B. H., Hurley, T. J., and Glusker, J. P. (1984). X-ray crystal structure of D-xylose isomerase at 4 A reso1ution.J. Biol. Chem. 259,3230-3236. Carrell, H. L., Glusker, J. P., Burger, V., Manfre, F., Tritsch, D., and Biellmann, J.-F. (1989). X-Ray analysis of o-xylose isomerase at 1.9 A: Native enzyme in complex with substrate and with a mechanism-designed inactivator. Proc. Nutl. Acud. Sci. U.S.A. 8 6 , 4440-4444. Carter C. W., Jr. (1977). New stereochemical analogies between iron-sulfur electron transport pr0teins.J. Biol. Chem. 252,7802-781 1. Chakrabarti, P. (1990). Systematics in the interaction of metal ions with the main-chain carbonyl group in protein structures. Biochemistry 29, 651-658. Chandrasekhar, K., and Burgi, H.-B. (1984). Dynamic processes in crystals examined through difference vibrational parameters AU: The low-spin-high-spin transition in tris(dithiocarbamato)iron(III)complexes. Actu Crystallogr., Sect. B 40, 387-397. Chuknyisky, P. P., Rifiind, J. M., Tarien, E., Beal, R. B., and Eichhorn, G. L. (1990). Structural studies on the active site of Escherichiu coli RNA polymerase. I. Interactions of metals on the i and i+ 1 sites. Biochemistry 29, 5987-5994. Cotton, F. A., Hazen, E. E., Jr., and Legg, M. J . (1979). Staphylococcal nuclease: Proposed mechanism of action based on structure of enzyme-thymidine 3’,5’-bisphosphatecalcium ion complex at 1.5-A resolution. Proc. Nutl. Acad. Scz. U.S.A. 76, 2551-2555. Dent-Glasser, L. S. (1981). Aluminum: When is it like silicon? Actu Crystallogr. Sect. A 3 7 , (2-162.

STRUCTURAL ASPECTS OF METAL LIGANDING

69

Dickerson, R. E., Eisenberg, D., Varnum, J., and Kopka, M. L. (1969). PtCI,’-: A methionine-specific label for protein crystallography.]. Mol. Biol. 45, 77-84. Dijkstra, B. W., Kalk, K. H., Hol, W. G. J., and Drenth, J. (1981). Structure of bovine pancreatic phospholipase A2 at 1.7 A reso1ution.J. Mol. Biol. 147,97-123. Edwards, S . L., Xuong, N. H., Hamlin, R. C., and Kraut, J. (1987). Crystal structure of cytochrome c peroxidase compound I. Biochemistry 26, 1503- 15 1 1 . Einspahr, H., and Bugg, C. E. (1980). The geometry of calcium-water interactions in crystalline hydrates. Acta Crystallogr. Sect. B 36, 264-271. Einspahr, H., and Bugg, C. E. (1981). The geometry of calcium-carboxylate interactions in crystalline complexes. Acta Crystallogr. Sect. B 37, 1044-1052. Elder, R. C., and Trkula, M. (1974). Structural trans effect in sulfur bound sulfitopentaarnminecobalt(II1) chloride. J . Am. Chem. SOC.96,2635. Elder, R. C., Heeg, M. J., Payne, M. D., Trkula, M., and Deutsch, E. (1978).trans Effect in octahedral complexes. 3. Comparison of kinetic and structural trans effects induced by coordinated sulfur in sulfito- and sulfinatopentaamminecobalt(II1) complexes. Inorg. Chem. 17,431-440. Fajans, K. (1923). Structure and deformation of electron sheaths and their significance for the optical and chemical properties of inorganic compounds. Natunuissenschaften 11, 165-172. Farber, G . K., Petsko, G. A., and Ringe, D. (1987). The 3.0 8, crystal structure of xylose isomerase from Streptomyces olivochromogenes. Protein Eng. 1,459-466. Fehlhammer, H., Bode, W., and Huber, R. (1977). Crystal structure of bovine trypsinogen at 1.8 8, resolution. 11. Crystallographic refinement, refined crystal structure and comparison with bovine trypsin.J. Mol. Biol. 111,415-438. Freeman, H. C. (1973). Metal complexes of amino acids and peptides. In “Inorganic Biochemistry” (G. L. Eichhorn, ed.), pp. 121-166. Elsevier, Amsterdam. Freeman, H. C., and Golomb, M. L. (1970). Model compounds for metal-protein interaction: Crystal structure of three platinum(I1) complexes of L- and DL-methionine and glycyl-L-methionine.Chem. Commun. pp. 1523-1524. Freer, S. T., Alden, R. A., Carter, C. W., Jr., and Kraut, J. (1975).Crystallographic structure refinement of Chromatium high potential iron protein at two angstroms resolution. J . Biol. Chem. 250,46-54. Frey, M., Sieker, L., Payan, F., Haser, R., Bruschi, M., Pepe, G., and LeGall, J. (1987). Rubredoxin from Desulfovibrio gigas. A molecular model of the oxidized form at 1.4 A reso1ution.J. Mol. Biol. 197,525-541. Fukui, K. (1971). Recognition of sterochemical paths by orbital interaction. Acc. Chem. Res. 4, 57-64. Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara, T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., and Matsubara, H. (1980).Structure of S. platemis [2Fe-2S] ferredoxin and evolution of chloroplast-type ferredoxins. Nature (London) 286, 522-524. Fung, C. H., Mildvan, A. S., Allerhand, A., Komoroski, R., and Scrutton, M. C. (1973). Interaction of pyruvate with pyruvate carboxylase and pyruvate kinase as studied by paramagnetic effects on 13C relaxation rates. Biochemistry 12, 620-629. Furey, W. F., Robbins, A. H., Clancy, L. L., Winge, D. R., Wang, B. C., and Stout, D. C. (1986).Crystal structure of Cd, Zn metallothionein. Science 231,704-7 10. Gandour, R. D., (1981). On the importance of orientation in general base catalysis by carboxylate. Bioorg. Chem. 10, 169-176. Gaykema, W. P. J., Hol, W. G. J., Vereijken, J. M., Soeter, N. M., Bak, H. J., and Beintema, J . J. (1984). 3.2 8, Structure of the copper-containing, oxygen-carrying protein Panulirus antemptus haemocyanin. Nature (London) 309,23-29.

70

JENNY P.GLUSKER

Gaykema, W. P. J., Volbeda, A., and Hol, W. G. J . (1985). Structure determination of Panulil-ls intemptus haemocyanin at 3.2 A resolution. Successful phase extension by sixfold density averaging. J . Mol. Biol. 187,255-275. Gelin, B. R., Lee, A. W.-M., and Karplus, M. (1983). Hemoglobin tertiary structural change on ligand binding. Its role in the co-operative mechanism.J. Mol. Biol. 171,489-559. Glusker, J. P. (1980). Citrate conformation and chelation: Enzymatic implications. Acc. Chem. Res. 13, 345-352. Goldschmidt, V. M. (1926). Geochemishe Verteilungsgesetze, VII: Die Gesetze der Krystallochemie (nach Untersuchungen gemeinsam mit T. Barth, G. Lunde, and W. H. Zachariasen). Skr. Nor. Vidensk.-Akad., [Kl.] I: Mat.-Naturvidensk. K1. 2. Goldschmidt, V. M. (1929). Crystal structure and chemical constitution. Trans. Faraday SOL. 25,253-283. Gurd, F. R. N., and Wilcox, P. E. (1956). Complex formation between metallic cations and proteins, peptides, and amino acids. A d a Protein Chem. 11,311-418. Guss, J. M., and Freeman. H. C. (1983).Structure of oxidized poplar plastocyanin at 1.6 A reso1ution.J. Mol. Biol. 169, 521-563. Guss, J. M., Harrowell, P. R., Murata, M., Norris, V. A., and Freeman, H. C. (1986). Crystal structure analyses of reduced (Cu’) poplar plastocyanin at six pH values./. Mol. Biol. 192,361-387. Hardman, K. D., Agarwal, R. C., and Freisner, M. J. (1982). Manganese and calcium binding sites of concanavalin A. J. Mol. Biol. 157,69-86. Hay, R. W. (1984). “Bio-inorganic Chemistry.” Halsted, New York. He, F.-C., Liu, L.-B., and Li, X.-Y. (1990). Molecular models constructed in an easy way. Part 1 . Models of tetrahedron, trigonal bipyramid, octahedron, pentagonal bipyramid and capped octahedron. J . Chem. Educ. 67,556-558. Henrick, K., Collyer, C. A., and Blow, D. M. (1989). Structures of D-xyloseisomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and ~-sorbitolat 2.5 A and 2.3 A resolution, respective1y.J. Mol. Bzol. 208, 129-157. Herriott, J. R., Sieker, L. C., Jensen, L. H., and Lovenberg, W. (1970). Structure of rubredoxin: An X-ray study at 2.5 A reso1ution.J. Mol. Biol.50, 391-406. Herzberg, O., and James, M. N. G. (1985). Structure of the calcium regulatory muscle protein troponin-C at 2.8 A resolution. Nature (London) 313,653-659. Higaki, J. N., Haymore, B. L., Chen, S., Fletterick, R. J., and Craik, C. S. (1990). Regulation of serine protease activity by an engineered metal switch. Biochemistry 29,8582-8586. Hoard, J. L. (1966). Stereochemistry of porphyrins. In “Hemes and Hemoproteins” (B. Chance, R. W. Estabrook, and T. Yonetani, eds.), pp. 9-24. Academic Press, New York. Holm, R. H. (1975). Iron-sulphur clusters in natural and synthetic systems. Endeavour 34, 38-43. Holmes, M. A., and Matthews, B. W. (1981). Binding of hydroxamic acid inhibitors to crystalline thermolysin suggests a pentacoordinate zinc intermediate in catalysis. BiochemGtry 20,6912-6920. Holmes, M. A., and Matthews, B. W. (1982). Structure of thermolysin refined at 1.6 A reso1ution.J. Mol. Biol. 160,623-639. Hossain, M. B., Jalal, M. A. F., Benson, B. A., Barnes, C. L., and van der Helm, D. (1987). Structure and conformation of two coprogen-type siderophores: Neocoprogen I and neocoprogen 11.J . Am. Chem. SOC.109,4948-4954. Hsu, L.-Y., and Nordman, C. E. (1983). Structures of two forms of sodium acetate, Na+*C2H302-.Acta Crystallop., Sect. C 39, 690-694. Hughes, M. N. (1981).“The Inorganic Chemistry of Biological Processes,” 2nd Ed. Wiley, Chichester, England.

STRUCTURAL ASPECTS OF METAL LIGANDING

71

Huheey, J. E. (1983).“Inorganic Chemistry. Principles of Structure and Reactivity,”3rd Ed. Harper & Row, New York. Irving, H., and Williams, R. J. P. (1948). Order of stability of metal complexes. Nature (London) 162,746. Irving, H., and Williams, R. J. P. (1953).Stability of transition metal comp1exes.J. Chem. Soc. pp. 3192-3210. Jahn, H. A., and Teller, E. (1937). Stability of polyatomic molecules in degenerate electronic states. I. Orbital degeneracy. Proc. R. Soc. London, Ser. A 161, 220-235. Jalal, M. A. F., Love, S. K., and van der Helm, D. (1988). N”-Dimethylcoprogens. Three novel trihydroxamate siderophores from pathogenic fungi. Biol. Met. 1,4-8. Johnson, C. K. (1965). X-Ray crystal analysis of the substrates of aconitase. V. Magnesium Acta Crystallogr. 18, citrate decahydrate [Mg(Hz0)6][MgC6H507(HzO)]2.2H20. 1004-1018. Jones, T . A., and Liljas, L. (1984).Structure of satellite tobacco necrosis virus after crystallographic refinement at 2.5 A reso1ution.J. Mol. Biol. 177, 735-767. Kannan, K. K., Liljas, A., Waara, I., Bergsten, P.-C., Lovgren, S., Strandberg, B., Bengtsson, U . , Carlbom, U., Fridborg, K., Jarup, L., and Petef, M. (1972). Crystal structure of human erythrocyte carbonic anhydrase C. VI. The three-dimensional structure at high resolution in relation to other mammalian carbonic anhydrases. Cold Spring Harbor Symp. @ant. Biol. 36,221-23 1 . Kauffman, G . B., Brock, W. H., Jensen, K. A., and JZrgensen, C. K. (1983). Ligand.J. Chem. E d w . 60,509-5 10. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R. G., and Davies, D. R. (1960). Structure of myoglobin. A three-dimensional Fourier synthesis at 2 A resolution. Nature (London) 185,422-427. Klopman, G. (1968).Chemical reactivity and the concept of charge- and frontier-controlled reactions. J. Am. Chem. Soc. 90,223-234. Klug, A., and Rhodes, D. (1987). Zinc fingers: A novel protein motif for nucleic acid recognition. Trends Biochem. Sci. 12,464-469. Kossiakoff, A. A., Chambers, J. L., Kay, L. M., and Stroud, R. M. (1977).Structure of bovine trypsinogen at 1.9 8, resolution. Biochemistry 16,654-664. Kretsinger, R. H. (1972). Gene triplication deduced from the tertiary structure of a muscle calcium binding protein. Nature (London)New Biol. 240,85-88. Kretsinger, R. H., and Nockolds, C. E. (1973)., Carp muscle calcium-binding protein. 11. Structure determinations and general descripti0n.J. Biol. Chem. 248,33 13-3326. Kruger, G. J., and Reynhardt, E. C. (1978). Hexaamminecobalt(II1) chloride. Acta Crystallogr., Sect. B 34,9 15-9 17. Kumar, V. D., Lee, L., and Edwards, B. F. P. (1990). Refined crystal structure of calciumliganded carp parvalbumin 4.25 at 1.5-A resolution. Biochemistry 29, 1404-1412. Lewinski, K., and Lebioda, L. (1986). Preferred geometry of cation-amide binding. Crystal structure of [Mg(NMA)2(H20)4(N0,)2 and Ca(NO& . 4NMA(NMA = N methylacetamide). J . Am. Chem. Soc. 108,3693-3696. Lewis, G . N. (1923). “Valence and the Structure of Atoms and Molecules.”Chem. Catalog Co., New York. Liao, D., Carrell, A. B., Carrell, H. L., and Glusker, J. P. (1992). Manuscript in preparation. Lin, S.-L., Stern, E. A., Kalb (Gilboa), A. J., and Zhang, Y. (1990). Evidence from X-ray absorption fine structure spectroscopy for significant differences in the structure of concanavalin A in solution and in the crystal. Biochemistry, 29,3599-3603. Loll, P. J., and Lattman, E. E. (1989). The crystal structure of the ternary complex of staphylococcal nuclease, CA2+,and the inhibitor pdTp, refined at 1.65 A. Proteins: Struct., Funct. Genet. 5, 183-201.

72

JENNY P.GLUSKER

Lovenberg, W. L., ed. (1973a). “Iron-Sulfur Proteins,” Vol. 1. Academic Press, New York. Lovenberg, W. L., ed. (1973b). “Iron-Sulfur Proteins,” Vol. 2 Academic Press, New York. Lovenberg, W. L., ed. (1977). “Iron-Sulfur Proteins,” Vol. 3. Academic Press, New York. Matthews, B. W., Weaver, L. H., and Kester, W. R. (1974). The conformation of thermolysin.J. Biol. Chem. 249,8030-8044. Mauk, A. G., Scott, R. A., and Gray, H. B. (1980). Distances of electron transfer to and from metalloprotein redox sites in reactions with inorganic complexes. J. Am. Chem. SOC. 102,4360-4363. McRee, D. E., Richardson, D. C., Richardson, J. S., and Siegel, L. M. (1986). The heme and Fe4S4 cluster in the crystallographic structure of Escherichia coli sulfite reductase. J. Biol. Chem. 261, 10277-10281. Melis, K. A., Carter, D. C., Stout, C. D., and Winge, D. R. (1983). Single crystals of cadmium, zinc metal1othionein.J. Biol. Chem. 258,6255-6257. Mildvan, A. S. (1970). Metals in enzyme catalysis. Enzymes 2, 445-536. Mildvan, A. S. (1987). Role of magnesium and other divalent cations in ATP-utilizing enzymes. Magnesium 6,28-33. Mildvan, A. S., and Serpersu, E. H. (1989). Genetic alteration of active site residues of staphylococcal nuclease: Insights into the enzyme mechanism. In “Metal Ions in Biological Systems” (H. Sigel and A. Sigel, eds.), pp. 309-334. Dekker, New York. Moews, P. C., and Kretsinger, R. H. (1975). Refinement of the structure of carp muscle calcium-binding parvalbumin by model building and difference Fourier analysis. J . Mol. Biol. 91, 201-228. Murray-Rust, P., Biirgi, H.-B., and Dunitz, J. D. (1975). Chemical reaction paths. V. The SN1 reaction of tetrahedral molecu1es.J. Am. Chem. SOC.97,921-922. Murray-Rust, P., Stallings, W. C., Monti, C. T., Preston, R. M., and Glusker, J. P. (1983). Intermolecular interactions of the C-F bond: The crystallographic environment of fluorinated carboxylic acids and related structures. J . Am. Chem. SOC.105, 32063214. Neilands, J. B. (1973). Microbial iron transport compounds (siderochromes). In “Inorganic Biochemistry” ( C . Eichhorn, ed.), p. 167-200. Elsevier, Amsterdam. Nordlund, P., Sjoberg, B.-M., and Eklund, H. (1990). Three-dimensional structure of the free radical protein of ribonucleotide reductase. Nature (London) 345,593-598. Norris, G. E., Anderson, B. F., and Baker, E. N. (1983). Structure of azurin from Alcaligenes denitnjicans at 2.5 A reso1ution.J. Mol. Biol. 165,501-521. Norris, G. E., Anderson, B. F., and Baker, E. N. (1986). Blue copper proteins. The copper site in azurin from Alcaligenes denitnjicans. J . Am. Chem. Soc. 108,2784-2785. OConnor, B. H., and Dale, D. H. (1966). A neutron diffraction analysis of the crystal structure of tetragonal nickel sulfate hexadeuterate. Acta C9stalLogr. 21, 705-709. Olson, A. J., Bricogne, G., and Harrison, S. C. (1983). Structure of tomato bushy stunt virus IV. The virus particle at 2.9 A reso1ution.J. Mol. Biol. 171,61-93. Omichinski, J. G., Clore, G. M., Appella, E., Sakaguchi, K., and Gronenborn, A. M. (1990). High-resolution three-dimensional structure of a single zinc finger from a human enhancer binding protein in solution. Biochemist9 29, 9324-9334. Orgel, L. E. (1966). “An Introduction to Transition-Metal Chemistry. Ligand-Field Theory,” 2nd Ed. Methuen, LondonIWiley, New York. Pauling, L. (1929). The principles determining the structure of complex ionic crystals. J . Am. Chem. SOC.51, 1010-1026. Pauling, L. (1948). “The Nature of the Chemical Bond.” Cornell Univ. Press, Ithaca, New York. Pavletich, N. P., and Pabo, C. 0.(1991).Zinc-finger DNA recognition: Crystal structure of a Zif268-DNA complex at 2.1 A. Science 252,809-817.

STRUCTURAL ASPECTS OF METAL LIGANDING

73

Pearson, R. G. (1966). Acids and bases. Science 151, 172-177. Pearson, R. G. (1968a). Hard and soft acids and bases. HSAB, Part I, Fundamental principles.J. Chem. Educ. 45,581-587. Pearson, R. G. (1968b). Hard and soft acids and bases. HSAB, Part 11, Underlying theories. J . Chem. Educ. 45,643-648. Pearson, R. G. (1986). Absolute electronegativity and hardness correlated with molecular orbital theory. Proc. Natl. Acad. Sci. U.S.A. 83, 8440-8441. Perutz, M. F. (1970). Stereochemistry of cooperative effects in haemoglobin. Nature (London) 228, 726-734. Perutz, M. F., Muirhead, H., Cox, J. M., Goaman, L. C. G., Matthews, F. S., McGandy, E. L., and Webb, L. E. (1968). Three-dimensional Fourier synthesis of horse oxyhaemoglobin at 2.8 8, resolution: ( 1 ) X-Ray analysis. Nature (London) 219, 29-32. Perutz, M. F., Fermi, G., Luisi, B., Shaanan, B., and Liddington, R. C. (1987). Stereochemistry of cooperative mechanisms in hemoglobin. Acc. Chem. Res. 20, 309-32 1 . Peterson, M. R., and Csizmadia, I. G. (1979). Determination and analysis of formic acid conformational hypersurface. J. Am. Chem. SOC.101, 1076- 1079. Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985).The 2.6 8, crystal structure of Pseudomonas putida cytochrome P-450.1. Biol. Chem. 260, 1612216130. Poulos, T. L., Finzel, B. C., and Howard, A. (1986). Crystal structure of substrate-free Pseudomonas putida cytochrome P-450. Biochemistry 25,5314-5322. Quiocho, F. A., and Lipscomb, W. N. (1971). Carboxypeptidase A: A protein and enzyme. Adv. Protein Chem. 25, 1-78. Rasmussen, H. (1990). The complexities of intracellular Ca2+ signalling (Ernst Klenk Lecture, November 1989). Biol. Chem. Hoppe-Seyler 371, 191-206. Rebek, J., Jr., Marshall, L., Wolak, R., Parris, K., Killoran, M., Askew, B., Nemeth, D., and Islam, N. (1985). Convergent functional groups: Synthetic and structural studies. J.Am. Chem. SOC.107,7476-7481. Rebek, J., Jr., Duff, R. J., Gordon, W. E., and Parris, K. (1986). Convergent functional groups provide a measure of stereoelectronic effects at carbonyl oxygen. J. Am. Chem. SOC.108,6068-6069. Rees, D. C., Lewis, M., and Lipscomb, W. N. (1983). Refined crystal structure of carboxypeptidase A at 1.56 8, resolution. J. Mol. Biol. 168,367-387. Regan, L., and Clarke, N. D. (1990). A tetrahedral zinc(I1)-binding site introduced into a designed protein. Biochemistry 29, 10878-10883. Rey, F., Jenkins, J., Janin, J., Lasters, I., Alard, P., Claessens, M., Matthyssens, G., and Wodak, S. (1988). Structural analysis of the 2.8 8, model of xylose isomerase from Actinoplanes missouriensis. Proteins: Struct., Funct. Genet. 4, 165- 172. Rosenfield, R. E., Parthasarathy, R., and Dunitz, J. D. (1977). Directional preferences of nonbonded atomic contacts with divalent sulfur. I. Electrophiles and nucleophiles. J.Am. Chem. SOC.99,4860-4862. Rosenfield, R. E., Jr., Swanson, S. M., Meyer, E. F., Carrell, H. L., and Murray-Rust, P. (1984). Mapping the atomic environment of functional groups: Turning 3D scatter plots into pseudo-density contours. J. Mol. Graphics 2,43-46. Scheidt, W. R., and Chiprnan, D. M. (1986). Preferred orientation of imidazole ligands in metalloporphyrins. J. Am. Chem. SOC. 108, 1163-1 167. Scheidt, W. R., and Lee, Y. J. (1987). Recent advances in the stereochemistry of metallotetrapyrroles. Struct. Bonding 64, 1-70. Schmidbaur, H., Classen, H. G., and Helbig, J. (1990).Aspartic and glutamic acid as ligands to alkali and alkaline-earth metals: Structural chemistry as related to magnesium therapy. Angew. Chem., Int. Ed. Engl. 29, 1090-1 103.

74

JENNY P.GLUSKER

Schneider, G., Eklund, H., Cedergren-Zeppezauer, E., and Zeppezauer, M. (1983). Crystal structures of the active site in specifically metal-depleted and cobalt-substituted horse liver alcohol dehydrogenase derivatives. Proc. Natl. Acad. Sci. U.S.A. 80, 5289-5293. Schultze, P., Worgotter, E., Braun, W., Wagner, G., VaSAk, M., Kagi, J. H. R., and Wiithrich, K. (1988). Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy. J. Mol. B i d . 203, 251-268. Schwarzenbach, G. (1961). The general, selective, and specific formation of complexes by metallic cations. Adv. Inorg. Radiochem. 3, 257-27 1 . Shannon, R. D. (1976). Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides. Acta Crystallop. Sect. A 32, 751-767. Shannon, R. D., and Prewitt, C. T. (1969). Effective ionic radii in oxides and fluorides. Acta Crystallop., Sect. B B25,925-946. Shepard, W. E. B., Anderson, B. F., Lewandoski, D. A., Norris, G. E., and Baker, E. N. (1990). Copper coordination geometry in azurin undergoes minimal change on reduction of copper(I1) to copper(I).J. Am. Chem. SOC.112,7817-7819. Sheriff, S., Hendrickson, W. A., and Smith, J. L. (1987). Structure of myohemerythrin in the azidomet state at 1.7/1.3 8, reso1ution.J. Mol. Biol. 197, 273-296. Silva, A. M., and Rossmann, M. G. (1985).The refinement of southern bean mosaic virus in reciprocal space. Acta Crystallop., Sect. B 41, 147-157. Smith, J. L., Hendrickson, W. A., and Addison, A. W. (1983). Structure of trimeric haemerythrin. Nature (London) 303, 86-88. Smith, S. O., Farr-Jones, S., Griffin, R. G., and Bachovchin, W. W. (1989). Crystal versus solution structures of enzymes: NMR spectroscopy of a crystalline serine protease. Science 244,96 1-964. Snyder, E. E., Buoscio, B. W., and Falke, J. J. (1990). Calcium(I1) site specificity: Effect of size and charge on metal ion binding to an EF-hand-like site. Biochemistry 29, 39373943. Stallings, W. C., Pattridge, K. A., Strong, R. K., and Ludwig, M. L. (1984). Manganese and iron superoxide dismutases are structural homo1ogs.J. B i d . Chem. 259,10695- 10699. Stallings, W. C., Pattridge, K. A., Strong, R. K., and Ludwig, M. L. (1985).The structure of manganese superoxide dismutase from Thermzcs thermophilus HB8 at 2.4-A resolution. J . Biol. Chem. 260, 16424-16432. Stenkamp, R. E., Siecker [sic], L. C., Jensen, L. H., and Sanders-Loehr, J. (1981). Structure of the binuclear ion complex in metazidohaemerythrin from Themiste dyscritum at 2.2. 8, resolution. Nature (London) 291,263-264. Stenkamp, R. E., Sieker, L. C., and Jensen, L. H. (1984). Binuclear iron complexes in methemerythrin and azidomethemerythrin at 2.0-8, reso1ution.J. Am. Chem. Sac. 106, 618-622. Strynadka, N. C. J., and James, M. N. G. (1989). Crystal structures of the helix-loop-helix calcium-binding proteins. Annu. Rev. Biochem. 58,95 1-958. Suguna, K., Bott, R. R., Padlan, E. A., Subramanian, E., Sheriff, S., Cohen, G. H., and Davies, D. R. (1987). Structure and refinement at 1.8 8, resolution of the aspartic proteinase from Rhizopzcs chinensis. J. Mol. Biol. 196,877-900. Szebenyi, D. M. E., and Moffat, K. (1986). The refined structure of vitamin D-dependent calcium-binding protein from bovine intestine. Molecular details, ion binding, and implications for the structure of other calcium-binding proteins. J. Biol. Chem. 261, 8761-8777. Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982). Determination and analysis of the 2 A structure of copper, zinc superoxide dismutase. J . Mol. B d . 160, 181-217.

STRUCTURAL ASPECTS OF METAL LIGANDING

75

Tainer, J. A., Getzoff, E. D., Richardson, J. S., and Richardson, D. D. (1983). Structure and mechanism of copper, zinc superoxide dismutase. Nature (London) 306, 284287. Tsuda, S., Ogura, K., Hasegawa, Y., Yagi, K., and Hikichi, K. (1990). ’H NMR study of rabbit skeletal muscle troponin C: Mg”-Induced conformational change. Biochemistry 29,4951-4958. Tsukihara, T., Homma, K., Fukuyama, K., Katsube, Y., Hase, T., Matsubara, H., Tanake, N., and Kakudo, M. (1981). Preliminary X-ray diffraction studies on a [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus. J. Mol. Biol. 152,82 1-823. Vainshtein, B. K., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A. A., Grebenko, A. I., Borisov, V. V., Bartels, K. S., Fita, I., and Rossmann, M. G. (1986).Three-dimensional structure of catalase from Penicillium d a l e at 2.0 A resolution. J . Mol. B i d . 188, 49-6 1. Valentine, J. S., Pantoliano, M. W., McDonnell, P. J., Burger, A. R., and Lippard, S. J. (1979). pH-dependent migration of copper(l1) to the vacant zinc-binding site of zinc-free bovine erythrocyte superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 76, 4245-4249. Vallee, B. L., and Auld, D. S. (1990a). Active-site zinc ligands and activated H2O of zinc enzymes. Proc. Natl. Acad. Sci. U.S.A. 87,220-224. Vallee, B. L., and Auld, D. S. (1990b). Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29,5647-5659. Vallee, B. L., and Williams, R. J. P. (1968). Metalloenzymes:The entatic state of their active sites. Proc. Natl. Acad. Sci. U.S.A. 59, 498-505. van der Helm, D., Jalal, M. A. F., and Hossain, M. B. (1987). The crystal structures, conformations and configurations of siderophores. I n “Iron Transport in Microbes, Plants and Animals” (G. Winkelmann, D. van der Helm, and J. B. Neilands, eds.), pp. 135-165. VCH Publ., Weinheim, Germany. Vedani, A., and Huhta, D. W. (1990). A new force field for modeling metalloproteins. J.Am. Chem. SOC. 112,4759-4767. Vedani, A., Dobler, M., and Dunitz, J. D. (1986). An empirical potential function for metal centers: Application to molecular mechanics calculations on metallo proteins. J. Comput. Chem. 7,701-710. Vedavathi, B. M., and Vijayan, K. (1977). Radioprotectant sodium fluoroacetate. Acta Crystallogr., Sect. B 33, 946-948. Voet, D., and Voet, J. G. (1990). “Biochemistry.” Wiley, New York. Ward, K. B., Hendrickson, W. A., and Klippenstein, G. L. (1975). Quaternary and tertiary structure of haemerythrin. Nature (London) 257,8 18-82 1. Wasastjerna, J. A. (1923). Radii of ions. SOC.Sci. Fenn., Commentut. Phys-Nat. 1 38, 1-25. Watenpaugh, K. D., Sieker, L. C., and Jensen, L. H. (1980). Crystallographic refinement of rubredoxin at 1.2 A resolution. J. Mol. Biol. 138,615-633. Werner, A. (1893). Beitrag zur Konstitution anorganischer Verbindungen. Z. Anorg. Allg. Chem. 3, 267-342. [Translated into English in Kauffman, G. B. (1968). “Classics in Coordination Chemistry, Part 1: The Selected Papers of Alfred Werner,” pp. 5-88. Dover, New York.] Williams, R. J. P. (1959). Coordination, chelation, and catalysis. Enzymes 1, 391-441. Williams, R. J. P. (1970). The biochemistry of sodium, potassium, magnesium, and calcium. Q. Rev. Chem. SOC.London 24,331-365. Williams, R. J. P. (1990). Overview of biological electron transfer. In “Electron Transfer in Biology and the Solid State. Inorganic Compounds with Unusual Properties” (M. K. Johnson, R. B. King, D. M. Kurtz, Jr., C. Kutal, M. L. Norton, and R. A. Scott, eds.), pp 3-23. Am. Chem. SOC.,Washington, D.C.

76

JENNY P. GLUSKER

Wyckoff, R. W. G., and Posnjak, E. (1921). The crystal structure of ammonium chloroplatinate.j. Am. Chem. Sac. 43,2291-2309. Yamashita, M., Wesson, L., Eisenman, G., and Eisenberg, D. (1990).Where metal ions bind in proteins. Proc. Natl. Acad. Sci. U.S.A. 87,5648-5652. Yong, G., Leone, C., and Strothkamp, K. G. (1990). Agaricus bisporus metapotyrosinase: Preparation, characterization, and conversion to mixed-metal derivatives in the binuclear site. Biochemistry 29,9684-9690. Zachariasen, W. H. (1931). A set of empirical crystal radii for ions with inert gas configuration. Z. Kristallogr. 80, 137-153. Zachariasen, W. H. (1954). Crystal chemical studies of the 5fseries of elements. XXIlI. On the crystal chemistry of uranyl compounds and of related compounds. Acta Crystallogr. I , 795-799. Zahrobsky, R. F., and Baur, W. H. (1968). On the crystal chemistry of salt hydrates. V. The determination of the crystal structure of CuSO4*3H20(bonattite). Acta Crystallogr., Sect. B 24, 508-513.

CALCIUM-BINDING SITES IN PROTEINS: A STRUCTURAL PERSPECTIVE By Catherine A. McPhaien, Natalie C. J. Strynadka, and Michael N. G. James Medical Research Council of Canada Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta TBG 2H7, Canada

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 11. Functional and Structural Overview of Protein Ca“-Binding Sites . . . . A. Helix-Loop-Helix Proteins . . . . . . . . . . . . . . . . . . B. Serine Proteinases . . . . . . . . . . . . . . . . . . . . . . C. Other Ca2+-BindingProteins . . . . . . . . . . . . . . . . . 111. Regularities and Recurrent Themes in Ca2+-Binding Sites . . . . . . . A. Continuous, Semicontinuous, and Discontinuous Coordinating Peptides B. Secondary Structural Elements and Main-Chain Conformations . . . C. Coordination Number . . . . . . . . . . . . . . . . . . . . D. Distribution of Ligand Types and Mean Ca2+-Ligand Distances . . . E. Ligand Geometry Compared to Ideal Polygons . . . . . . . . . . F. Stereochemistry of Ca2+-Ligand Interactions . . . . . . . . . . . G. Networks of Hydrogen Bonds among Ca2+-Ligand Residues . . . . H. Lack of Interaction between Ca2+and Helix Dipoles . . . . . . . . I. Ca2+-BindingAffinities and Structural Characteristics . . . . . . . J. Selectivity of Ca*+-BindingSites . . . . . . . . . . . . . . . . K. Ca2+-FreeSites . . , . . . . . . . . . . . . . . . . . . . . IV. Discussion and Summary . . . . . . . . . . . . . . . . . . . . A. A “Regular” Ca*+-BindingSite . . . . . . . . . . . . . . . . B. Structure-Function Relationships . . . . . . . . . . . . . . . C. Prediction and Design of Ca2+-BindingSites . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

. .

.

77 86 87 91 99 108 108 109 112 113 115 118 124 130 131 134 135 136 136 138 139 140

I. INTRODUCTION Calcium is the fifth most abundant element on earth and is ubiquitous in biological organisms, processes, and structures. It is, of course, an essential component in the biomineralization of teeth, bones, and shells. In these structures Ca2+ is in the form of single crystals of hydroxyapatite, calcite, or aragonite (reviewed by Mann, 1988). Intracellularly, Ca2+ has an extraordinary number of diverse roles in metabolic regulation, nerve transmission, muscle contraction and cell motility, cell division and growth, secretion, and membrane permeability. In the cytoplasm of a resting cell, the level of Ca2+ is normally several orders of magnitude lower than that outside the cell (0.2 p M versus 2 mM).Ca2+is thus able to act as an intracellular second messenger, its level changing rapidly in ADVANCES IN PROTEIN CHEMISTRY, Val. 42

77

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

78

CATHERINE A. MCPHALEN ET AL.

response to extracellular stimuli. Ca2+homeostasis in eukaryotic cells has been reviewed by Pietrobon et al. (1990). Even within the cell, local concentrations of Ca2+can vary widely to allow fine tuning and differentiation of cellular response to a stimulus (Rose and Loewenstein, 1975). The characteristics and functions of oscillating Ca2+ waves in cells have been reviewed by Berridge (1990). Most often, Ca2+-dependent intracellular functions are mediated o r regulated by Ca'+-binding proteins. T h e Ca2+ affinity of these proteins must be carefully tuned to initiate biological responses only at appropriately high (or low) levels of Ca'+. In addition the proteins must be very selective in ion binding in order to distinguish Ca2+ from other biologically important ions, such as Mg2+, Na+, and K+. Outside the cell many extracellular proteins also require bound Ca2+ to function. In the relatively constant Ca" levels of the extracellular milieu, the protein-bound Ca2+ is most often a stabilizing factor, rather than an action signal. Several earlier reviews have been written on structural aspects of Ca2+ binding to proteins and small molecules (Einspahr and Bugg, 1980,198 1, 1984; Kretsinger, 1976, 1980). Strynadka and James (1989) dealt extensively with crystal structures of the helix-loop-helix (HLH) family of Ca'+-binding proteins. Recently, the crystal structures of additional Ca*+-binding proteins have been solved and fully refined at high resolution, providing a greatly expanded data base of accurate structures for the analysis of Ca'+-binding sites. Table I provides information on most of the currently known crystal structures of proteins with bound Ca2+ (although multiple determinations of some structures have not been included in the table). Coordinates for the majority of these structures were obtained from the Brookhaven Protein Data Bank (PDB) (Bernstein et al., 1977). Some structures that have been reported in the literature are not yet part of the PDB (please see Acknowledgments for sources of other coordinates). One important class of Ca2+-binding proteins missing from Table I is that of proteins containing the modified amino acid y-carboxyglutamic acid (Gla). On Cy of the side chain, Gla residues have two carboxylate groups capable of binding Ca2+ ions simultaneously. Two crystal structures have been reported for one Gla-containing protein-fragment 1 of bovine prothrombin (Olsson et al., 1982; Tulinsky et al., 1985,1988).This fragment has 10 Gla residues near its amino terminus. Unfortunately, one of the structures was determined only at low resolution and the bound Ca2+ was not located in the electron density maps (Olsson et al., 1982). The amino terminus is badly disordered in the other structure and neither protein nor Ca2+ is visible in the electron density maps at higher resolution (Tulinsky et al., 1985, 1988). The structures of some small-

'

Helix-loop-helix (HLH) or "EF-hand"motif in Ca2+-bindingsites. The Ca2+ligands are part of a 12-residue loop flanked by orthogonal a helices.

TABLE I X-Ray Crystal Structures of Calcium-Binding Proteins

Protein

PDB code"

Resolution (A)

R factolb

No. of Ca2+sites"

Troyonin C Turkey

5TNC

2.0

0.155

4

Chicken

4TNC

2.0

0.172

4

Calnmdulin

3CLN

2.2

0.175

4

CalbindingK Parvalbumin Carp

3ICB

2.3

0.178

2

5CPV

1.6

0.187

2

4CPV

1.5

0.215

2

3CPV

1.9

0.25

2

Pike

-

1.9

0.176

2

Rat

-

2.0

0.181

2

Role of Ca2+ion Muscle contraction and regulation Muscle contraction and regulation Intracellular enzyme regulation Calcium transport

Oncomodulin

lOMD

1.85

0.166

3

P-Trypsin S . griseur trypsin Elastase Subtilisin Novo (BPN')

2PTN 1SGT 3EST 2SNI

1.55 1.7 1.65 2.1

0.193 0.161 0.169 0.154

1 1 2

Intracellular Ca2+ buffer? Intracellular Ca2+ buffer? Intracellular Ca2+ buffer? Intracellular Ca2+ buffer? Intracellular Ca2+ buffer? Intracellular Caz+ buffer? Stabilizing Stabilizing Stabilizing Stabilizing

lS0l

1.3

0.14

2

Stabilizing

1

Reference Herzberg and James (1985a, 1988) Sundaralingam et al. (1985), Satyshur et al. (1988) Babu et al. (1985, 1987, 1988) Szebenyi and Moffat (1986, 1987) Swain et al. (1989) Kumar et al. (1990) Moews and Kretsinger (1975) Declerq et al. (1988) McPhalen et al. (1991) Ahmed et al. (1990) Bode and Schwager (1975) Read and James (1988) Meyer el al. (1988) McPhalen and James (1988) Pantoliano et al. (1988) (continued)

TABLE I (Continued)

Protein

PDB code"

Resolution (A)

R factop

No. of Ca2+sites"

Role of Caz+ion

Reference Bott et al. (1988) McPhalen et al. (1985), McPhalen and James (1988) Bode et al. (1987) Neidhart and Petsko (1988) Betzel et al. (1988), Bajorath et al. (1989) Gros et al. (1989) Dauter et al. (1988), Teplyakov et al. ( 1990) Gros et al. (1989), Gros (1990) Holmes and Matthews (1982) Suguna et al. (1987) Loll and Lattman (1989)

Subtilisin Carlsberg

1ST2 2SEC

2.0 1.8

0.17 0.136

2 2

Stabilizing Stabilizing

Proteinase K

lCSE 1SBC 2PRK

1.2 2.5 1.5

0.178 0.206 0.167

2 1 2

Stabilizing Stabilizing Stabilizing

Thermitase

lTEC

2.2 1.4

0.179 0.149

3 3

Stabilizing Stabilizing

3TLN 2APR lSNC

2.0 1.6 1.8 1.65

0.165 0.213 0.143 0.161

3 4 1 1

Stabilizing Stabilizing Stabilizing Catalytic?

2SNS -

1.5 2.0 2.8

0.157 0.19

1 3 4

Catalytic? Catalytic, stabilizing Actin polymerization

Cotton et al. (1979) Ofner and Suck (1986) Kabsch et al. (1990)

1BP2 1P2P 3P2P 5P2P 1ALC 2GBP

1.7 2.6 2.1 2.4 1.7 1.9

0.171 0.241 0.186 0.189 0.22 0.146

1 2 2 2 1 1

Stabilizing,catalytic? Stabilizing,catalytic? Stabilizing,catalytic? Stabilizing,catalytic? Stabilizing,cofactor Stabilizing?

Dijkstra et al. (1981b) Dijkstra et al. (1983) Thunnissen et al. (1990a) Thunnissen et al. (1990b) Acharya et al. (1989) M. N. Vyas et al. (1989), N. K. Vyas et al. (1987)

W

0

Thermolysin Rhizopus pepsin Staphylococcus nuc1ease DNase I Actin:DNase I Phospholipase A2 Bovine Porcine a-Lactalbumin D-Galactose-binding protein

-

Dihydrofolate reductase a-Am ylase

Concanavalin A

Pea lectin

m

4DFR

1.7

0.155

2TAA lAAA 3TAA 2CNA

3.0 2.9 2.1 2.1 2.0

0.36 0.169 0.195

-

1 1 2 2 1

Stabilizing, catalytic? Matsuura et al. (1984) Stabilizing, catalytic? Buisson et al. (1987) Stabilizing, catalytic? Boel et al. (1990) Stabilizing, catalytic? Boel et al. ( 1990) Cell binding Becker et al. (1975)

3CNA

2.4

-

1

Cell binding

Hardman and Ainsworth (1972)

-

2.9

0.19

1

Cell binding

Derewenda et al. (1989)

3.0d

0.24

1

Einspahr et al. (1986)

Jones and Liljas (1984) Silva and Rossmann (1985)

-

2LTN

-

Bolin et al. (1982)

1

Favin

-

2.8

0.24

1

Lectin IV

-

2.8

0.2 1

1

2STV

2.5

0.27

3

Carbohydrate binding Carbohydrate binding Carbohydrate binding Stabilizing

4SBV

2.8

0.26

3

Stabilizing

Satellite tobacco necrosis virus Southern bean mosaic virus

Reeke and Becker (1986) Delbaere et al. (1990)

Proteins are referred to by their identifying codes for the coordinate set in the Brookhaven Protein Data Bank (PDB) (Bernstein et al., 1977). R factor (C 11 F, I - I F, 11 /B I F, 1 ) is a measure of agreement between observed (F,) and calculated (Fc) structural data. Lower values indicate better agreement. Some known Ca2+-binding sites are not filled in some of the X-ray structures. The 2LTN structure has now been refined to an R factor of 0.177 at 1.7 A resolution (PDB data).

82

CATHERINE A. MCPHALEN ET AL.

molecule models for Gla residues binding to Ca2+ have been solved, however (Karipedes et al., 1977; Briggman and Oskarsson, 1977; Albertsson et al., 1978; Zell et al., 1985). From the hundreds of X-ray crystal structures determined for complexes of Ca2+ with small-molecule oxygen ligands, a clear picture has emerged of preferred Ca2+-ligandtypes, ligand geometry, and distances (Martin, 1984; Einspahr and Bugg, 1984; Brown, 1988; Carrel1 et al., 1988). Ca2+ ions in small-molecule structures are able to accommodate four to 12 oxygen ligands in their primary coordination sphere, but coordination numbers of six, seven, or eight are by far the most common (Brown, 1988). T h e mean Ca2+-ligand distance varies slightly with coordination number, but hovers around 2.4 8, (Einspahr and Bugg, 1984). Ligand atoms are generally arranged regularly around the Ca2+ ion; seven ligands (heptacoordinate Ca2+)tend to form a pentagonal bipyramid (Color Plate la) with five atoms in an equatorial plane. Six ligands (hexacoordinate Ca2+)tend to lie close to the vertices of an ideal octahedron (Color Plate lb). A priori, one would not expect protein Ca2+binding sites to be strikingly different from those observed with small molecules. In particular, an efficient protein Ca2+-bindingsite is likely to require at least six ligands. In crystal structure analyses of proteins, the presence of Ca2+ ions is usually determined indirectly. Ions, being larger and more electron dense, are differentiated from water molecules based on their peak heights in electron density maps, and their high occupancies and low thermal motion parameters during least-squares refinement of the COLORPLATE1. (a) A stereo view of Ca2+-binding site 3 of troponin C (5TNC) (Herzberg and James, 1988). Main-chain atoms are white, aspartate and glutamate side-chain atoms are red, asparagines are pink, hydrophobic side chains ate green, and basic amino acids are blue. The Ca2+ion is a magenta sphere. The conserved Ca2+-coordinatingwater molecule is a small blue sphere. The Ca2+ ion is heptacoordinate (seven ligands). The spatial disposition of the coordinating oxygen ligands defines a pentagonal bipyramid (outlined in thin yellow lines). Asn- 108 Osl,Asp-1 10 Osl,the main-chain carbonyl oxygen atom of Phe-112 and the two side-chain carboxylate oxygen atoms of Glu-117 define the pentagonal plane. The water molecule and Asp- 106 0 ' 'form the apices of the bipyramid. A pentagonal bipyramidal arrangement of the Ca2+ ligands is by far the most prevalent coordination geometry observed in the Ca2+-bindingdomains of proteins (Section 111,C). (b) A stereo view of the Ca2+-binding domain of bovine trypsin (2PTN) (Bode and Schwager, 1975). Residue coloring as in (a). See Fig. 2b for numbering of the amino acids shown here. The Ca2+ ion is hexacoordinate (six ligands). The spatial disposition of coordinating oxygen ligands defines an octahedron (shown in yellow lines). This type of Ca2+coordination is observed in only three protein Ca2+-bindingsites of Table 11: PPTN (Fig. 2b), elastase (3EST, Fig. 2c), and thermolysin (STLN) site 2 (Fig. 3a). Special thanks to Protos Corporation (Emeryville, CA) for a grant to include these color plates.

COLORPLATE1

COLOR PLATE2

CALCIUM-BINDING SITES I N PROTEINS

83

protein structure. Interatomic distances and angles are rarely restrained for ions during structure refinement, thus final refined ion-ligand distances are good indicators of chemical identity for a protein-bound ion. In a few cases the identification of a particular ion as Ca2+ has been checked by repeating the structure analysis at different Ca2+ concentrations (Pantoliano et al., 1988; Gros et al., 1989; Gros, 1990), or with other ions expected to replace the Ca2+ directly (Herzberg and James, 1986; Vyas et al., 1989; Swain et al., 1989). From the proteins in Table I we have selected 17 representatives containing a total of 27 unique Ca*+-binding sites for analysis and comparison (Table 11). T h e sites in Table I1 were chosen on the basis of resolution of the protein structure determination (2.3 A or better), mean ion-ligand distances of around 2.4 A, a reasonable number of ligands observed, and coordination geometry approximating one of the ideal polygons (Color Plate 1). Two sites listed in Table I had very unusual coordination geometry, although they met the other criteria above: thermolysin (3TLN) site 4 and Rhizopus pepsin (2APR). These two were omitted from the following analysis, but 3TLN site 4 is discussed in Section I1,C. The first four proteins in Table I1 [turkey troponin C (5TNC) to carp parvalbumin (5CPV)l are members of the classical HLH family. They are intracellular proteins, with well-characterized Ca*+binding properties (Kretsinger, 1980) and relatively high Ca2+ affinities. T h e others [trypsin (2PTN) to concanavalin A (2CNA)I are mainly extracellular proteins with generally lower Ca2+affinities. Many are known to require Ca2+ for stability and/or catalytic activity, but few have been characterized thoroughly as Ca2+-binding proteins. The proteins in Tables I and I1 are a very small subset of the hundreds known to require Ca2+,but their diversity of function emphasizes the broad importance of Ca2+ in biological systems. In this chapter we describe and compare in detail the Ca2+-binding COLOR PLATE2. (a) A stereo view of Ca*+-bindingsite 4 in troponin C (STNC) highlighting the intricate hydrogen-bonding patterns among side- and main-chain atoms. Residue coloring as in Plate l a (see Fig. 7a for residue numbers). Hydrogen bonds are depicted as dashed yellow lines. Note the large number of interactions that involve aspartate and glutamate residues. The system of hydrogen bonds is highly conserved throughout the helix-loop-helix (HLH) Ca2+-binding proteins. (b) A stereo view of the Ca2+-bindingsite of S.aurew nuclease (1SNC) (Loll and Lattman, 1989).Coloring of residues is as for Plate l a (see Fig. 7b for residue numbers). The pdTp that coordinates the Ca2+ ion with one phosphate oxygen atom is shown in magenta. Hydrogen bonds are depicted as dashed yellow lines. Several features of the illustrated hydrogen-bonded interactions are proposed to stabilize the Ca2+-coordinationsphere (Section 111,G). Special thanks to Protos Corporation (Emeryville, CA) for a grant to include these color plates.

TABLE I1 Calcium-Binding Characteristics of Sites in Moderate- to High-Resolution X-Ray Crystal Structures"

Protein TroponinC Calmodulin

PDB code

Coordinating Site peptideb

5TNC

3

3CLN

4 1

2 3 CalbindingK

3ICB

Parvalbumin

5CPV

4 1 2 1

2 Bovine trypsin S . griseus trypsin Elastase Subtilisin Carlsberg Proteinase K

2PTN 1SGT 3EST 2SEC 2PRK

1 1 1 1 2 1 2

C C C C C C C C C C C D C S C D D

No. of coordinating ligands

No. of coordinating waters

No. of bidentate ligands

7 7 7 7 7 7 7 7 7 7 6 7 6 7 5 8 5

1 1 1 1 1 1 1 1 0 1 2 2 1 0 2 4

1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 1 1

2

Root mean square deviation of ligands from ideal polygon' (A) 0.41

0.53 0.40 0.41

KdCa2+ (M)d

Reference for affinity data

10-7 10-7 10-5 10-5

Zot and Potter (1987) and references therein Klee et al. (1986) and references therein

10-6 10-6- 10-8 10-6- 10-8 -10-9 10-9 3 x 10-5

Shelling and Sykes (1985) and references therein Wnuk et al. (1982) and references therein Cliffe and Grant (1981)

0.37 0.49

0.53 0.53 0.4 1

0.50 0.39 0.36 0.30 0.41 0.49' 0.27'

-

1.4 x 10-4 -10-10

Dimicoli and Bieth (1977) Briedigkeit and Frommel

-10-4 7.6 X

Bajorath et al. (1988)

-

(1989)

lSNC 1BP2 lALC 2GBP

1 2 1 2 3 1 1 1 1

S C S C C D S

2CNA

1

C

Thermitase

lTEC

Thermolysin

JTLN

S. aureus nuclease Phospholipase A2 a-Lactalbumin D-Galactosebinding protein Concanavalin A

C S

7 6 7 6 7 7

0.36 1.27' 0.48 0.15 0.31 0.30 0.40 0.19 0.25

7 7 7

7

2

1

0.64

10- 10 -10-10 1 x lo+ 2 x 10-5 5.5 x 10-5 2.5 x 10-4 4 x 10-9 4.7 x 10-6

3

X

Briedigkeit and Frommel ( 1989) Voordouw and Roche (1974) Cuatrecasas et al. (1968) De Haas et al. (1971) Permyakov et al. (1987) Vyas et al. ( 1989) Kalb and Levitzki (1968)

'Refined structures at 12.3 A resolution. From Table I the following sites from unique high-resolution structures are not included: lOMD [coordinates not yet released from the Brookhaven Protein Data Bank (PDB)]; lTEC site 3 (probably occupied by Na+); 3TLN site 4 (unusual coordination geometry); 2APR (unusual coordination geometry); DNase I (coordinates not deposited with the PDB); 4DFR (only three ligands observed); lAAA and 3TAA (coordinates not yet available from the PDB); and 2LTN (coordinates released from the PDB after completion of our analysis). In the cases of multiple determinations of a single protein structure, one representative structure has been included. C, All ligands are part of a continuous local segment of the protein chain; S (semicontinuous), the majority of ligands are from a continuous segment of the protein chain; D (discontinuous), several separated segments. Ligand atoms were superposed on either an octahedron (six coordinate) or a pentagonal bipyramid (seven coordinate) with all vertices 2.4 A from the central Ca2+ion. Sites 2 in PSEC, SPRK, and lTEC were compared to modified pentagonal bipyramids, with appropriate ligands omitted. Although the root mean square deviation for lTEC site 2 is high when the ligands are superposed on a modified pentagonal bipyramid, the deviation is even higher for superposition on an octahedron. Calcium-binding affinities have not been measured for all sites in each protein. In some cases the identification of the bound ion as Ca2+ is based primarily o n characteristic bond lengths in the refined structure. Binding affinities were too weak to be measured by the techniques used for 2PRK site 2 and JTLN site 3.

86

CATHERINE A. MCPHALEN ET AL.

sites listed in Table 11, attempting to extract common features from them. T h e immediate goal of this analysis is to determine structural parameters of a “regular” Ca2+-binding site. Relating structure to function is a much more difficult task, and questions abound. What are the structural correlates of stronger or weaker Ca2+binding? How d o Ca2+binding sites distinguish between Ca2+ and the very similar Mg2+ or other ions? How does Ca2+ binding elicit structural changes that alter protein activity? How does Ca2+binding enhance protein stability? Why is Ca2+ the ion of choice in so many biological roles? Is it possible to predict where Ca2+ will bind to a protein and how tightly? Complete answers to such questions are scarce as yet, but we attempt to deal with some of them here.

11. FUNCTIONAL AND STRUCTURAL OVERVIEW OF PROTEIN Ca2+-BINDINGSITES

We begin with an introduction to the Ca2+-bindingsites in the proteins of Table I1 and the functions of Ca2+ in binding to them. Three broad classes of function are apparent from Table I: Ca2+ modulation of protein action, Ca2+ stabilization of protein structure, and involvement of the Ca2+ ion in enzymatic catalysis. T h e Ca2+-modulated proteins have altered interactions with other proteins on binding of Ca2+.Binding Ca2+ stabilizes some proteins against thermal or chaotropic denaturation, or proteolytic degradation. In a few cases a Ca2+ ion is thought to participate directly in enzymatic catalysis via electrostatic interactions with substrate. The following three sections discuss three structural classes of proteins and the function(s) of Ca2+ in the proteins of each class. The HLH proteins troponin C and calmodulin are Ca2+modulated in their biological interactions with other proteins (references in Section 11,A). As possible Ca2+ transport or buffer proteins, calbindingK and parvalbumin might also be considered Ca2+ modulated, but little evidence is available on their interactions with other proteins in vivo. T h e serine proteinases of Section II,B are all stabilized by Ca2+ binding. In some cases the bound ion increases stability against thermal or chaotropic denaturation. I n others it seems to provide protection against proteolytic degradation, although this can be closely related to denaturation in autolytic enzymes. T h e remaining proteins in our survey have varying functions for Ca2+ (references in Section 11,C). a-Lactalbumin with Ca2+ bound acts as a modulator protein in an enzymatic complex. Concanavalin A may be considered Ca2+ modulated, because Ca2+ binding induces structural changes necessary to form the saccharide-binding site on the

CALCIUM-BINDING SITES IN PROTEINS

87

protein. Thermolysin is stabilized against thermal denaturation when it binds Ca2+. The D-galactose-binding protein is also thought to be stabilized in some fashion by its tightly bound Ca2+ ion. Phospholipase A2 and Staphylococcus aureus nuclease both have their catalytic activity modulated directly by Ca2+ interaction with substrates.

A . Helix-Loop-Helix Proteins Troponin C is part of the troponin complex of muscle thin filaments, the molecular switch that triggers muscle contraction in response to a Ca2+ signal (Leavis and Gergely, 1984; Zot and Potter, 1987). Troponin C is an 18K protein with four Ca2+-bindingsites: two low-affinity Ca2+specific sites in the amino-terminal domain and two high-affinity Ca2+/ Mg2+sites in the carboxy-terminal domain (Fig. l a and Table 11). In the crystal structures of the avian troponin C molecules, only the highaffinity sites are occupied (Herzberg and James, 1985a, 1988; Sundaralingam et al., 1985; Satyshur et al., 1988). Under physiological conditions the high-affinity sites bind Ca2+ o r Mg2+ at all times, and these bound ions are required to maintain protein structural stability. Binding of Ca2+ to the low-affinity regulatory sites induces a conformational change that is transmitted to other protein components of the thin filament, resulting in contraction. This conformational change in troponin C had been modeled based on the crystal structures and solution data (Herzberg et al., 1986a); a change in the relative disposition of helices flanking the regulatory Ca2+-binding loops exposes hydrophobic patches on the surface of the amino-terminal domain. These patches may enhance the interaction of troponin C with other components of the troponin complex. This proposed model has been supported by recent biochemical and site-directed mutagenesis experiments (Grabarek et al., 1990; Fujimori et al., 1990). Calmodulin is 5 1% identical in sequence and similar in tertiary structure to troponin C. It has a molecular weight of 16.7K and also binds four Ca2+ ions; the two carboxy-terminal ion-binding sites are of slightly higher affinity (Table 11). All four sites are occupied by Ca2+in the crystal structure (Babu et al., 1985, 1987, 1988). Calmodulin is a ubiquitous intracellular Ca2+ receptor and modulator protein in eukaryotic cells (Klee et al., 1980; Means et al., 1982). In its active Ca2+-saturated form calmodulin binds to an amazing variety of other proteins, altering their activity in response to a Ca2+influx signal. Binding of Ca2+to calmodulin is proposed to cause a conformational change that exposes hydrophobic patches on the protein surface (Babu et al., 1988; Strynadka and James, 1988). T h e proposed conformational change for calmodulin is similar to

.SER 2

VRL 161

VRL 161

14 1

141

FIG. 1. Stereo views of Ca2+-bindingsites in helix-loop-helix (HLH) proteins. (a) The a-carbon backbone trace of troponin C (5TNC) (Herzberg and James, 1988) highlighting the locations of the Ca2+-bindingsites. Ca2+ positions are marked with circles. The aminoand carboxy-terminal residues of the protein are labeled, as well as every 20th amino acid residue. In 5TNC the two structural high-affinity sites have Ca2+ bound. The two empty regulatory low-affinity sites lie in the amino-terminal domain (top) of the dumbbell-shaped molecule. (b) Ca2+-bindingsite 3 of calmodulin (3CLN) (Babu etal., 1988).The Ca2+ion is a large circle, oxygen atoms of water molecules (HOH)are small circles, and ligand coordination bonds are indicated by dashed lines. Main-chain (N, Ca, C, and 0)atoms are shown for all residues in thin lines. Only side chains of Ca2+-ligand residues are shown (thick lines). Ca2+-binding residues are labeled, plus the amino and carboxy termini of this segment of polypeptide chain. The two helices flanking the Ca2+-bindingloop (E and F) are included in this view. Apical ligands in the pentagonal bipyramidal coordination of this site (Plate la) are Asp-93 and HOH-155; equatorial ligands are Asp-95, Asn-97, Tyr-99 (peptidecarbonyl oxygen atom), and Glu-104 (bidentate). (c) Ca2+-binding site 1 of calbindingK (3ICB) (Szebenyi and Moffat, 1987), drawn and labeled as in (b). Apical ligands are Ala-14 (peptide carbonyl oxygen) and HOH-5; equatorial ligands are Glu-17, Asp-19, Gln-22 (all peptide carbonyl oxygens), and Glu-27 (bidentate). Residues Ala- 15 and Pro-20 are insertions relative to the canonical HLH loop. (d) Ca2+-binding site 1 of carp parvalbumin (5CPV) (Swain et al., 1989). The usual apical water ligand of the HLH sites is replaced by the long side chain of Glu-59. The second apical ligand is Asp-51, and equatorial ligands are Asp-53, Ser-55, Phe-57 (peptide carbonyl oxygen), and Glu-62 (bidentate).

CALCIUM-BINDING SITES IN PROTEINS

89

b 81

112

C 2

2

5

5

AS

FIG. 1. (continued)

90

CATHERINE A. MCPHALEN ET AL.

d

FER

rSER

39

39

RSP

FIG. 1. (continued)

that postulated for the amino-terminal domain of troponin C on binding of regulatory Ca2+ (Herzberg et al., 1986a). As with troponin C, these patches may enhance interaction of calmodulin with target proteins. Ca2+-binding site 3 of calmodulin (3CLN) is depicted in Fig. l b as an example of a typical HLH Ca2+-bindingsite. Asp-93 and the ligand water molecule sit opposite each other at the apices of a pentagonal bipyramid (Plate 1 illustrates the two kinds of observed ligand geometries). Along with the bidentate Glu-104, these ligands are absolutely conserved in all of the Ca2+-bindingsites of troponin C and calmodulin. Tyr-99 of calmodulin is not conserved, but the equivalent residue in all HLH sites contributes its main-chain carbonyl oxygen to the Ca2+-ligand sphere. Asp-95 of calmodulin contributes its side chain to the pentagonal plane of the ligand sphere. The nature of this side chain is not invariant at this position among the HLH sites, but the side chain is always a Ca2+ligand. T h e pentagonal plane of the ligand sphere is completed by the side chain of the residue equivalent to 97 in calmodulin (Fig. lb). The geometries and interactions of all 12 residues in the HLH Ca2+-bindingloops have been reviewed comprehensively by Strynadka and James (1989).

CALCIUM-BINDING SITES I N PROTEINS

91

CalbindingK (also known as intestinal Ca"-binding protein), with a molecular weight of 9K, contains only two Ca2+-bindingsites. Both Ca2+ sites are of relatively high affinity (Table 11) and are occupied in the crystal structure. T h e protein has been localized to the absorptive cells of the small intestine (Taylor, 1983). Its exact function is uncertain, but it has been proposed that it participates in Ca2+translocation or absorption at the intestinal wall (Wasserman and Fullmer, 1982; Wasserman et al., 1983; Levine and Williams, 1982). Ca'+ binding is thought to cause only minor conformational changes in calbindings, unlike troponin C and calmodulin (Dalgarno et al., 1983; Chiba and Mohri, 1987; Skelton et al., 1990). Parvalbumin is another protein in search of a function. It contains three HLH motifs ( M , 1lK), but only the second and third are functional Ca2+-binding sites. These are high-affinity Ca2+/Mg2+sites, and both are filled with Ca2+ in the known crystal structures (references in Table I). In fast twitch muscle, where most parvalbumins are found, the protein is postulated to act as a Ca2+buffer (Haich et al., 1979; Gillis et al., 1982). As Ca2+ is released from troponin C after muscle contraction, the Ca'+ may be bound by parvalbumin to prevent reinitiation of contraction. In resting cells parvalbumin likely binds Mg2+, rather than Ca2+ (Haiech et al., 1979). The second Ca2+-binding sites in calbindingK and parvalbumin are both regular HLH sites, with the same pattern of conserved ligands and similar geometry to calmodulin (3CLN) site 3 (Fig. lb). Site 1 of calbindingK is also composed of two flanking helices and a loop that provides ligands to a heptacoordinate Ca2+,but the first three ligands from the loop are peptide carbonyl oxygen atoms, rather than side-chain atoms (Fig. lc). The first half of the loop also contains two single-residue insertions that change the backbone conformational angles of this segment. The second half of the loop more nearly resembles a regular HLH site in its Ca2+ ligands and conformation. Ca"-binding site 1 in carp parvalbumin (5CPV) is a regular HLH site in most respects, but it lacks the single coordinating water molecule present in all of the other HLH sites. Instead, the carboxylate group on the long side chain of Glu-59 coordinates directly to the Ca2+ at this ligand position without distorting the loop conformation (Fig. Id).

-

B . Serine Proteinases Members of both structural families of serine proteinases, the trypsinlike and the subtilisin-like, have been found to bind Ca2+ (references in Tables 1 and 11). T h e role of Ca2+ in all of these proteolytic enzymes appears to be one of stabilization of structure and/or maintenance of

92

CATHERINE A. MCPHALEN E T AL.

a .Y

78

FIG.2. Stereo views of Ca2+-bindingsites in serine proteinases, drawn and labeled as in Fig. la and b. (a) The a-carbon backbone trace of bovine trypsin (2PTN) (Bode and Schwager, 1975). Side chains of the catalytic triad (Ser-195, His-57, and Asp-102) are labeled and highlighted with thick lines. The Ca2+-binding site is a loop on the surface of the protein, distant from the catalytic residues and substrate-binding site. (b) The Ca'+binding site of 2PTN (Bode and Schwager, 1975), with its six ion ligands forming an octahedral coordination sphere (Color Plate lb). The ligand residues are Glu-70, Asn-72, and Val-75 (peptide carbonyl oxygen atoms), Glu-80, and two water molecules. (c) The Ca2+-binding site of elastase (JEST) (Meyer et al., 1988), showing the substitution of Asn-770" for one of the water ligands in the 2PTN site. The sites are quite similar otherwise, despite several amino acid differences in the sequence of the Ca2+-bindingloops. (d) The Ca2+-bindingsite of S. p e w trypsin (ISGT) (Read and James, 1988). Unlike 2PTN and JEST, this site is discontinuous and heptacoordinate. The apical ligands of the pentagonal bipyramid are Ala-l77A (peptide carbonyl oxygen atom) and HOH-13. The equatorial ligands are Asp-165 (bidentate), Glu- 180 (peptide carbonyl oxygen), Glu-230, and HOH15. (e) The a-carbon backbone trace of subtilisin Carlsberg (2SEC) (McPhalen and James, 1988), showing two Ca*+-binding sites. Side chains of the catalytic triad (Ser-22 1, His-64, and Asp-32) are drawn in thick lines to mark the active-siteregion. (f) Ca2+-bindingsite 1 of 2SEC (McPhalen and James, 1988). This is a semicontinuous heptacoordinate site, with the majority of ligands part of a protein loop that interrupts a long helix (residues 64-85). His-64 is part of the catalytic triad. The apical ligands are Leu-75 and Thr-79 (peptide carbonyl oxygens); equatorial ligands are Gln-2, Asp-4 1 (bidentate), Asn-77, and Val-81 (peptide carbonyl oxygen). (9) Ca2+-bindingsite 1 of proteinase K (2PRK) (Bajorath et al., 1989), one of the rare eight-coordinate sites observed in protein structures. The protein ligands to the Ca2+ are from discontinuous segments of polypeptide chain. The ligands include four water molecules, two peptide carbonyl oxygens, and one bidentate aspartic acid. (h) The polygon formed by the eight ligand atoms of 2PRK site 1, outlined in dashed lines. The coordination geometry at this site is essentially a pentagonal bipyramid, with one vertex shared by two ligand atoms. The two carboxylate oxygens of Asp-200 may be considered to share one apical position; alternatively, HOH-4 and Val-177 (peptide carbony1 oxygen) may be considered to share one equatorial position, depending on which

CALCIUM-BINDING SITES IN PROTEINS

93

b

C

atoms are defined as forming the pentagonal plane around the Ca4+ion. (i) Ca2+-binding site 2 of thermitase in 100 mM CaCI2 (Gros, 1990). This ion-binding site is unique to thermitase among the serine proteinase structures. It contains eight Ca2+ ligands, one of them an uncommon bidentate Thr-64 (both O y and the peptide carbonyl oxygen are ligands). This continuous eight-coordinate site is more crowded than 2PRK site 1 , with only one water molecule, three aspartates (one bidentate), one glutamine, and the bidentate threonine as ligands. Cj) The polygon formed by the eight ligand atoms of thermitase site 2, outlined in dashed lines. The coordination geometry is very similar to 2PRK site 1 (Fig. 2h), that of a pentagonal bipyramid with one vertex split between two ligand atoms.

d__

177R GLU 180

-._

HOH 15

"

Ca2t

,

,

f

ASP 165

ASP 165

Is

77

FIG. 2. (continued) 94

17

HOH

FIG. 2. (continued) 95

96

CATHERINE A. MCPHALEN ET AL.

i

FIG. 2. (continued)

CALCIUM-BINDING SITES IN PROTEINS

97

catalytic activity (Bier and Nord, 1951). The Ca2+ does not participate directly in catalysis, and the Ca2+-bindingsites are all at least 10 A from the catalytic residues (Fig. 2a). Interestingly, the Ca2+-bindingsites are in different positions in each of the serine proteinases in Table I1 (trypsinlike: 2PTN, lSGT, and 3EST; subtilisin-like: BSEC, 2PRK, and 1TEC). The diversity in the Ca2+-bindingsites of these serine proteinases seems to derive from sequence variability of surface loops, especially nonconserved aspartic acid residues. The single Ca2+-bindingsite in the trypsin (2PTN) structure is formed by a 12-residue surface loop (Fig. 2a and b). The sequence and positions of ligand residues in the loop d o not follow the canonical HLH pattern, however. The secondary structural elements flanking the loop are /3 strands, rather than a helices, and this binding site can be classified as an (n loop (Leszczynskiand Rose, 1986; Section 111,B). Four ion ligands are contributed by loop residues and two are water molecules. Apart from the HLH-binding sites, continuous coordinating peptides such as this one (with all protein ligands from a short single segment of polypeptide chain) are relatively rare (Table 11). The 2PTN site is one of the few truly octahedral Ca'+-coordination spheres found in protein structures to date; the majority are heptacoordinate with pentagonal bipyramidal geometry (Table I1 and Color Plate 1). None of its ligands is bidentate, although two of them are oxygen atoms of glutamate side chains. The glutamate ligands in all of the non-HLH sites are unidentate, in contrast to the bidentate glutamate residues found in all of the HLH sites discussed above. Although the same loop of residues provides the Ca*+-binding site in 2PTN and elastase (SEST), one of the water ligands in 2PTN is replaced by the side chain of Asn-77 in 3EST (Fig. 2c). The site in 3EST shares all of the unusual features of the PPTN site: a continuous coordinating peptide, octahedral ligand geometry, and a lack of bidentate ligands. In the bacterial Streptomyces griseus trypsin (ISGT) structure (33% sequence identity to bovine trypsin) the Ca2+-bindingsite involves a different surface loop almost diametrically across the molecule from the site in 2PTN. Three ligand residues are widely spaced along the 15-residue methionine loop (Asp-165 to Glu- 180); two contribute main-chain carbony1 oxygen atoms and one is a bidentate aspartate side chain (Fig. 2d). The lSGT site is discontinuous, in contrast to 2PTN and 3EST; the fifth protein ligand is from the side chain of Glu-230. The other two ligand positions in this heptacoordinate site are filled by water molecules. Proteolytic autolysis of S. griseus trypsin in solution is prevented by millimolar concentrations of Ca2+ (Olafson and Smillie, 1975). One common Ca2+-binding site is found in all of the subtilisin-like serine proteinase structures, except proteinase K (PPRK). Four of the

98

CATHERINE A. MCPHALEN ET AL.

ligands at this site are part of a nine-residue surface loop [Leu-75 to Gly-83 in the subtilisin Novo numbering (Wells et al., 1983; Olaitan et al., 1968)] that interrupts the helix containing the active-site His-64 (Fig. 2e and f ). The other ligands are from two separate segments of polypeptide that pass on opposite sides of the Ca2+-bindingsite. The carbonyl oxygen atoms of Leu-75 and Thr-79 are the apical ligands in this heptacoordinate site (Fig. 2f). The five equatorial ligands are: the carbonyl oxygen atom of Val-81, Osl of Asn-77, 0"'of Gln-2, and the side-chain oxygen atoms of Asp-41 (bidentate). This is one of the five Ca2+-binding sites in our survey with no water molecules coordinating the ion (Table 11). Ca2+ binding at this site may stabilize the surface loop and its preceding helix toward proteolysis; the entire loop is absent in the proteolysis-resistant proteinase K (2PRK). Ion-binding site 2 of the subtilisins is in a surface crevice close to the primary specificity site for substrate binding (Fig. 2e). The exact location of this site and the identity of the bound ion differ among the subtilisin structures listed in Table I. In subtilisin Carlsberg (2SEC) the site is best described as a pentagonal bipyramid that is missing one equatorial and one axial li and (McPhalen and James, 1988); the mean ion-ligand distance is 2.6 slightly long for Ca2+.The same site is found in the subtilisin Carlsberg (1CSE) structure, with a mean ionligand distance of 2.4 8,(Bode el al., 1987). No ion was observed at this site in the native subtilisin Carlsberg structure, lSBC (Neidhart and Petsko, 1988). Site 2 in the subtilisin Novo (BPN') structures (2SN1, 1SO1, and 1ST2) is even more variable. I n 2SNI the ion is hexacoordinate, but with pentagonal bipyramidal geometry lacking one equatorial ligand (McPhalen and James, 1988). This site has three protein ligands in common with site 2 of subtilisin Carlsberg (2SEC), but the mean ion-ligand distance is 2.9 A, more characteristic of K+ than Ca2+.I n 1ST2 the same ligands and mean ion-ligand distance are observed, plus one additional water ligand (Bott et al., 1988). Site 2 in lSOl is shifted in position relative to the other structures, with four water ligands, and only two protein ligands in common with the other sites 2 (Pantoliano et al., 1988). T h e shifted site is still heptacoordinate, with penta onal bipyramidal geometry and a mean Ca2+-ligand distance of 2.4 . Pantoliano et al. (1988) showed that the occupancy of lSOl site 2 (but not site 1) increases with an increasing concentration of CaC12 in crystallization buffers. When the CaC12 was replaced with NaOH or KCl, the position of the bound ion changed to resemble more closely the SSNI and 1ST2 sites. These authors showed that increasing amounts of CaC12 provide increased stability to thermal denaturation for subtilisin Novo. Site 1 of proteinase K (2PRK) is located in the same region as site 2 of the subtilisins, but has different ligands again (Fig. 2g and h). It is one of the few octacoordinate protein Ca2+-bindingsites observed to date, with

1,

1

CALCIUM-BINDING SITES IN PROTEINS

99

four protein and four water ligands at a mean distance of 2.5 A. The geometric arrangement of the ligands resembles a pentagonal bipyramid with one vertex shared by two oxygen atoms of a bidentate ligand (Fig. 2h). T h e crystal structure of EDTA-treated proteinase K shows small conformational changes in the region of this Ca2+-binding site that may affect substrate binding and thus reduce catalytic activity (Bajorath et al., 1989). EDTA treatment does not alter the protein conformation around the weakly bound Ca2+ at site 2 of 2PRK. The location of 2PRK site 2 on the protein surface far from the active site is unique among the subtilisinlike proteinases. T h e five Ca2+ ligands of this site superpose well on an ideal pentagonal bipyramid that lacks two equatorial vertices. Thermitase is known to bind u p to three Ca2+ ions in solution (A mme1 and Hohne, 198l), and possible sites for all of these ions were locared in the crystal structures (lTEC and others in Table I). Site 1 is similar to site 1 in the subtilisins, discussed above, with Asp-5 in thermitase substituted for Gln-2 in the subtilisins as a Ca2+ ligand. Site 2 in thermitase is novel; it is formed by a continuous loop of 10 residues (Asp-57 to Gln-66), providing all of the coordinating protein ligands. In the subtilisin structures this loop is not well ordered and contains the single amino acid deletion between Carlsberg and Novo. The coordination geometry of site 2 in lTEC is somewhat irregular; it is roughly pentagonal bipyramidal, but lacks one axial ligand (Table 11).Crystals for the lTEC structure were grown with no added Ca2+,and the mean ion-ligand distance is 2.6 A. A second structure of thermitase crystallized from a solution of 5 mM Ca'+ shows similar geometr at this site, but a more characteristic mean ionligand distance of 2.5 for the Ca2+ (Gros et al., 1989). Recently, a third structure of thermitase was solved with crystals grown from 100 mM Ca2+ (Gros, 1990). In this third structure the Ca2+ of site 2 is eight coordinate with a mean ion-ligand distance of 2.4 8, (Fig. 2i and j). As with proteinase K (2PRK) site 1, the geometry of this eight-coordinate site is best described as pentagonal bipyramidal with two ligands sharing one vertex (Fig. 2j). Site 3 of lTEC is analogous to site 2 of the subtilisins and appears to bind Na+ or K+ in the lTEC structure, with a mean ionligand distance of 2.9 A. In the thermitase structure from 100 mM Ca2+, site 3 is a regular Ca2+-binding site with seven ligands arranged in a pentagonal bipyramid. Four of the ligands are water molecules, and the mean ion-ligand distance is 2.4 8, (Gros, 1990).

1

C . Other Ca2+-BindingProteins Thermolysin is a zinc-dependent proteinase ( M , = 34.613) that requires Ca2+ for optimal thermal stability. The thermolysin (STLN) crystal structure contains four bound Ca2+ ions. Two of these lie only 3.8 A

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CATHERINE A. MCPHALEN ET AL.

a /

9

T I R 193

T R S P 185

,l&y IE3/

“\/

TYR 133

FIG.3. Stereo views of other Ca2+-bindingproteins, drawn and labeled as in Fig. la and b. (a) The unusual double-Ca2+site of thermolysin (3TLN) (Holmes and Matthews, 1982), with both ions bound by the same segment of protein, Glu-177 to Tyr-193. Site 1 is heptacoordinate, with Asp-138 and Asp-185 as apical ligands and Glu-177, Glu-187 (peptide carbonyl oxygen), Glu-190 (bidentate), and HOH-346 as equatorial ligands. Site 2 is only hexacoordinate, with octahedral geometry. Ca2+2 shares three ligand side chains with Ca2+ 1: Asp-185, Glu-177, and Glu-190. (b) Ca2+-bindingsite 3 of 3TLN (Holmes and Matthews, 1982). This is a regular heptacoordinate site, with Gln-61 (peptide carbonyl oxygen) and HOH-503 as apical ligands and Asp-57 (bidentate), Asp-59, and two other water molecules as equatorial ligands. (c) Ca2+bound at the active site of S. aureus nuclease (1SNC) (Loll and Lattman, 1989).The apical ligands of this heptacoordinate site are Asp-2 1 and HOH-87, and the equatorial ligands are Asp-40, Thr-41 (peptide carbonyl oxygen), HOH-70, HOH-79, and one phosphate oxygen from the pdTp substrate analog bound to the enzyme. HOH-87 is also hydrogen bonded to the side chain of the proposed catalytic residue Glu-43. (d) The a-carbon backbone trace of bovine phospholipase A2 (1BP2) (Dijkstra et al., 1981b), with a single Ca2+ ion bound at the active site. The highlighted residues (thick lines) are the catalytic His-48 and the adjacent Asp-49, a bidentate ligand to the Ca2+ ion. (e) An enlarged view of the Ca*+-bindingsite in 1BP2 (Dijkstra et al., 1981b), including the catalytic residue His-48. The Ca2+ ligands are the peptide carbonyl oxygen of Tyr-28 and HOH-12 (apical), plus the peptide carbonyl oxygens of Gly-30 and Gly-32, Asp-49 (bidentate), and HOH-5 (equatorial). (f) The a-carbon backbone trace of alactalbumin (1ALC) (Acharya et d., 1989).The long helix following the Ca‘+-binding loop forms one side of the sugar- binding cleft. Thr-33 and Glu-49, on the opposite side of the molecule, are the residues positioned analogously to the two catalytic residues in the structurally similar lysozyme molecule (Glu-35 and Asp-52). (9) The Ca2+-binding site of 1ALC (Acharya et al., 1989),including the two helices that flank the ion-binding loop,joined by a tight turn, or “elbow.” This heptacoordinate site lacks a bidentate carboxylate ligand. Apical ligands are Lys-79 and Asp-84 (both peptide carbonyl oxygens); equatorial ligands are Asp-82, Asp-87, Asp-88, and the two water molecules. (h) The Ca2+-bindingsite of the o-galactose-binding protein (ZGBP) (Vyas et al., 1989). Note the conformational and sequence similarities of the ion-binding loop to those of the helix-loop-helix (HLH) sites

101

CALCIUM-BINDING SITES IN PROTEINS

\

\

C

(Fig. 1). In particular, 2GBP resembles site 1 of parvalbumin (5CPV, Fig. Id), with the long side chain of Gln-142 replacing the usual water molecule as one of the apical ligands. A segment of the p strand following the loop in PGBP is shown (Gln-142 to Leu-145); this strand replaces the usual helix following the HLH sites. (i) The Ca'+-Mn2+ binding site of concanavalin A (2CNA). As in the double-Ca'+ site of thermolysin (JTLN, Fig. 3a), one segment of polypeptide chain provides almost all of the ligands to both ions. The Ca'+binding site is heptacoordinate, with Asp-19 and HOH-6 as the apical ligands; Asp-10 (bidentate), Tyr-12 (peptide carbonyl oxygen), Asn-14, and HOH-5 are the equatorial ligands. Asp-10 and Asp-19 are also Mn'+ ligands, as are Glu-8, His-24, and two water molecules.

102

CATHERINE A. MCPHALEN ET AL.

d

e

l

I FIG. 3. (continued)

n

FIG.3. (continued)

v

104

CATHERINE A. MCPHALEN ET AL.

h

k

ASN I30

& 130

140 ASN 1

1\!

LEU 145

FIG.3. (continued)

\

LEU 145

CALCIUM-BINDING SITES IN PROTEINS

105

apart in an unusual double-Ca2+ site, with the side chains of three residues as ligands to both ions (Fig. 3a). Site 1 is a regular heptacoordinate site with pentagonal bipyramidal geometry and one bidentate glutamate side-chain ligand. Site 2 is more unusual; it is hexacoordinate, with all four protein ligands derived from the same continuous segment of 13 residues that also provides most of the ligands for site 1 (Fig. 3a). This is the second genuinely octahedral site among the proteins of Table 11, and the root mean square (rms) deviation of the ligand atoms from an ideal octahedron is only 0.15 A. Three negatively charged side chains provide ligands to both Ca2+ sites: Asp-185 (Ospto site 1 and Osl to site 2), Glu-177 (OE1 to site 1 and O"*to site 2), and Glu-190 (OE1 to site 1 and O"* to both sites 1 and 2). The double site is close to a substrate specificity pocket of the enzyme, but plays no direct role in catalysis. The four protein ligands of site 3 in 3TLN are part of a five-residue loop (Fig. 3b). The three ligands completing the pentagonal bipyramidal coordination sphere are water molecules. Site 4 is also formed by a short surface loop, but has five protein and two water ligands. The geometry of site 4 is that of a distorted pentagonal bipyramid, although the ion-ligand distances are quite reasonable for Ca2+.Some of the distortion may result from the unusual bidentate threonine coordinating the ion; both the 0"'and the carbonyl oxygen of Thr-194 are ligands. The Ca2+ ions bound at the individual sites 3 and 4 may contribute to thermostability by stabilizing and protecting from autolysis the exposed loops forming the sites (Voordouw and Roche, 1974; Holmes and Matthews, 1982). Staphylococcus aureus nuclease and phospholipase A2 both require Ca2+ for catalytic activity. The nuclease catalyzes DNA and RNA hydrolysis at the 5' position of a phosphodiester bond. Enzymatic studies of the nuclease show that two Ca2+ions are necessary for catalysis (Cuatrecasas et al., 1968), but only one is found in the S. aureus nuclease (1SNC) crystal structure (Cotton et al., 1979; Loll and Lattman, 1989). The single Ca2+is intimately bound at the active site, with the 5'-phosphate group of a deoxythymidine 3',5'-bisphosphate (pdTp) substrate analog as one ligand and the side chain of the proposed catalytic general base, Glu-43, close by (Cotton et al., 1979; Loll and Lattman, 1989) (Fig. 3c). This is a heptacoordinate Ca2+-binding site, with good pentagonal bipyramidal geometry. Only three ligands are protein atoms, however; the others are three waters and one phosphate oxygen from the bound pdTp. One of the ligand waters is also hydrogen bonded to the side chain of the catalytic Glu-43. T h e role of the Ca2+ ion is postulated to be polarization of the phosphate at the scissile phosphoester bond of a substrate (Cotton et al., 1979).

106

CATHERINE A. MCPHALEN ET AL.

Phospholipase A2 hydrolyzes the 2-acyl bond of phosphoglycerides (van Deenen and De Haas, 1964). In phospholipase A2 the Ca’+ ion bound near the active site (Fig. 3d) may function similarly to the Ca2+ of the S. aurem nuclease (Dijkstra et al., 1981a). The Ca2+-binding site of bovine phospholipase A2 (1BP2) is a fairly regular pentagonal bipyramid including two water molecules and one bidentate aspartate side chain (Fig. 3e). Based on the 1BP2 crystal structure and mechanistic analogy with the serine proteinases (Dijkstra et al., 1981a,b), the Ca2+ ion is proposed to serve as part of an oxyanion hole. This is the positively polarized binding pocket of serine proteinases that accommodates the carbonyl oxygen atom of a scissile peptide bond (Henderson, 1970). It acts to orient and polarize the carbonyl bond, leaving the carbonyl carbon atom with a partial positive charge and thus more susceptible to nucleophilic attack. A second possible role for the Ca2+in lBP2 is in binding of the substrate phosphate group. Both of these suggestions for the function of Ca2+in phospholipase A2 have received support from the recently solved structure of porcine phospholipase A:! (5P2P) with an inhibitory substrate analog bound at the active site (Thunnissen et al., 1990b). In this structure two water ligands of the Ca2+ are replaced by a phosphate oxygen and a carbonyl oxygen atom of the inhibitor. A second Ca“binding site is observed in the crystal structure of porcine phospholipase A2 (3P2P), with six ligands contributed by two molecules packed together in the crystals (Dijkstra et al., 1983; Thunnissen et al., 1990a). a-Lactalbumin is a modulator component of the lactose synthase complex (Brew et al., 1968). T h e overall fold of the a-lactalbumin (1ALC) structure is very similar to that of hen egg white lysozyme (Acharya et al., 1989) (Fig. 3f ), although their “catalytic” residues and substrate-binding sites are different in detail (a-lactalbumin has no enzymatic activity). Ca2+ binding to a-lactalbumin may affect the protein conformation and its modulator activity toward the lactose synthase complex within the Golgi apparatus (Permyakov et al., 1981; Musci and Berliner, 1985). In this respect the possible modulator role of Ca2+in a-lactalbumin is similar to that in troponin C and calmodulin. On the other hand, lysozymes from some species bind Ca2+ at the same site as in a-lactalbumin, but have no known modulator function (Acharya et al., 1989, and references therein). T h e Ca2+-binding site in the lALC structure is formed by two helices with a loop joining them (Fig. 3g), but the arrangement of the structural units is quite unlike a classical HLH site (Acharya et al., 1989). The first helix in 1ALC is a single turn of 3 1helix, ~ followed by a tight loop of only four residues and a longer a helix. The angle between the helices is approximately 135” (with the helix dipoles close to antiparallel), compared to around 110”in HLH proteins. Acharya et al. (1989) refer to this

CALCIUM-BINDING SITES I N PROTEINS

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helix-loop-helix arrangement as an “elbow,” rather than an EF hand. The second helix of the lALC Ca2+-binding site forms one wall of the deep substrate-binding cleft (Fig. 3f ). The seven Ca2+-binding ligands (five from the continuous segment of protein plus two water molecules) form a regular pentagonal bipyramid (rms deviation of 0.19 A) (Table I1 and Fig. 3g). The lack of a bidentate ligand at this site (contributing two oxygen ligands much closer to each other than ideal) explains in part the regularity of the ligand geometry. The D-galactose-binding protein is one member of a large family of periplasmic binding proteins participating in bacterial chemotaxis and active transport (reviewed by Furlong, 1987). It binds one Ca2+ ion in a nine-residue loop (residues 134- 142) that strongly resembles the HLH loop in its ligands and conformation (Vyas et al., 1987, 1990) (Fig. 3h). The nine residues of the D-galactose-binding protein (2GBP) Ca2+binding loop can be aligned nicely with the first nine residues of the canonical HLH loop (Vyas et al., 1987).The rms deviation between the 36 main-chain atoms of the two nine-residue segments is 0.55 A [PGBP residues 134- 142 versus calmodulin (3CLN) residues 93-1011. The main-chain torsion angles 4 and t / ~of the 2GBP segment are very close to the mean values found for the equivalent residues in the HLH loops (Strynadka and James, 1989). T h e 2GBP segment is preceded by a reverse turn (residues 131-134), begins with a reverse turn (residues 134137) and a y turn (residues 133-135), and the eighth and ninth residues (Ile-141 and Gln-142) are part of a /3 strand, all characteristic features of the HLH loops (Figs. 1 and 3h). Sequence alignment of the 2GBP and HLH loops reveals that the Ca2+-ligand residues are all conserved among them, except the side chain of the residue at position 7 that coordinates the Ca2+ with a main-chain carbonyl oxygen atom (Vyas et al., 1987). Other conformationally important residues are also maintained (e.g., the highly conserved glycine at position 6, and the hydrophobic side chain at position 8 that appears to anchor these loops to the protein core). In 2GBP the invariant glutamic acid at position 12 of the HLH loop is replaced by Glu-205, making the PGBP site only semicontinuous. Conformationally, though, the Ca2+ligands are positioned as in a regular HLH site. The 2GBP site contains no water molecules as ion ligands; as in carp parvalbumin (5CPV) site 1, the side chain of Gln-142 is long enough to coordinate directly to the Ca2+ ion without an intervening water. Unlike the HLH loops, however, the 2GBP loop is not preceded immediately by a helix, and it ends in an extended /3 strand, rather than a helix (Fig. 3h). Most unusual, compared to all of the HLH loops, the 2GBP site is not one of a pair of Ca’+-binding loops. The paired HLH loops have likely evolved from a common ancestor

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(Kretsinger, 1980), but the 2GBP loop has probably evolved independently. The proposed role for Ca2+ in 2GBP is structural stabilization (Vyas et al., 1987). The Ca2+-binding site is 30 8, or more from known protein functional regions for ligand binding and interaction with signal transducers. Concanavalin A is another saccharide-binding protein, structurally and functionally unrelated to the D-galactose-binding protein. It is a mitogenic lectin that binds to cell surface carbohydrates and can agglutinate a variety of cell types (Wang etal., 1971).Both Mn’+ and Ca2+must bind sequentially before saccharide will bind to concanavalin A (Sumner and Howell, 1936; Agrawal and Goldstein, 1967; Kalb and Levitzki, 1968; Yariv et al., 1968). The two metal ions share two aspartate sidechain carboxylate groups as ligands, in an arrangement reminiscent of the double-Ca2+ site in thermolysin (3TLN) (Hardman and Ainsworth, 1972; Becker et al., 1975) (Fig. 3i). The saccharide-binding site is about 10 8, from the metals (Derewenda et al., 1989), thus small structural rearrangements on metal ion binding may order or adapt the protein for subsequent saccharide binding. Such adaptation is indicated by comparison of the metal-binding and metal-free protein structures (Shoham et al., 1979).

111. REGULARITIES A N D RECURRENT THEMES I N Ca*+-BINDINGSITES A. Continuous, Semicontinuous, and Discontinuous Coordinating Peptides One of the most obvious similarities among the Ca2+-binding sites of Table I1 is that the protein ligands in many sites are all part of the same local segment of polypeptide chain (termed a “continuous” binding site). A similar observation was made by Einspahr and Bugg (1984) for the smaller number of proteins in their survey. Outside of the 10 HLH sites listed, all of which have continuous coordinating peptides (Fig. l), eight other sites are continuous (Table 11; 2PTN in Fig. 2b; 3EST in Fig. 2c; 2SEC site 2; lTEC site 2 in Fig. 2i; 3TLN sites 2 and 3 in Fig. 3a and b; lALC in Fig. 3g; and 2CNA in Fig. 39. T h e HLH loops are 12-residue segments [14 for calbindingK (3ICB) site 11 contributing six or seven ligands to the Ca2+-coordinationsphere. The other continuous binding loops range from five to 14 residues in length and provide three to six coordinating ligands. An additional five Ca*+-bindingsites listed in Table I1 have continuous local peptide segments, providing the majority of the Ca2+ ligands from protein [Table 11; 2SEC site 1, four of seven ligands (Fig. 2f); lTEC site

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1, four of seven ligands; 3TLN site 1, five of six ligands (Fig. 3a); 1BP2, three of five ligands (Fig. 3d); and 2GBP, five of seven ligands (Fig. 3h)l. These semicontinuous sites resemble the fully continuous binding loops in their irregular secondary structure and lack of conformational similarity. T h e one exception is the loop in the D-galactose-binding protein (2GBP), which is highly similar to the first nine residues of an HLH loop (Vyas et al., 1987; Section 11,C). Four of the Ca2+-bindingsites in Table I1 can be considered completely discontinuous [ lSGT (Fig. 2d), 2PRK sites 1 and 2 (Fig. 2h), and lSNC (Fig. 3c)l. The S. griseus trypsin (1SGT) site comprises five protein ligands: the bidentate Asp- 165 and the main-chain carbonyl oxygen atoms of Ala-177A and Glu-180. The 0"' of Glu-230 completes the ligand sphere, along with two water molecules. Site 1 of proteinase K (2PRK) contains only four protein ligands; site 2 of 2PRK and the S. aureus nuclease (1SNC) site each contain only three protein ligands. Water molecules comprise the remaining ligands in each site (and an inhibitor phosphate oxygen in the case of ISNC).

B . Secondary Structural Elements and Main-Chain Conformations Conformationally, there is little in common among the Ca*+-binding sites listed in Table 11, except their lack of regular repeating secondary structure (ahelices o r /3 strands). The HLH sites all begin with a y turn, contain a three-residue /3 strand, and finish with the three aminoterminal residues of an a helix (Strynadka and James, 1989). Thermolysin (3TLN) sites 1 and 2 begin with four residues of a helix (Fig. 3a). T h e a-lactalbumin (1ALC) site begins with three residues of 310 helix and ends with four residues of a helix (Fig. 3g). All of the remaining sites are composed of only turns, bends, and nonregular structure, with the occasional isolated /3 bridge observed (analysis and terminology as in Kabsch and Sander, 1983). Given the variability in length of the coordinating peptides, the different positions of ligands along the peptides, differences in the use of main-chain versus side-chain atoms as ligands, and the positions of most coordinating peptides on the surfaces of proteins, the lack of structural similarity among the binding sites is not at all surp r isi n g . Although the continuous and semicontinuous Ca2+-bindingpeptides lack regular repeating secondary structure, several of them can be classified as fl loops (Leszczynskiand Rose, 1986).An fl loop is any segment of polypeptide chain between six and 16 residues in length; the termini of the loop must lie close together, and the loop must contain no regular secondary structure (ahelices or /3 strands). fl loops are usually compact structures, with side chains of loop residues packed tightly in the center

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of the loop. As stated by Leszczynski and Rose (1986), the HLH loops are excellent examples of fl loops (Fig. 1) if the restriction on inclusion of regular secondary structure is relaxed slightly (each HLH loop contains a three-residue j3 strand). Of the 12 non-HLH continuous or semicontinuous sites in Table 11, nine could be considered fl loops. The Ca2+binding loops of 2PTN (Fig. 2b), 3EST (Fig. 2c), 2SEC site 1 (Fig. 2f), lTEC site 1, lTEC site 2 (Fig. 2i), 3TLN site 3 (Fig. 3b), 1BP2 (Fig. 3e), 1ALC (Fig. 3g), and 2CNA (Fig. 3i) are all Q loops or segments of fl loops by the strict definition of this structural unit (single turns of helix within a loop are not considered regular secondary structure). The remaining three of the 12 Ca2+-binding loops do not qualify as fl loops on grounds of lack of compactness [subtilisin Carlsberg (2SEC) site 21 or distance between loop termini [thermolysin (3TLN) sites 1 and 2 (Fig. 3a)l. T h e high frequency of fl loops at Ca2+-bindingsites is reasonable, given that such sites are often formed by loops of protein averaging 10 residues in length, with side chains directed to the interior of the loop available to coordinate the Ca2+ ion. An Asx turn is a secondary structural interaction involving hydrogen bonding between the side-chain oxygen atom of an aspartate, asparagine, serine, or threonine residue at position n with the main-chain NH of a residue at position n+2 (Richardson, 1981; Rees et al., 1983; Baker and Hubbard, 1984). T h e values of the main-chain torsion angles b, and J, of residue n+ 1 in an Asx turn are equivalent to b, and J, of residue n+2 in a 310 reverse turn; thus, Asx turns are classified by their analogy to reverse turns (Rees et al., 1983). The importance of Asx turns within the framework of a Ca2+-binding site was first described in the crystallographic analysis of turkey troponin C (5TNC) (Herzberg and James, 1985b). Contiguous Asx turns occur within each of the 12-residue high-affinity Ca2+-binding loops in this structure (Asn-108, Asp-110, and Asp-114 of site 3; Asn-144, Asp-146, and Asp-150 of site 4). These turns, in conjunction with two reverse turns and a conserved glycine residue, are critical in maintaining the conformation of the ligand loop (see Color Plate 2a). Five of the six side chains involved in initiating these Asx turns are also direct Ca2+ligands. I n each case the side-chain conformation required to form the Asx turn also appears to assist in positioning the side-chain oxygen ligand correctly for effective coordination to the Ca2+ ion. In addition these hydrogen-bond interactions serve to stabilize, in part, the cluster of negatively charged ligand oxygen atoms in the Ca2+-coordination sphere. Analysis of the structures of other HLH proteins indicates that the series of Asx turns observed in 5TNC is a highly conserved feature of the Ca2+-bindingsites for members of this protein family [e.g., calmodulin (3CLN) Asp-95 and Asn-97 (Fig. lb), calbindingK (3ICB) Asp-19

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(Fig. lc), and carp parvalbumin (5CPV) Asp-53 and Ser-55 (Fig. Id)] (Strynadka and James, 1989). Examination of the non-HLH protein structures listed in Table I1 indicates that Asx turns are components of most Ca2+-bindingloops. For example, the Ca2+-binding sites of 2PTN [ A m 7 2 (Fig. 2b)], 3EST [Asn72 (Fig. 2c)], 2SEC [Asn-77 (Fig. 2f)], lTEC [Asp-57 and Asp-62 (Fig. 2i)], 3TLN [Asp-185 (Fig. 3a) and Asp-57 and Asp-59 (Fig. 3b)], lALC [Asp-84 (Fig. 3g)], and 2GBP [Asp-138 (Fig. 3h)l all contain Asx turns. Other examples are not illustrated. The only Ca*+-bindingsites of Table I1 lacking an Asx turn are S. grzieus trypsin (ISGT), subtilisin Carlsberg (2SEC) site 2, proteinase K (2PRK) site 2,phospholipase A:, (1BP2), and concanavalin A (2CNA). The aspartate or asparagine side chains forming an Asx turn also often provide one or two oxygen atoms to coordinate the Ca2+ ion. As in the HLH proteins, the role of the Asx turn in the non-HLH proteins appears to be both stabilization of main-chain conformation and correct positioning of a ligand oxygen atom. In addition over 80%of the proteins with Asx turns in their Ca2+-bindingloops have a proline or glycine residue adjacent to the Asx turn (within the three residues preceding o r following the Asx). The combination of the Asx turn and the restricted range of d, and 9 values associated with a proline favors a reversal of the polypeptide chain direction following the turn. T h e presence of a glycine residue close to the Asx turn provides additional flexibility in the main chain that enables chain reversal. In many cases the chain reversal is essential to allow either a main-chain carbonyl oxygen atom or another aspartate side chain to be positioned favorably as a Ca2+ligand. Martin (1984) suggested that proline residues often follow immediately after residues that act as Ca2+ ligands via their peptide carbonyl oxygen atoms; the reduced acidity of the proline peptide nitrogen is proposed to enhance the basicity of the preceding carbonyl oxygen. In our larger sample of protein Ca2+-bindingsites, proline residues follow only 14% of peptide carbonyl oxygen ligands. Their overrepresentation in Ca2+-bindingloops (Martin, 1984; Einspahr and Bugg, 1984) is thus more likely due to their unusual conformational properties than to special chemical properties. Baker and Hubbard (1984) point out that n to n+2 hydrogen bonding occurs in protein structures with serine, threonine, and cysteinyl residues in addition to aspartate or asparagine side chains. In the Ca2+-binding sites of the proteins analyzed, aspartate side chains are clearly the most common type of residue forming the Asx turns, with asparagine residues a distant second. Aspartate residues are also the most common type of Ca2+ ligand in these sites (Section 111,D).Two serine residues form Asx turns in Ca2+-binding sites: Ser-55 of carp parvalbumin (5CPV) and

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Ser-24 of calbindingK (3ICB). Two glutamate residues also form highly unusual n to n+2 hydrogen bonds (Baker and Hubbard, 1984): Glu-174 of proteinase K (2PRK) and Glu-43 of S. aurew nuclease (ISNC). T h e involvement of Asx turns in metal binding sites is not unique to the Ca2+-bindingproteins. The iron-sulfur protein family, including ferredoxin, rubredoxin, and the high potential iron protein, exhibits conserved cysteine residues that participate both in coordination to the iron and in n to n+2 interactions with main-chain nitrogen atoms (Adman et al., 1975). C. Coordination Number Studies of small-molecule Ca2+ complex structures have shown that the Ca2+-coordinationnumber can vary from four to 12 (Brown, 1988). The numbers of coordinating ligands observed in the protein structures of Table 11 range from five to eight. The geometric distributions of the ligands and the ligand-Ca2+-ligand angles of the binding sites indicate, however, that most sites are best modeled as either octahedral (six coordinate) or pentagonal bipyramidal [seven coordinate (Color Plate l)]. In the sites with five or six ligands that display pentagonal bipyramidal geometric paramaters [subtilisin Carlsberg (2SEC) site 2, proteinase K (2PRK) site 2, and thermitase (1TEC) site 21, the “missing” ligands are usually presumed to be disordered water molecules that are not observed in electron density. If these three sites are treated as incomplete heptacoordinate, then all but four sites in Table I1 are best modeled as pentagonal bipyramids. Three of the exceptions are trypsin [SPTN (Fig. 2b)], the almost identical site in elastase [SEST (Fig. 2c)], and site 2 of thermolysin [3TLN (Fig. 3a)]; the six ligands of these sites fit best to the ideal octahedron (Color Plate lb). None of these three sites contains a bidentate ligand. Having two ligands very close together, as in a bidentate carboxylate side chain, may be one way to permit seven protein ligands to crowd around a single Ca‘+ ion. Subtilisin Carlsberg (2SEC) site 2 and a-lactalbumin [IALC (Fig. 3g)l are heptacoordinate sites that lack a bidentate ligand; the first, however, presumably has four water ligands. Th e sheer steric and topological difficulty of arranging more than seven relatively bulky amino acid groups within reasonable distance of a Ca2+ ion may explain the relative lack of Ca*+-binding sites in proteins with coordination numbers of eight or higher. Indeed, Einspahr and Bugg (1984) observed an increase of mean Ca2+-ligand distance with increasing coordination number in their survey of small-molecule Ca2+complex structures. One eight-coordinate Ca2+-binding site has been reported recently in

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the proteinase K (2PRK) structure. Site 1 of 2PRK (Fig. 2g and h) is located in the same depression on the protein surface as site 2 of the subtilisins and thermitase. The eight ligands at this site include four from the protein (one bidentate) and four water molecules. The geometry of the site is somewhat irregular, and can best be described as a pentagonal bipyramid with one vertex shared by two ligands (Fig. 2h). Ca2+-binding site 2 in the structure of thermitase at 100 mM Ca2+is also reported to be eight coordinate (Gros, 1990) (Fig. 2i and j). Although only one of the ligands at this site is a water molecule, two protein residues provide bidentate coordination: the side-chain carboxylate group of an aspartate and the side-chain Oy plus the peptide carbonyl oxygen of a threonine (Fig. 2i). Bidentate coordination by a threonine residue is rare, but has been observed also in thermolysin (3TLN) site 4 (not included in Table 11).A third eight-coordinate protein Ca2+-bindingsite has been reported recently (Boel et al., 1990) in two a-amylase structures (1AAA and 3TAA). T h e ligands are a bidentate aspartate side chain, an asparagine side chain, two main-chain carbonyl oxygens, and three water molecules. The mean Ca2+-ligand distances in these eight-coordinate sites are 2.58(14)A (1AAA and STAA), 2.49(9)A (PPRK), and 2.44(17)A (thermitase at 100 mM Ca2+),slightly longer than the averages of 2.38( 12)A and 2.42( 17)A for the hexacoordinate and heptacoordinate sites, respectively (see Section 111,D).With the current small populations of six- and eight-coordinate sites, these small differences are merely suggestive that mean Ca2+-ligand distances may increase with coordination number in proteins, as they d o in small molecules. D. Distribution of Ligand Types and Mean Ca"+ -Ligand Distances The great majority of Ca2+ligands in small-molecule crystal structures are oxygen atoms. In protein crystal structures oxygen atoms are the only Ca2+ligands observed to date. Of the 182 ligands in the 27 sites of Table 11,53 (29%) are aspartate side chains, 11 (6%)are asparagines, 33 (18%) are glutamates, three (2%)are glutamines, 43 (23%)are peptide carbonyl oxygens, 37 (20%) are water molecules, one is a serine, and one is a phosphate oxygen. Two threonine residues are found as Ca2+ ligands in sites not included in Table 11 [thermolysin (3TLN) site 4 and site 2 of thermitase at high Ca2+ concentration]. The high proportion of aspartate and glutamate side chains is not surprising; the carboxylate groups of these residues provide charge balance for the Ca2+ ion at most sites. As well, the side chains are able to participate in the network of hydrogenbond interactions that stabilize most Ca*+-binding sites (see Section 111,G).Asparagine and glutamine bind to Ca2+via their side-chain amide

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oxygen atoms and can only donate a hydrogen bond from their sidechain amide nitrogen atoms when they are Ca2+ ligands. The paucity of serine and threonine side chains as Ca2+ligands may be because they lack additional functional groups in their side chains to participate in stabilizing hydrogen-bond networks. Possibly, too, the more polar carboxylate, carbonyl, and water oxygen atoms are able to interact more strongly with the Ca2+ ion by means of negative charges or permanent dipoles. T h e only absolutely invariant Ca2+ ligand in all the sites of Table I1 is a peptide carbonyl oxygen; all sites contain at least one. The position of this oxygen in the ligand geometry is not conserved, however. Peptide carbony1 oxygens occur as both axial and equatorial ligands in the pentagonal bipyramidal sites. All but five of the sites contain at least one water molecule, but the position of the water oxygen in the ligand geometry is not conserved either. If the Ca2+-binding sites of Table I1 are divided into HLH-like sites (5TNC, SCLN, 3ICB, 5CPV, and 2GBP) and non-HLH sites, some interesting differences between the types of ligands in the two classes are apparent. All of the HLH-like sites contain a bidentate glutamate side chain, one of the invariant residues of the HLH loop. Only five of the 16 non-HLH sites contain glutamates (Table 11; 2PTN in Fig. 2b; 3EST in Fig. 2c; lSGT in Fig. 2d; and STLN sites 1 and 2 in Fig. 3a), and only one of these is bidentate (Glu-190 of 3TLN site 1). None of the aspartates in the HLH-like sites are bidentate, but nine non-HLH sites have bidentate aspartates (Table 11; lSGT in Fig. 2d; 2SEC site 1 in Fig. 2f; 2PRK sites 1 and 2 in Fig. 2g; lTEC sites 1 and 2 in Fig. 2i; STLN site 3 in Fig. 3b; 1BP2 in Fig. 3e; and 2CNA in Fig. 3i). These differences between the two classes of sites reflect primarily the highly conserved sequences and modes of ligand binding in all of the HLH-like sites. Non-HLH sites have much greater sequence and structural variability, but the most common amino acid side-chain ligands for the Ca2+ ions of these sites are still aspartate and glutamate. T h e non-HLH sites incorporate more peptide carbonyl oxygens and water molecules as ligands than d o the HLH sites. Each HLH-like site has one carbonyl oxygen ligand [except site 1 of calbindingK (3ICB), which has four (Fig. lc)] and one water ligand [except carp parvalbumin (5CPV) site 1 (Fig. Id) and D-galactose-binding protein (2GBP) (Fig. 3h), with none]. The non-HLH sites each have from one to three carbonyl oxygen ligands: 44% with one, 3 1% with two, 25% with three, and an average of 1.8 per site. They also contain zero to four water molecules: 19% with zero, 12% with one, 51% with two, 12% with three, 6% with four, and an average of 1.8 water ligands observed per site. The greater numbers of peptide carbonyl oxygen and water ligands in non-HLH sites might be correlated with the locations of these sites in

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relatively unstructured loops on the protein surfaces. In the case of those sites with a greater number of water ligands, the surface loops of nonHLH sites may be more accessible to solvent than the HLH sites, having generally fewer secondary structural interactions with the body of the protein than HLH loops. In the case of the sites with more carbonyl oxygen atom ligands, having more of them bound to the Ca2+would tend to stabilize the backbone conformations of the loops. The HLH loops acquire extra conformational stability through their secondary structural interactions in pairs. Tethering the backbone conformation of surface loops by coordinating peptide carbonyl oxygen atoms to Ca2+ ions in non-HLH proteins may be one mechanism for the Ca2+ stabilization of proteins against thermal denaturation or proteolysis. Einspahr and Bugg (1984),in their review of Ca'+-ligand interactions, found that average Ca2+-ligand distances differed very slightly for different ligand types in small-molecule structures. T h e mean distances that they observed were 2.42(7)8, for Ca'+-water interactions, 2.36(6)8, for Ca'+-carbonyl interactions, and 2.38(7)8, and 2.53(7)8, for unidentate and bidentate Ca2+-carboxylate interactions, respectively. No statistically significant differences are found among the various ligand types for the Ca2+-bindingsites in the proteins of Table 11. The mean Ca'+-ligand distance for Ca'+-carboxylate (aspartate and glutamate, uni- and bidentate each calculated separately), Cay+-asparagine and Ca*+-glutamine, Ca'+-peptide carbonyl oxy en, and Ca2+-water interactions for the 182 ligands of Table I1 is 2.4(2) . Distances range from extremes of 2.01 and 2.05 8, [calmodulin (SCLN) site 31 to 2.99 and 3.15 8, [concanavalin A (SCNA), unrefined structure]. Overall coordinate errors in refined protein structures are usually around 0.2 A, although they may be as low as 0.1 A for clearly defined regions such as most of these Ca2+-binding sites. Coordinate errors in small-molecule crystal structures are typically an order of magnitude lower; thus, small variations in distances, such as those seen by Einspahr and Bugg, are more easily observed. Any such variations in protein Ca'+-binding sites are likely masked by the lower accuracy of the protein coordinates.

1

E. Ligand Geometry Compared to Ideal Polygons

It is clear from the data in Table I1 that protein Ca2+-bindingsites do not have perfectly octahedral or pentagonal bipyramidal geometry (see also Color Plate 1). Figure 4 provides some clues as to which ligand types deviate most from ideal geometry, and the nature of the deviations. First, it can be seen that the observed deviations of ligands from their ideal positions are not due entirely to coordinate errors. The majority of the

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1.4

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deviations are much larger than the estimated coordinate errors of 0.1-0.2 A for the Ca2+-bindingresidues in most structures of Table 11. Second, the distributions for uni- and bidentate ligands are different (Fig. 4).The mean deviation from ideal position for all unidentate ligands from sites with complete coordination spheres (122 in total) is 0.3(2)w, significantly smaller than the mean deviation of 0.6(I)A for bidentate ligands. In part, this is due to structural constraints that prevent the simultaneous positioning of both bidentate carboxylate oxygens on two vertices of an ideal polygon. The two oxygen atoms of a

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normal carboxylate group are only 2.2 A apart, whereas the vertices of an ideal polygon for Ca2+ ligands are 2.82 A (equatorial ligands of a pentagonal bipyramid) or 3.39 A apart (apical ligands of a pentagonal bipyramid, all ligands of an octahedron). Third, the deviation of ligands from ideal position is a combination of deviations from the standard Ca2+ligand distance of 2.4 A and deviations from regular ligand-Ca2+ligand angles in the ideal polygons. If the deviations were only the result of variations in the Ca'+-ligand distance, the data points of Fig. 4 would all lie on the dotted lines. If the deviations were entirely in the ligandCa2+-ligand angles, the data points would all lie on a vertical line at a ligand distance of 2.4 A. Instead, the deviations are quite uniformly distributed between these two extremes. Physically, this means that the geometric criteria for a good Ca2+-bindingsite in a protein are not rigid. T o accommodate up to seven amino acid side chains and peptide groups clustered around a single Ca2+ion, ligand distances and dispositions can be adjusted somewhat from ideal geometry. No correlations are apparent between the rms deviation from the ideal polygon for a site (Table 11) and characteristics such as resolution of the structure, R factor, number of water ligands, number of bidentate ligands, number of peptide carbonyl oxygen ligands, or numbers of aspartate or glutamate ligands. There is no difference between the mean deviations from ideal position for axial versus equatorial ligands. As discussed by Strynadka and James (1989), one oxygen atom of the bidentate glutamate in the HLH Ca2+-binding sites consistently lies out of the equatorial pentagonal plane by approximately 1.2 8, (Fig. 1). They speculate that increasing this deviation from planarity may provide a mechanism for adapting the sites between Mg2+and Ca2+binding. Small changes in the side-chain torsional angles (x',x2,and x3) of the glutamate would allow the carboxylate group to reorient its plane to be approximately perpendicular to the equatorial ligand plane, with only one carboxylate oxygen atom then coordinating the bound ion. This potential switch from a seven- to six-coordinate ligand sphere would favor Mg'+ binding, as this ion has a strong preference for octahedral coordination. The non-HLH Ca2+-binding sites with bidentate ligands have a similar deviation of the carboxylate group from the equatorial plane, although the deviation is somewhat smaller [0.8(3)A]. T h e one exception is the bidentate glutamate in the HLH-like site of the D-galactose-binding protein [SGBP (Fig. 3h)l. Its carboxylate group lies perfectly in the equatorial plane (0.09 8, deviation), well within the coordinate errors of this highly reliable structure. The 2GBP site does bind Mg2+with reasonM) (Vyas et al., 1989). This site may still make able affinity ( K d = 3 x use of a reorientation of the bidentate carboxylate to adapt for Mg2+

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CATHERINE A. MCPHALEN ET AL

binding, but if the above model were to apply, it would require larger changes in the side-chain torsion angles of the glutamate residue.

F. Stereochemistry of Ca" -Ligand Interactions I n general the geometry of ion-ligand interactions is not restrained to particular values during refinement of protein structures; thus, the relatively narrow distributions of distances and angles observed are not an artifact of the structure refinement procedure. One of the most striking features in the interactions of protein Ca2+-ligand groups with the ion is that the Ca2+does not generally lie in the plane of the ligand group (Fig. 5; diagrams of angles defining Ca2+-ligand interactions are given in Fig. 5a and b). This observation is in contrast to that by Einspahr and Bugg (1984) for small-molecule ligands, but is in agreement with the finding by Carrel1 et al. (1988) that Ca2+ ions are less likely to lie in the plane of a small-molecule ligand group than other metals. Unidentate carboxylate groups have the most variable interaction geometry with Ca2+ (Fig. 5c). The mean distance of the Ca2+ to the plane of the carboxylate is 1.1(5)A for the 44 ligands of this type in the proteins of Table 11. Ca2+ ions in HLH-like sites are only marginally further from the carboxylate plane [ 1.2(3)A on average] than those in non-HLH sites [0.8(5)A on average]. T h e angular distribution of the Ca2+ ions relative to the carboxylate group is also the widest for unidentate carboxylate ligands; up to 60" on either side of the Cy-081 vector in the carboxylate plane (angles 6 in Fig. 5a and c). The distribution of Ca2+ ions around asparagine and glutamine side chains (Fig. 5d) is similar to the carboxylates: mean distance of Ca2+ out of the ligand plane, 1.0(6)& angular range of up to 55" out of the ligand plane (angle 6 in Fig. 5b) and up to 35" from the Cy-081vector in the ligand plane (angle 6). There is no significant difference between the distributions for HLH-like and non-HLH sites. The bidentate carboxylate ligands are oriented most directly and consistently toward the Ca2+ion. The mean distance of the Ca2+out of the bidentate carboxylate plane is 0.4(3)A,with a correspondingly small 4 angle distribution out of the plane of less than 30" (Fig. 5e). The angular distribution in the carboxylate plane is, of course, limited by requiring the Ca2+ ion to interact similarly with both carboxylate oxygen atoms. Interactions of metal ions with peptide carbonyl oxygen atoms have been surveyed by Chakrabarti (1990a). Of the 7 1 metal-carbonyl oxygen interactions studied, nine were with metals other than Ca2+.Only refined protein structures were included in the Chakrabarti survey, but structures at both lower and high resolution were accepted. Despite the differences in the proteins sampled, we have obtained values for the geometry

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CALCIUM-BINDING SITES IN PROTEINS

of Ca'+-carbonyl oxygen interactions that are very similar to those obtained by Chakrabarti. Our mean Ca2+-0 distance for a sample size of 43 is 2.3( l)A, compared to 2.4(2)A found by Chakrabarti (Fig. 5f). Our mean distance for the Ca2+ out of the pe tide plane is 0.7(4)& with minimum and maximum distances of 0.02 [thermitase (ITEC) site 11

x

a

b

0 I

ca2+

I

aca2+ FIG. 5 . Distributions of Ca'+ ions around ligand oxygen atoms for Ca"-binding sites in the proteins of Table 11. For each type of ligand group (unidentatecarboxylate, asparaghe/ glutamine, bidentate carboxylate, and peptide carbonyl oxygen), the atoms defining the plane containing the ligand oxygen were superposed on a standard ligand group. The Ca'+ ion position was then transformed by the matrix relating its ligand group to the standard to give the ion distributions. All Cae+ ions are indicated by filled circles. Protein atoms other than those in the standard ligand groups (e.g., Ca's) are shown only for illustrative clarity; they are not necessarily in the same position in all ligand residues. (a) The angle 0 describes the position of a Ca2+ ion in the plane of the ligand group, relative to the C-0 vector. (b) The angle 4 describes the deviation of the Ca" ion from the plane of the ligand group. (c) Distribution of Ca"' ions around unidentate aspartate and glutamate side-chain carboxylate groups. The standard ligand group for superpositions consisted of Cp, Cy, Osl(OD l), and Os2 (OD2) (aspartate) or Cy, C6, W2 and 0"' (glutamate). The Ca2+ ions are more widely distributed around these carboxylate side chains than around other ligand types. The distributions for all ligand types are continuous; they do not consist of separated Ca2+ ion clusters at distinct preferred positions. (d) Distribution of Ca'+ ions around asparagine and glutamine side chains. The standard ligand group was CP, Cy, 0'' (ODl), and Ns2 (ND2) (asparagine), or Cy, C6, W', and Ne2 (glutamine). (e) Distribution of Ca2+ ions around bidentate aspartate and glutamate side chains [standard groups as for (c)1. The Ca2+ions are most tightly clustered about this ligand type, in both their planar (angle 0) and out-of-plane (angle 4) distributions. (f) Distribution of Ca'+ ions around peptide carbonyl oxygen atoms. The standard group was C,: C,, On, N,, , and C:+, .

,

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CATHERINE A. MCPHALEN ET AL.

C

/

0 .

58

0 . OD2

0

0

PIo::+ *

002

0

0

d

m2

NO2

@&

: FIG. 5. (continued)

and 1.98 [subtilisin Carlsberg (PSEC) site 21, respectively. In angular terms the maximum angle (4)of the Ca2+out of the peptide plane is 55", and the maximum angle from the C-0 vector in the plane (6) is 30". T h e corresponding values from Chakrabarti (1990a) are a mean 4 angle out of the peptide plane of about 35" and a mean 6 angle in the peptide plane of about 10". As Chakrabarti indicates, there is no preference for the Ca2+ ions to be located in the directions of the lone pair orbitals on the carbonyl oxygen atoms, in contrast to the clustering of water molecules around carbonyl oxygens (Thanki et al., 1988; Baker and Hubbard,

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e

OD2

OD2

\

a

\

0 '

a a

0'

a m

FIG.5 . (continued)

1984; Rees et al., 1983). We observe a small tendency for the Ca2+to lie on the C a side of the C - 0 vector (Fig. 5f); 58%of the ions in our survey are closest to the Ca, as are 72% of the ions in the survey by Chakrabarti ( 1990a).

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T h e stereochemistry of the Ca2+ interaction with unidentate side chains (aspartate, asparagine, glutamate, and glutamine) is quite specific in one respect. T h e Ca2+may be positioned either syn [on the same side of the ligand C-0 bond as the second carboxylate oxygen (Fig. 6a)l or anti [on the opposite side of the ligand C-0 bond as the second carboxylate oxygen (Fig. 6b)l with respect to the carboxylate oxygen atoms. If the Ca2+ ion lies between the two side-chain carboxylate oxygens in the syn

CB 106

CB 106

CG 106

CG 106

OD2 106

OD2 106

FIG.6. Examples of the positioning of Ca2+ions relative to nonligand side-chain atoms of unidentate ligand residues. The Ca2+ions are filled circles. (a) Asp-106 from troponin C (STNC) site 3. The Ca'+ ion lies between the two C-0 vectors of the carboxylate group (or between the analogous C-0 and C-N vectors of asparagine and glutamine side chains). The Ca2+and the nonligand oxygen atom are on the same side of the ligand oxygen atom C - 0 vector (i.e., in the syn conformation). The torsion angle (highlighted by thick lines) between the ligand oxygen atom and Ca (aspartate and asparagine) or C/3 (glutamate and glutamine) of the amino acid residue is cis (x2 = 0'). The Caz+ ion is approximately 1 8, away from the ideal syn position in this example. (b) Glu-80 from bovine trypsin (PPTN). The Ca'+ ion lies outside the two C-0 vectors of the carboxylate group. The Ca2+and the nonligand oxygen atom are on opposite sides of the ligand oxygen atom C-0 vector (i.e., in the anti conformation). The torsion angle (highlighted by thick lines) between the ligand oxygen atom and Ca (aspartate or asparagine) or CP (glutamate or glutamine) is trans (x3 = 180"). This Ca2+ is approximately in the plane of the carboxylate group (4 = 0'). Examples may also be found in the sites of Table I1 of synltrans and antilcis conformations.

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b

80

80

80

80

Ca2-t

Ca2+

FIG.6. (continued)

conformation (or between 0 and N of asparagine and glutamine side chains), the oxygen that is the Ca2+ ligand is usually cis to the Ca (aspartate or asparagine) or Cp (glutamate or glutamine) atom of the side chain (Fig. 6a). Of the 47 side chains with the Ca2+ oxygen ligand cis to the appropriate side-chain carbon, 40 have the Ca2+ ion in the syn conformation. Sterically, this is certainly a favorable gemoetry that avoids short van der Waals contacts between the Ca2+and the side-chain carbon atom. In the 11 cases in which the oxygen ligand is trans to the side-chain carbon atom (Fig. 6b), seven (64%)of the Ca2+ions are found in the anti

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conformation. With the oxygen ligand and side-chain carbon trans, the Ca'+ can be accommodated comfortably on either side of the oxygen atom. Chakrabarti (1990b) also examined the geometry of metal ion binding to carboxylate and carboxamide groups. Some of the results are similar to our analysis presented here. An alternative or additional influence on the position of the Ca2+ ion relative to the two carboxylate oxygens of aspartate and glutamate side chains may be the inherent basicity of the syn and anti lone pairs of the ligand oxygen atom. Gandour (1981) has proposed that the syn lone pair is more basic than the anti in general base catalysis by carboxylate groups. Carrell et al. (1988), in a survey of metal ion-carboxylate interactions from small-molecule structures, determined that the syn lone pair is generally preferred for cation binding. Ca2+ is one of the larger cations that they examined, however, and larger cations had a higher percentage of anti binding. Cations with mean metal-oxygen distances between 2.3 and 2.6 A, such as Ca2+at 2.42 A, also showed a significant percentage of "direct" or bidentate binding to carboxylate groups. Thus, Carrell et al. found that Ca'+ ions in small-molecule structures were divided almost equally into syn (32%),anti (34%),and bidentate (34%)binding modes with carboxylate groups. In the protein Ca2+-binding sites of Table 11, there is a marked preference for the Ca2+ ion to bind to aspartate and glutamate side-chain carboxylate groups in the syn conformation [53% (Fig. 5c)l. The bidentate binding mode is less common [31% (Fig. 5e)], and the anti conformation is least common [ 16%(Fig. 5c)l. Although the observed preference for Ca2+binding in the syn conformation in proteins may reflect the greater basicity of the carboxylate oxygen syn lone pair, the Ca2+ ions d o not cluster strongly in the immediate direction of the lone pair orbital [i.e., in the plane of the carboxylate group, with a C-O-Ca'+ angle of 120" (Fig. 5c)l. Overall, a combination of steric hindrance from C a or CP of the aspartate or glutamate side chain plus the higher basicity of the syn lone pair produces the observed preference for the syn conformation in protein Ca'+-carboxylate interactions.

G . Networks of Hydrogen Bonds among Ca"

-Ligand Residues The predominance of aspartate and glutamate residues in the Ca2+coordination spheres of proteins was quantified in Section II1,D. They constitute 29% and 18%,respectively, of the Ca'+-coordinating ligands from the proteins presented in Table 11. An analysis of the hydrogenbonding networks around the Ca'+-binding sites of these proteins (Color Plate 2 and Fig. 7) indicates several reasons for the prevalence of these negatively charged amino acids. The side-chain carboxylate moiety of

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aspartate or glutamate not only provides one o r two oxygen atoms with which to bind directly to Ca2+, but also allows for the formation of favorable interactions with main-chain nitrogen atoms, with amide, hydroxyl, o r basic groups on side chains, and with ordered solvent molecules. In the Ca2+-bindingsites of the proteins analyzed, every aspartate or glutamate that coordinates Ca2+ is invariably involved in at least one, and often two, three, or more, hydrogen-bonded interactions. The majority of the interactions involve hydrogen bonds from a carboxylate oxygen to a main-chain nitrogen. As discussed in Section III,B, many side chains in Ca2+-bindingloops take part in the n to n+2 hydrogen-bond pattern of an Asx turn (e.g., Asn-144 and Asp-146 in Color Plate 2a and Fig. 7a; Glu-43 in Color Plate 2b and Fig. 7b). As well, some side-chain carboxylate groups form hydrogen bonds to main-chain nitrogen atoms even farther along the same peptide chain [e.g., the n to n + 4 and n+5 hydrogen bonding of the invariant aspartate in position 1 of the HLH Ca2+-bindingloops (see Figs. l b and c and 7a and Color Plate 2a)] or to a main-chain nitrogen atom from a distant segment of polypeptide [e.g., Glu-205 of D-galactose-binding protein (2GBP) (Fig. 2h)l. Water is the second most common hydrogen-bond donor for the carboxylate oxygen atoms of aspartate and glutamate residues that coordinate to Ca2+ ions. Most of the waters involved in these interactions are Ca2+ ligands themselves (e.g., 0-174 in Color Plate 2a and Fig. 7a). In a few cases the water forms a hydrogen-bonded bridge between the aspartate or glutamate carboxylate oxygen atom and adjacent main-chain or side-chain atoms (e.g., 0-171 in Color Plate 2b and Fig. 7b). There are also several examples of the acidic oxygens of aspartate or glutamate interacting directly with the side chains of nearby asparagine, glutamine, lysine, o r arginine residues (e.g., Asp-146 in Color Plate 2a and Fig. 7a). The multiple hydrogen bonds formed by acidic Ca2+-ligand residues serve to position effectively the side-chain carboxylate oxygen(s) for coordination to the Ca2+ ion, to stabilize neighboring Ca2+ ligands (water molecules or amide side chains), and to stabilize the main-chain conformation of the binding loop required for the correct orientation of other ligand groups. Furthermore, the extensive involvement of both of the carboxylate oxygen atoms in various hydrogen-bonded interactions aids in counteracting the repulsive electrostatic forces associated with juxtaposing two or more negatively charged amino acids within the relatively limited volume of the Ca2+-coordination sphere (Strynadka and James, 1989; Vyas et al., 1990). Asparagine and glutamine residues constitute only 6% and 2% of the Ca2+-coordinating ligands in the sites of Table 11. In theory the amide side chains of these amino acids can provide one oxygen for unidentate coordination to Ca2+ as well as interaction with hydrogen-bond donors;

126

CATHERINE A. MCPHALEN ET AL

b

FIG.7. (a) A stereoscopic view of the hydrogen bonding around Ca2+-bindingloop 4 of troponin C (5TNC). The protein backbone is represented by thick lines; the side chains, by thin lines. All residues are labeled. The extensive hydrogen-bonding pattern shown (dashed lines) is preserved among the Ca2+-binding loops of the helix-loop-helix (HLH) family. Conserved water molecules are small circles, and the Ca2+ion is a large circle. The view is identical to that of Color Plate 2a. (b) The hydrogen bonding around the discontinuous Ca'+-binding site of S.aurew nuclease (lSNC, drawn as in Fig. 7a). The inhibitor pdTp group is drawn with the thicker lines. The view is identical to that of Plate 2b.

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the amide nitrogen atom may donate a hydrogen bond to other groups. Examination of the hydrogen-bonding interactions of asparagine and glutamine residues coordinating to Ca'+ indicates that in the majority of cases the amide oxygen atom acting as a Ca2+ ligand also rece'ives a hydrogen bond from a main-chain nitrogen atom. Most of the asparagine ligands to Ca'+ in this situation form the n to n+2 hydrogen-bonding pattern of an Asx turn (e.g., Asn-144 in Color Plate 2a and Fig. 7a). In two of the 14 asparagine/glutamine residues analyzed, the amide oxygen atom also interacts with a Ca'+-coordinating water molecule. T h e primary interaction of the side-chain nitrogen atom of the Ca'+-coordinating asparaginelglutamine residues is a hydrogen bond to the side chain of an adjacent aspartate or glutamate residue (e.g., Asn-144 to Asp-146 in Color Plate 2a and Fig. 7a). There are no examples in our survey of side-chain amide nitrogen atoms interacting with Ca'+-coordinating water molecules or main-chain carbonyl oxygen atoms. Due to the hydrogen-bonding limitations of the amide nitrogen atom, the number of stabilizing interactions involving a Ca2+-bindingasparagine or glutamine residue is much lower than for an aspartate or a glutamate. Within the Ca'+-binding sites of Table 11,there is a single serine residue that acts as a Ca'+ ligand [carp parvalbumin (5CPV) site 1 (Fig. Id)]. Two examples of threonine ligands to Ca" ions are not included in Table 11 [thermolysin (STLN) site 4 and thermitase at high Ca'+ concentration (see Section 11) 1. Th e limitations of serine and threonine hydroxyl groups relative to amide and carboxylate side chains in forming multiple hydrogen-bonded interactions are obvious. These polar side chains d o play a less direct role in the Ca'+-coordination sphere in that they often form part of a "second shell" of polar and charged residues that interact with and stabilize direct Ca2+-ligand residues (examples given below). Although it is clearly impossible to detail all of the hydrogen-bonding networks around the Ca'+-binding sites of every protein in Table 11, the following two examples illustrate the general features common to many of these networks. Figure 7a and Color Plate 2a show many of the hydrogen bonds around Ca2+-binding site 4 of turkey troponin C (5TNC) (Herzberg and James, 1988). A detailed analysis of hydrogen bonding within the HLH Ca'+-binding loops has been presented (Strynadka and James, 1989); thus, only the major features are highlighted here. Proceeding from the amino terminus of the loop, the first Ca'+-coordinating residue is Asp-142. T h e carboxylate of this side chain plays a prominent role in Ca2+ binding and in providing a focus around which the first six residues of the loop fold. One of the oxygen atoms of the Asp-142 carboxylate is a direct Ca2+ ion ligand; it also forms hydrogen bonds to main-chain NH groups at positions 4 and 5 of the 12 residue

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CATHERINE A. MCPHALEN ET AL.

loop (Asn-145 and Asp- 146, respectively). The other carboxylate oxygen atom is the recipient of strong hydrogen bonds from the main-chain NH of the conserved glycine in position 6 and from a conserved water. Position 1 of the Ca2+-bindingloop is an aspartate in all of the HLH sites except calbindingK (3ICB) site 1. In light of the extensive hydrogen bonding to both side-chain oxygen atoms, it is clear that even the conservative change to an asparagine would be difficult to accommodate at this position. Continuing along the Ca*+-binding loop of 5TNC site 4, the second Ca'+-coordinating ligand is Asn-144 (Fig. 7a and Color Plate 2a). T h e side-chain oxygen of this residue coordinates Ca2+and also forms an Asx turn with the main-chain nitrogen of the n+2 residue, Asp-146. T h e side-chain nitrogen atom of Asn-144 donates a hydrogen bond to the non-Ca'+-coordinating side-chain oxygen atom of Asp- 146, the next Ca'+-coordinating residue in the loop. This oxygen atom of Asp-146 is also hydrogen bonded to the side chain of Arg-148 and to the conserved water molecule 0-174 (which, in turn, coordinates the Ca2+ ion). Asp146 uses its other carboxylate oxygen atom to coordinate the Ca2+ ion and to form an Asx turn with the main-chain NH of Arg-148. T h e Asx-turn hydrogen bond of Asp- 146 is important in stabilizing the conformation for the peptide unit of Arg-148, the main-chain carbonyl oxygen atom of which forms the next ligand along the Ca2+-binding loop. T h e chain reversal that brings this peptide carbonyl oxygen atom into the correct orientation for binding Ca2+ requires the go", 0" (4, 4) values of the invariant glycine at position 6 and relies on stabilization of the resulting main-chain fold through the interactions with the carboxylates of Asp-142 and Asp-146 mentioned above. T h e final Ca2+coordinating amino acid along the loop is Glu-153. T h e residue at this position is always a glutamate in the HLH-binding sites. The carboxylate group of Glu- 153 contributes both oxygen atoms for coordination to the Ca'+. Additionally, the oxygen atoms of the carboxylate group are involved in hydrogen bonds to the main-chain nitrogens of Lys- 143, Asn147, and Asp-150. Color Plate 2a and Fig. 7a indicate that, in addition to the acidic and amide residues that coordinate Ca*+ directly, there are additional charged and polar side chains surrounding the primary Ca2+coordination shell. Ser-141, Lys-143, Asn-145, Arg-148, Asp-150, and Asp-152 form a second shell of oxygen and nitrogen atoms that participates in hydrogen bonds with Ca2+-ligand groups and aids in stabilization of ligand residues and loop conformation. For example, one carboxylate oxygen atom of Asp-150 shares a hydrogen bond with the conserved Ca'+-ligand water, while the other initiates an Asx turn to the main-chain nitrogen atom of Asp-152. This turn redirects the main chain so that the side chain of the bidentate Glu-153 is correctly oriented to

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interact with the Ca2+ ion. Overall, this first example of the hydrogenbonding network surrounding a Ca*+-binding site illustrates nicely the multiple interactions of side-chain carboxylate oxygen atoms with both the CA2+ ion and other groups in the binding loop. The importance of the hydrogen-bonding network in directing the mainchain conformation of the binding loop is also apparent in this example. The S. aureus nuclease provides our second example of a hydrogenbonding network surrounding a Ca2+-bindingsite [ lSNC, Color Plate 2b and Fig. 7b (Loll and Lattman, 1989)l. The Ca2+-ligand residues and their coordination bonds in this enzymatic active site are shown in Fig. 3c. One carboxylate oxygen atom of Asp-21 interacts with the Ca2+ and also receives a hydrogen bond from the main-chain nitrogen atom of Thr-41. The second carboxylate oxygen of Asp-2 1 interacts with the side-chain hydroxyl group of Thr-22 and with water 170 (which, in turn, binds to the Ca2+ ion). The side chain of Asp-40 binds the Ca2+ ion also, and its ligand carboxylate oxygen atom binds a second water molecule (0-187) that is a Ca2+ ligand. The second carboxylate oxygen atom of Asp-40 binds to water 171, which, in turn, binds to the hydroxyl group of Tyr-113 and a phosphate oxygen of the pdTp inhibitor bound at the active site. Both the Ca*+-ligand carboxylate oxygen atoms of Asp-21 and Asp-40 form a long electrostatic interaction with the positively charged guanidinium group of Arg-35. Jointly, the multiple hydrogenbond interactions of Asp-2 1 and Asp-40 help to stabilize these negatively charged side chains in close proximity. The main-chain carbonyl oxygen atom of Thr-41 is the third protein ligand of the Ca2+ ion in ISNC. This residue is adjacent to Pro-42. The following Glu-43 (the proposed catalytic residue) forms an Asx turn to the main-chain nitrogen atom of Lys-45. T h e main-chain nitrogen atom of Thr-4 1 is stabilized by a hydrogen bond to the side-chain carboxylate group of Asp-40. The hydrogen-bonding interactions of the three Ca2+coordinating waters-0- 170, 0-179, and 0-187-highlight the importance of nonligand polar and charged residues in stabilizing Ca*+coordinating groups. Tyr-13, Asp-19, Thr-22, Arg-35, Glu-43, Lys-49, and Glu-52 together form a second shell of polar amino acids around the Ca2+ ligands. The side chains of all of these residues participate in hydrogen bonding with Ca2+-coordinating water molecules and other side-chain groups. Replacement of one of these second-shell amino acids, Glu-43, by an aspartate in a recent site-directed mutagenesis experiment resulted in the loss of a Ca'+-ligand water (0-187 in Fig. 7a) (Loll and Lattman, 1990). Presumably, the side chain of the mutant aspartate was too short to form a good hydrogen bond and stabilize the water ligand (Loll and Lattman, 1990).

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CATHERINE A. MCPHALEN ET AL.

In summary, Ca2+-ligand residues almost invariably participate in extensive further hydrogen-bonding interactions around the ionbinding site. Aspartate and glutamate residues are especially suited to this role, with their capacity for multiple hydrogen bonds via side-chain carboxylate oxygen atoms. Surrounding the direct Ca2+-ligand residues is a second shell of polar residues, adding another layer to the network of hydrogen bonds. T h e hydrogen-bonding network provides additional stabilization of ligand orientation and of main-chain conformation in the ion-binding loop.

H . Lack of Interaction between Ca2+ and Helix Dipoles Helices are situated close to Ca2+-bindingsites in 12 of the 16 proteins of Table I1 (5TNC, SCLN, SICB, 5CPV, lSGT, 2SEC, 2PRK, lTEC, lALC, STLN, 1BP2, and 1SNC). The strongest interaction of any ion with a helix dipole is clearly when the ion lies on the helix axis close to the oppositely charged end of the helix. In this position the ion effectively “sees” only the charged end to which it is closest. The farther the position of the ion away from the helix dipole axis, and the more equally the ion sees both ends of the dipole, the weaker the interaction is. T h e strength of the interaction decreases as a function of r3 with distance from the dipole. Surprisingly, none of the negative ends of the helix dipoles in the proteins of Table I1 interacts directly o r strongly with the positively charged Ca2+ ions. This is in direct contrast to the binding of negatively charged phosphate ions to proteins; Hol(l985) found that about 60% of low-molecular-weight phosphate compounds bound to proteins interacted closely with an a-helix dipole, binding within 5 8, of the positive end of a helix. Hol(1985) notes, however, that helix dipoles are not involved in binding high-molecular-weight compounds such as DNA or RNA. Richardson and Richardson (1988) described the structural similarity between the Ca2+-binding HLH motif and the DNA-binding helix-turn -helix motif. Helix dipoles d o not interact strongly with the bound ion in either motif. In all of the Ca2+-binding proteins of Table 11, the longitudinal axes of neighboring helices pass at least 6 8, to one side of the Ca2+ position; most Ca2+ ions lie 7 or 8 A from a helix axis. The HLHbinding sites illustrate this phenomenon nicely (Fig. 1). Each HLH site, of course, contains two helices in proximity to the Ca2+ position. As is clear from Fig. 1, the axis of neither helix points directly at the Ca“. T h e negative dipole of the first helix, at its carboxy terminus, is aimed well away from the positive ion. The dipole of the second helix is also aimed well away from the Ca2+;this is more intuitively understandable, since the positive dipole of the second helix, at its amino terminus, is closest to

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the Ca2+-binding site. The same factors are apparent in the non-HLH sites that have helices close by (e.g., Figs. 2e and f, 3d, f, and g, and 8). The helix dipoles are all too far from the Ca2+ to provide significant charge balance and stabilization. Of the 13 helices close to Ca2+-bindingsites in the non-HLH proteins, eight are positioned with the positive ends of their dipoles closest to the binding site (Table 11; ISGT, two helices; 3TLN sites 1 and 2 in Fig. 3a; 3TLN site 3 in Fig. 3b; ISNC; 1BP2 in Fig. 3d; and lALC in Fig. 3g). Many of the helices near Ca'+-binding sites d o interact strongly with the Ca2+ ion, but not via their helix dipoles. Instead, the side chains of negatively charged residues at the ends of the helices are frequently Ca2+ ligands (Fig. 8). For example, the invariant aspartate ligand at the beginning of the HLH loop and the bidentate glutamate at the end of the loop are at the carboxy terminus of the first helix and the amino terminus of the second helix, respectively (Fig. 1). Similar situations are also found in non-HLH proteins (Figs. 2d, 3a and g, and 8). The side chains of these Ca2+-ligand residues project outward and away from their respective helix axes so that they are able to interact with the Ca2+,although the helix dipoles are too far from the ion to influence binding significantly. In the Ca2+-binding sites of Table 11, formal charge balance for the Caz+ is provided by aspartate or glutamate side-chain ligands and/or charge dissipation via access to bulk solvent. Extra charge stabilization by helix dipoles is not required. Direct interaction with protein atoms may provide a better-defined ligand geometry than a helix dipole, which is important to an ion-binding site that requires selectivity in its biological function. I. Ca2+-Binding Affinities and Structural Characteristics

One of the goals in the analysis of protein three-dimensional structures is the eventual prediction of biochemical properties from a knowledge of structural characteristics. For Ca2+-bindingsites we would hope to correlate particular structural features with ion affinity and selectivity in particular. Some attempts have been made to derive quantitative structureactivity relationships between binding affinity and sequence for the HLH proteins (Sekharudu and Sundaralingam, 1988; Marsden et al., 1988). Such relationships are extremely difficult to derive completely and rigorously for a family of related proteins, however. Sequence differences, resulting in conformational and physicochemical differences within and around the site of interest introduce a large number of additional variables to the problem. Dissecting out all of the relevant variables is difficult, although some correlations may be seen between affinities and

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CATHERINE A. MCPHALEN ET AL.

3 ASN 181

I

FIG. 8. An example of the lack of strong interaction between Ca‘+ ions bound to proteins and a-helix dipoles. Shown is the double-Ca’+-binding site of thermolysin (STLN), with two associated helices (residues Gly-136 to Asn-181). Side chains are drawn only for Asp-138 and Glu- 177 (thick lines), two Ca2+-ligand residues from the helical regions. Only main-chain atoms are shown for other residues. The Ca“ ions are circles. The positive amino terminus of the dipole from the first helix passes to one side of the Ca‘+ positions. The negative carboxy terminus of the dipole from the second helix bypasses the Ca’+ positions at some distance. The only interaction between the ions and the helices is with the side chains of Asp-I38 and Glu-177 that protrude from their respective helix axes.

specific structural characteristics. The situation is further complicated by not knowing, in most cases, how well the conformation of the Ca2+binding site is maintained in the absence of Ca2+ (see Section ILK). Ion binding to a “preorganized” site (i.e., one requiring minimal conformational change to bind the ion) is tighter and more favorable entropically than binding to a more flexible site. In the case of Ca2+-binding sites, determining any correlation between structure and affinity is even further complicated by difficulties in determining the correct affinity. T h e literature is full of measured dissociation constants for Ca2+-binding

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proteins differing by up to four orders of magnitude for the same site. Some of the variation arises from the use of different measurement techniques. In addition several of the proteins of Table I1 have altered Ca2+ affinities in vitro and when they interact with their biological targets. Very few of the structural characteristics of Ca2+-binding sites described in previous sections correlate with Ca2+-bindingaffinities. There is no correlation between affinity and goodness of fit for the ligand atom to ideal geometry (Table 11) or coplanarity of Ca2+ with ligand groups. Among all of the sites of Table 11, net formal charge of Ca2+-ligand groups is not a predictor of affinity. A trend can be discerned between number of ligand water molecules and affinity; the five highest-affinity sites use zero or one water ligand, while the five sites with lowest affinity use one, two, or three water ligands. The trend is not monotonic, however. There is no correlation between affinity and whether the binding loop is completely continuous, semicontinuous, or discontinuous. Overall, Ca*+-bindingaffinity does not seem to be related directly to the geometry of ligand binding. This is in agreement with observations on Ca2+ coordination in small-molecule structures; Ca2+-coordination geometry is more variable and irregular than that of similar ions (Einspahr and Bugg, 1984; Martin, 1984; Williams, 1986). Energetically, the affinities of the strongest and weakest sites differ by only a few kilocalories. This implies that structural differences influencing affinities are very subtle and may pass unnoticed in the general variability of the sites. T h e affinities are also influenced by more than just the structure of the immediate Ca'+-binding site. In the studies by Grabarek et al. (1990) and Fujimori et al. (1990), for example, the Ca2+-bindingaffinity of troponin C was altered by mutations of single residues distant from the Ca2+.The mutations were designed to interfere with the proposed conformational change in the Ca2+-binding domain of troponin C (Herzberg et ul., 1986a). Other influences from the rest of the protein, such as electrostatic environment, are also likely important, but are more difficult to analyze. Linse et al. (1990) and Martin et al. (1990), for example, mutated aspartate or glutamate residues to asparagine or glutamine in calbindingK ; the mutated residues were near the Ca2+-bindingsite, but were not direct Ca2+ ligands. Mutant proteins containing various combinations of the charged wild-type and neutral mutated residues showed reduced affinity for Ca2+.Thus, the variability of protein Ca2+-bindingsites, the subtlety of local geometric effects on affinity, and the influence on affinity of factors more distant from the binding site all conspire to make general prediction of Ca*+-bindingaffinities from structural parameters difficult at best.

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J . Selectivity of Ca2+-Binding Sites Two distinct aspects of selectivity in Ca2+-binding sites are apparent. One concerns the way in which Ca2+-binding sites prevent or inhibit binding of other metal ions found in the protein environment. T h e other aspect is the ability of some Ca2+-bindingsites to adapt their structure for the binding of other functionally important metal ions. Threedimensional crystal structures of proteins with other metals substituted at Ca2+-bindingsites are rare to date. Solution studies on metal substitution are abundant, however. They attempt to determine which ions will bind to a given site and how the physical properties of an ion affect its binding affinity. Several factors are believed to contribute to the selectivity of protein Ca2+-binding sites in the first sense mentioned above (i.e., preventing binding of metals other than Ca2+) (Snyder et al., 1990, and references therein; Vyas et al., 1989), although three-dimensional structural evidence is still lacking on these factors for proteins. Coordination by seven oxygen atoms arranged approximately in a pentagonal bipyramid favors Ca2+binding over ions with other, more rigid, geometric requirements. T h e high net negative charge of the ligands at most Ca2+binding sites favors divalent and trivalent cation binding over monovalent cations. T h e cavity size and deformability of a Ca2+-bindingsite affect the size range of ions that can be accommodated within it. T h e free energy of metal-ion dehydration varies with hydration number of the free ion and ionic radius, and thus may contribute to binding selectivity. Some of these factors have also been noted in small-molecule-ion complexes (Martin, 1984, and references therein; Einspahr and Bugg, 1984). Selectivity in the second sense (i.e., how some Ca2+-bindingsites adapt their structures to bind other functionally important metals) is less well explored as yet. Two high-resolution refined structures of proteins with Cd2+ substituted for Ca2+ are available: carp parvalbumin (ICDP) (Swain et al., 1989) and the D-galactose-binding protein (Vyas et al., 1989). In both structures the Cd2+ coordination is identical to that seen in the corresponding Ca2+structure. The subtilisins (site 2) and thermitase (site 2) appear to bind different ions, depending on the concentration of Ca2+ in the crystallization buffer (Pantoliano et al., 1988; Gros, 1990). Based on characteristic ion-ligand distances, subtilisins and thermitase are proposed to bind monovalent cations at certain sites at low levels of endogenous Caz+ (Drenth et al., 1972; Pantoliano et al., 1988; Gros et al., 1989; Gros, 1990). T h e adaptation from monovalent cations to Ca2+appears to be accomplished by an increase in the number of ion ligands at a given site, particularly aspartates, and a tightening of the ion-ligand distances

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in these relatively flexible loop regions. The monovalent cations at these sites have not been identified chemically, though, and their possible biological function in the protein is unknown. Until more threedimensional structures of other ions in Ca*+-binding sites are available, the best we can do is to continue speculating about how Ca2+-binding sites adapt structurally to the observed coordination preferences of other metals (see, e.g., the discussion in Section III,E on the adaptation of HLH sites from Ca2+ to Mg2' binding).

K . Ca2+-Free Sites With the numbers of negatively charged groups that converge at most Ca'+-binding sites, it would be reasonable to expect considerable disruption of the local protein structure on removal of the Ca2+.This may be the situation with proteins that lose stability or function with loss of Ca'+. Such disordered or denatured proteins are generally not suitable for crystallographic analysis, however. Two proteins of Table I1 have been crystallized with empty Ca2+-binding sites: troponin C (4TNC and 5TNC) and proteinase K (2PRK) (Bajorath et al., 1989). Each of the two Ca2+-bindingsites in 2PRK contains only one negatively charged ligand, an aspartate side chain, and several water ligands (Table I1 and Fig. 2g). In the Ca2+-free structure the protein around site 2 is minimally disturbed. Water molecules replace the Ca2+ of site 1, and the protein ligands change position only slightly. The small shifts appear to be propagated through the protein to the catalytic residues and substrate-binding site. The changes in the active site may be sufficient to account for the observed loss of catalytic activity in proteinase K with Ca2+ removed. The Ca*+-freesites in the amino-terminal domain of troponin C have a conformation that is different from that of the carboxy-terminal Ca2+filled sites (Herzberg and James, 1985b, 1988; Herzberg et al., 1986b). Small changes in main-chain dihedral angles of the Ca*+-free loops position negatively charged residues farther from each other than in the loops with Ca*+bound. Interhelix angles also change slightly, indicatin that longer-range interactions are probably important in defining Ca2 affinity. Overall, the Ca2+-free sites appear to be somewhat more open and flexible than the Ca2+-filled sites. The Ca2+-free sites maintain a network of hydrogen bonds among loop residues similar to the network stabilizing the Ca2+-filled sites. Stabilizing networks of hydrogen bonds surround most &*+-binding sites in the proteins of Table I1 (see Section 111,G).T h e networks and the accessibility of charged ligands to bulk solvent may reduce the disruption of protein structure on loss of bound Ca2+.

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IV. DISCUSSION AND SUMMARY

A . A “Regular” Ca*+-Binding Site We have carried out the analysis and comparison of the structural parameters in a number of protein Ca2+-binding sites to determine the common features of a “regular” Ca2+-bindingsite in proteins. One of the most interesting aspects of protein Ca2+-bindingsites is their variability; among just the proteins listed in Table 11, nature has devised at least 16 strikingly different ways to fold a polypeptide chain around a Ca2+ ion (with occasional assistance from solvent molecules). T h e diversity of structure among Ca*+-binding sites likely reflects the diversity of Ca2+ functions in biology. A number of common features are still found in these binding sites, even amid the diversity. The majority of protein ligands for a Ca2+ionare most likely to be part of the same local segment of polypeptide chain, all within a loop of 10-15 residues in length (Section 111,A). These continuous or semicontinuous binding loops contain little or no regular repeating secondary structure (ahelices or 0 strands), but almost always contain some nonrepeating secondary structure (Section 111,B).Asx turns are the most frequent motif, and 310 reverse turns are often found, allowing the polypeptide chain to wind around the Ca2+ ion. Most of the Ca2+-binding site peptides are n loops or segments of loops. Ca2+-coordinationnumbers in proteins vary from five to eight (Section 111,C).The most usual coordination number is seven, and most sites with lower coordination number still exhibit pentagonal bipyramidal ligand geometry (Plate la). Eightcoordinate sites also resemble the pentagonal bipyramid, with one vertex shared between two ligand atoms. A few six-coordinate sites do have proper octahedral coordination geometry (Color Plate Ib). All of the ligands at protein Ca2+-binding sites are oxygen atoms (Section 111,D). Almost 50% of the ligands are from side-chain carboxylate groups, and another 43% are provided by peptide carbonyl oxygen atoms or water molecules. Coordination by serine or threonine side-chain hydroxyl groups is extremely rare. All of the sites of Table I1 have at least one peptide carbonyl oxygen atom ligand in their Ca2+-coordinationsphere. Ca2+-binding sites with coordination numbers of seven or eight tend to employ more bidentate and water ligands, possibly to relieve ligand crowding around the ion. The mean Ca2+-ligand distance for all types of ligand oxygen atoms is 2.4(2)w. The geometry of the coordinating ligands differs significantly from the ideal pentagonal bipyramid or octahedron for almost all of the

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sites, both in Ca2+-ligand distances and in ligand-Ca2+-ligand angles (Section 111,E). Unidentate ligands deviate less from ideal geometry, on average, than bidentate ligands. One source of the geometric deviations of ligands is the frequent positioning of the Ca2+ ion out of the mean plane of a ligand group [i.e., carboxylate or amide (Section III,F)]. Mean distance of the ion out of the ligand plane varies with the type of ligand, from 1.1(5)A for unidentate carboxylate groups to 0.4(3)A for bidentate carboxylate groups. In general Ca2+ ions are distributed through a wide angular range around ligand oxygen atoms and have little tendency to cluster at the positions of the ligand lone pair orbitals. This is in direct contrast to the clustering observed for water molecules interacting with protein oxygen atoms (Rees et al., 1983; Baker and Hubbard, 1984; Thanki et al., 1988). Ca2+ ions do bind preferentially to the syn lone pair orbital of protein carboxylate groups, probably as a result of the higher basicity of the syn lone pair and to avoid steric clashes with side-chain carbon atoms of ligand residues. All Ca*+-binding sites in the proteins surveyed have extensive networks of hydrogen bonds among ligand and binding loop residues (Section 111,G).The majority of the groups participating in the networks are aspartate and glutamate side-chain carboxylates. The acidic side chains dominate the networks because they provide a charge balance for the Ca2+ ion, and because they can participate in more hydrogen bonds per residue than asparagine, glutamine, serine, or threonine side chains. Asx turns are a frequent feature of the hydrogen-bonding networks. Many hydrogen bonds in the networks are formed among Ca2+-ligand groups (e.g., side-chain carboxylate and amide groups o r water molecules). The hydrogen-bonding networks around Ca*+-bindingsites probably serve at least four purposes: they position side-chain groups effectively to coordinate the Ca*+ ion, they stabilize adjacent Ca2+ ligands (especially water molecules), they stabilize changes in direction for the main chain of the Ca2+-binding loop to bring other ligands into Ca*+-binding position (in conjunction with proline and glycine residues), and they help to disperse the formal net negative charge concentrated at most Ca*+-binding sites (Vyas et al., 1990). None of the Ca2+ ions in the proteins of Table I1 interacts at all strongly with helix dipoles in order to stabilize their binding (Section 111,H). Instead, the Ca2+ ions are often coordinated by aspartate and glutamate residues from helices, with their side-chain carboxylate groups protruding from the helix axis. From this summary we are able to produce a reasonably detailed picture of a “regular” protein Ca2+-bindingsite with regard to types and geometry of ligands. We are also able to define, at least on a statisti-

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cal basis, the normal variability of many structural parameters in protein Ca2+-binding sites. In terms of the immediate Ca2+-ligand sphere, protein Ca2+-binding sites closely resemble small-moleculeCa2+complexes. With these characteristics of a regular protein Ca2+binding site as a basis, unusual Ca2+-binding sites such as thermolysin (3TLN) site 4 and Rhizopus pepsin (2APR) may be examined for structural features contributing to their different geometries. Differentiation in X-ray crystal structures among Ca2+,other ions, and water molecules bound strongly to proteins may also be aided by characteristic differences in their binding modes (although, ultimately, chemical confirmation of ion binding should be obtained).

B . Structure-Function Relationships T h e results of our analysis provide at least partial answers to some of the questions posed in Section I. The relationships between the protein structure at a Ca2+-binding site and the function of the ion within the protein are both subtle and complex, however. Disentangling the complexities is not aided by the tremendous structural diversity of protein Ca2+-binding sites outside the immediate ligand sphere. A great deal of data is still missing on the structural features of other ions in Ca2+binding sites and of ion-free sites. Despite these problems, we have a few tantalizing glimpses of how structure affects function in protein Ca2+ binding. The ion affinity of a Ca2+-binding site is not simply correlated with ligand type or geometry (Section II1,I). It is clear from our analysis and the work by others that both proximal and distal protein structure affects the affinity of a site. Longer-range electrostatic effects, protein flexibility, and stability may be more important for Ca2+-binding affinity than ideality of ligand geometry. Proposed structural correlates of selectivity in protein ion binding are multiple and generally are not yet shored up by three-dimensional structural evidence (Section IIIJ). Yamashita et al. (1990) presented a novel description of where metal ions bind in proteins, based on an analysis of the hydrophobicity contrast of the regions around known metal-binding sites. Simplistically, proteins bind ions selectively at certain sites or adapt sites to bind different ions by providing a complementary environment to chemical characteristics and preferred ligand geometry of a particular ion. Currently, we have one example in the subtilisin-like serine proteinases of structural changes in a Ca2+-bindingsite that adapt it to bind monovalent cations (Section IIIJ). Although chemical characteristics and preferred ligand geometry differ between Ca2+ and Cd2+,two structures of proteins with Cd2+ bound in place of Ca2+show no significant differences in their ion-binding param-

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eters. Of particular biological interest would be future three-dimensional structures of Mg2+ bound to HLH proteins in place of Ca2+. T w o examples are available of the structural changes elicited by Ca2+ binding to proteins (Section 111,K): comparison of the Ca2+-free and Ca2+-bound structures of proteinase K (2PRK), and comparison of the amino-terminal domains of troponin C (5TNC) and calmodulin (3CLN). In both cases relatively small adjustments of the Ca2+-ligand residues are propagated through the protein to affect the conformation of a site for interaction with other molecules, This sort of mechanism may be applicable to Ca2+-binding proteins in a rather general sense. T h e structural adjustments observed in 2PRK and in 5TNC versus 3CLN are subtle, however, and are likely difficult to predict a pm'ori for other proteins. Again, more three-dimensional structural data are needed. Ca2+ binding appears to enhance protein stability by promoting extra energetically favorable interactions within the protein structure. In some cases the positively charged Ca2+ ion provides charge balance to a group of negatively charged side chains clustered together in a Ca2+-binding loop. Having the protein ligands arranged around the Ca2+ ion allows the formation of additional energetically favorable hydrogen bonds among the ligands (Section 111,G). The use of peptide carbonyl oxygen atoms as Ca2+ion ligands reduces the flexibility of the polypeptide chain in specific regions, thus contributing to order and stability to denaturation in the protein structure. Many Ca2+-binding sites include ligands from distant segments of polypeptide chain, supplying structural cross-linking and stability similar to that from disulfide bridges. For the future, quantitation of these proposed factors is desirable to compare the amount of stabilization predicted from such effects with the actual amount of stabilization realized by a protein on Ca2+ binding. C . Prediction and Design of Ca2+-BindingSites

Ca2+ may be the ion of choice in so many biological roles because of its relative flexibility in preferred coordination number and ligand geometry. This chapter shows that there are many possible ways to form a good Ca2+-bindingsite in a protein. This diversity in structure and sequence of binding sites, however, makes general prediction of Ca2+-binding positions a daunting (if not impossible) task. Outside of the HLH group of proteins, the frequent and variable use of water molecules as Ca2+ ligands complicates the task of prediction further. Even within a structurally related family of proteins, prediction of Ca2+-binding sites is not straightforward. HLH binding sites have been successfully predicted in new protein sequences from the highly conserved HLH consensus se-

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quence (Tuffy and Kretsinger, 1975; Hardin et al., 1987; Kobayashiet al., 1988). Among the subtilisin-like serine proteinases, with more variable sequences than HLH proteins, it may be possible to predict a conserved Ca2+-binding site or the lack of a site if crucial ligand residues are missing. The sequence variability of sites, and the partly discontinuous nature of many Ca2+-bindingpeptides make successful prediction of a novel site (e.g., site 2 of thermitase) rather unlikely. Similar problems are encountered with other protein families. Using protein sequence to predict the Ca2+-binding affinity of a postulated binding site is even more fraught with risk. In the future improvements in techniques for modeling structures of similar proteins may provide better predictions of ionbinding sites within structural families, but a priori prediction of sites from sequences of novel proteins seems remote at best.

ACKNOWLEDGMENTS We thank A. L. Swain, E. L. Amma, and R. H. Kretsinger for providing atomic coordinates of the refined carp parvalbumin (5CPV) before their release and for preprints of related papers; F. A. Quiocho, M. N. Vyas, and N. K. Vyas for atomic coordinates of the refined o-galactose-binding protein (PGBP),and a preprint of their review on the structure; E. E. Lattman for the coordinates of the refined S. aureus nuclease (1SNC); and P. Gros for coordinates of the thermitase (ITEC) structure at different Ca“ concentrations and for a copy of his Ph.D. thesis. We apologize to anyone whose Ca2+-bindingprotein structure has inadvertently been omitted from this chapter. Special thanks to Protos Corporation for a grant enabling us to include Color Plates 1 and 2 in color. David Eisenberg provided useful comments on the manuscript. Mark Israel and Perry d’Obrenan gave valuable assistance in the preparation of figures. Mae Wylie, as usual, cheerfully handled all our revisions of this manuscript and produced a most professional product. N.C.J.S. acknowledges the support of Alberta Heritage Foundation for Medical Research for a studentship. This research was supported by the Medical Research Council of Canada.

REFERENCES Acharya, K. R.,Stuart, D. L., Walker, N. P. C., Lewis, M., and Phillips, D. C. (1989)./. Mol. Biol. 208,99- 127. Adman, E., Watenpaugh, K. D., and Jensen, L. H. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 4854-4858. Agrawal, B. B. L., and Goldstein, I. J. (1967). Bzochzm. Biophys. Acta 147,262-271. Ahmed, F. R.,Przybylska, M., Rose, D. R., Birnbaum, G. I., Pippy, M. E., and MacManus, J. P. (1990).J. Mol. Biol. 216, 127-140. Albertsson, J., Oskarsson, A., and Svensson, C. (1978). Acta Cqstallogr. Sect. B 34, 27372743. Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means, A. R., and Cook, W. J. (1985). Nature (London) 315,37-40. Babu, Y. S., Bugg, C. E., and Cook, W. J. (1987).In “Methods in Enzymology”(A. R.Means and P. M. Conn, eds.) Vol. 139, pp. 632-642. Academic Press, Orlando.

CALCIUM-BINDING SITES IN PROTEINS

141

Babu, Y. S., Bugg, C. E., and Cook, W. J. (1988).J.Mol. Biol. 203, 191-204. Bajorath, J., Hinrichs, W., and Sanger, W. (1988).Eur.J. Biochem. 176,441-447. Bajorath, J., Raghunathan, S., Hinrichs, W., and Sanger, W. (1989). Nature (London) 337, 481-484. Baker, E. N., and Hubbard, R. E. (1984). Prog. Biophys. Mol. Biol. 44,97-179. Becker, J. W., Reeke, G. N., Jr., Wang, J. L., Cunningham, B. A., and Edelman, G. M. (1975).J.B i d . Chem. 250, 1513-1524. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977). J. Mol. Bzol. 112, 535-542. Berridge, M. J. (199O).J.Biol. Chem. 265,9583-9586. Betzel, C., Pal, G. P., and Sanger, W. (1988). Actu Crystallogr. Sect. B 44, 163-172. Bier, M., and Nord, F. F. (1951). Arch. Biochem. Biophys. 33, 320-332. Bode, W., and Schwager, P. (1975).J.Mol. Biol. 98,693-71 7. Bode, W., Papamokos, E., and M u d , D. (1987). Eur. J. Biochem. 166,673-692. Boel, E., Brady, L., Brzozowski, A. M., Derewenda, Z.,Dodson, G. G., Jensen, V. J., Petersen, S. B., Swift, H., Thim, L., and Woldike, H. F. (1990). Biochemistry 29, 6244-6249. Bolin, J. T.,Filman, D. J., Matthews, D. A., Hamlin, R. C., and Kraut, J. (1982).J.Biol.Chem. 257,13650-13662. Bott, R., Ultsch, M., Kossiakoff, A,, Graycar, T., Katz, B., and Power, S. (1988).J.Biol. Chem. 263,7895-7906. Brew, K., Vanaman, T. C., and Hill, R. L. (1968).Proc. Natl. Acad. Sci. U.S.A.59,491-497. Briedigkeit, L., and Frommel, C. (1989). FEBS Lett. 253,83-87. Briggman, B., and Oskarsson, A. (1977).Actu Crystallogr. Sect. B 33, 1900-1906. Brown, I. D. (1988). Actu Crystallogr., Sect. B 44, 545-553. Buisson, G., Duee, E., Haser, R., and Payan, F. (1987).E M B O J . 6,3909-3916. Carrel], C. J., Carrel], H. L., Erlebacher, J., and Glusker, J. P. (1988).J.Am. Chem. SOC.110, 865 1-8656. Chakrabarti, P. (1990a). Biochemistry 29,651-658. Chakrabarti, P. (1990b). Protein Eng. 4,49-56. Chiba, K., and Mohri, T. (1987).Biochemistry 26,711-715. Cliffe, S. G. R., and Grant, D. A. W. (1981).Bi0chem.J. 193,655-658. Cotton, F. A., Hazen, E. E.,Jr., and Legg, M. J. (1979). Proc. Natl. Acud. Sci. U.S.A. 76, 2551-2555. Cuatrecasas, P., Fuchs, S., and Anfinsen, C. B. (1968).J.Biol. Chem. 242,3063-3067. Dalgarno, D. C., Levine, B. A., Williams, R. J. P., Fullmer, C. S., and Wasserman, R. H. (1983). Eur. J . Biochem. 137,523-529. Dauter, Z., Betzel, C., Hohne, W. E., Ingelman, M., and Wilson, K. S. (1988).FEBSLett.236, 171-178. Declercq, J.-P., Tinant, B., Parello, J., Etienne, G., and Huber, R. (1988).J.Mol. Biol. 202, 349-353. De Haas, G . H., Bonsen, P. P. M., Pieterson, W. A., and van Deenen, L. L. M. (1971). Biochim. Biophys. Acta 239, 252-266. Delbaere, L. T. J., Vandonselaar, M., Prasad, L., Quail, J. W., Pearlstone, J., Carpenter, M. J ., Smillie, L. B., Nikrad, P. V., Spohr, U.,and Lemieux, R. U. (1990). CanJ . Chem. 68, 1116-1121. Derewenda, Z.,Yariv, J., Helliwell, J. R., Kalb (Gilboa), A. J., Dodson, E. J., Papiz, M. Z., Wan, T., and Campbell, J. (1989). E M B O J . 8,2189-2193. Dijkstra, B. W., Drenth, J., and Kalk, K. H. (1981a). Nature (London) 289,604-606. Dijkstra, B. W., Kalk, K. H., Hol, W. G.J., and Drenth, J. (1981b).J.Mol. Biol. 147,97-123.

142

CATHERINE A. MCPHALEN ET AL.

Dijkstra, B. W., Renetseder, R., Kalk, K. H., Hol, W. G. J., and Drenth, J. (1983).J.MoLBiol. 168,163-179. Dimicoli, J. L., and Bieth, J. (1977). Biochemistry 16,5532-5537. Drenth, J., Hol, W. G. J., Jansonius, J. N., and Koekoek, R. (1972). Eur. J . Biochem. 26, 177-181. Einspahr, H., and Bugg, C. E. (1980). Acta Crystallogr., Sect. B 36,264-271. Einspahr, H., and Bugg, C. E. (1981). Acta Crystallogr., Sect. B 37, 1044-1052. Einspahr, H., and Bugg, C. E. (1984). In “Metal Ions in Biological Systems” (H. Sigel, ed.), Vol. 17, pp. 51-97. Dekker, Basel, Switzerland. Einspahr, H., Parks, E. H., Suguna, K., Subramanian, E., and Suddath, F. (1986).J. Biol. Chem. 261,16518-16527. Frommel, C., and Hohne, W. E. (1981). Biochim. Biophys. Acta 670,25-31. Fujimori, K., Sorenson, M., Herzberg, O., Moult, J., and Reinach, F. C. (1990). Nature (London)345, 182-184. Furlong, C. E. (1987). In “Escherichia coli and Salmonella typhimurium” (F. C. Neidhart, ed.), Vol. 1 , p. 768-796. Am. SOC.Microbiol., Washington, D.C. Gandour, R. D. (198 1). Bioorg. Chem. 10, 169- 176. Gillis,J. M., Thomason, P., Lefevre, I., and Kretsinger, R. H. (1982).J.MuscleRes. CellMotil. 3,377-398. Grabarek, Z., Tan, R.-Y., Wang, J., Tao, T., and Gergely, J. (1990). Nature (London) 345, 132- 135. Gros, P. (1990). Ph.D. thesis. Rijksuniversiteit Groningen, Groningen, The Netherlands. Gros, P., Betzel, C., Dauter, Z., Wilson K. S., and Hol, W. G. J. (1989).J.Mol. Biol. 210, 347-367. Haiech, J., Derancourt, J., Pechkre, J.-F., and Demaille, J. G., (1979). Biochemistry 18, 2752-2758. Hardin, S. H., Keast, M. J., Hardin, P. E., and Klein, W. H. (1987). Biochemistry 26, 3518-3523. Hardman, K. D., and Ainsworth, C. F. (1972).Biochemistry 11,4910-4919. Henderson, R. (1970).J.Mol. Biol. 54,351-354. Herzberg, O., and James, M. N. G. (1985a). Nature (London) 313,653-659. Herzberg, O., and James, M. N. G. (1985b). Biochemistry 24,5298-5302. Herzberg, O., and James, M. N. G. (1986). FEBS Lett. 199,279-282. Herzberg, O., and James, M. N. G. (1988).J.Mol. Biol. 203,761-769. Herzberg, O., Moult, J., and James, M. N. G. (1986a).J.Biol. Chem. 261,2638-2644. Herzberg, O., Moult, J., and James, M. N. G. (1986b).In “Calcium and the Cell” (D. Evered and J. Whelan, eds.), pp. 120-139. Wiley, Chichester, England. Hol, W. G. J. (1985). Prog. Biophys. Mol. Biol. 45, 149-195. Holmes, M. A., and Matthews, B. W. (1982).J.Mol. Biol. 160,623-639. Jones, T. A., and Liljas, L. (1984).J.Mol. Biol. 177,735-767. Kabsch, W., and Sander, C. (1983). Biopolymers 22,2577-2637. Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990).Nature (London) 347,37-44. Kalb, A. J., and Levitzki, A. (1968). Biochem. J. 109,669-672. Karipedes, A., Auk, J., and Reed, A. J. (1977). Inorg. Chem. 16,3299. Klee, C. B., Crouch, T. H., and Richman, P. G. (1980). Annu. Rev. Biochem. 49,489-515. Klee, C. B., Newton, D. L., Ni, W. C., and Haiech, J. (1986). In “Calcium and the Cell” (D. Evered and J. Whelan, eds.), pp. 162-182. Wiley, Chichester, England. Kobayashi, T., Tagaki, T., Konishi, K., Ohnishi, K., and Watanabe, Y. (1988). Eur. J . Biochem. 174,579-584. Kretsinger, R. H. (1976). Annu. Rev. Biochem. 45,239-266.

CALCIUM-BINDING SITES IN PROTEINS

143

Kretsinger, R. H. (1980). CRC Crit. Rev. Biochem. 8, 119-174. Kumar, V. D., Lee, L., and Edwards, B. F. P. (1990).Biochemistry 29, 1404-1412. Leavis, P. C., and Gergely, J. (1984). CRC Crit. Rev. Biochem. 16,235-305. Leszczynski,J. F., and Rose, G. D. (1986). Science 234, 849-855. Levine, B A., and Williams, R. J. P. (1982). In “Calcium and Cell Function” (W. Y. Cheung, ed.), Vol. 2, pp. 1-38. Academic Press, New York. Linse, S., Brodin, P., Johansson, C., Thulin, E., Grundstrom, T., and Forsen, S. (1990). Nature (London) 335,651-652. Loll, P. J., and Lattman, E. E. (1989). Proteins: Struct., Funct. Genet. 5, 183-201. Loll, P. J., and Lattman, E. E. (1990).Biochemistry 29,6866-6873. Mann, S. (1988). Nature (London) 332, 119-124. Marsden, B. J., Hodges, R. S., and Sykes, B. D. (1988). Biochemistry 27,4198-4206. Martin, R. B. (1984).In “Metal Ions in Biological Systems” (H. Sigel, ed.), Vol. 17, pp. 1-50. Dekker, Basel, Switzerland. Martin, S. R., Linse, S., Johansson, C., Bayley, P. M., and Forsen, S. (1990).Biochemistry 29, 4188-4193. Matsuura, Y., Kunusoki, M., Harada, W., and Kakudo, M. (1984).J. Biochem. (Tokyo) 95, 697-702. McPhalen, C. A., and James, M. N. G. (1988). Biochemistry 27,6582-6598. McPhalen, C. A., Schnebli, H. P., and James, M. N. G. (1985). FEBS Lett. 188,55-58. McPhalen, C. A., Sielecki, A. R., Santarsiero, B. D., and James, M. N. G. (1991).Manuscript in preparation. Means, A. R., Tash, J . S., and Chafouleas, J. G. (1982). Physiol. Rev. 62, 1-39. Meyer, E., Cole, G., Radahakrishnan, R., and Epp, 0. (1988). Acta Crystallogr. Sect. B 44, 26-38. Moews, P. C., and Kretsinger, R. H. (1975).J.Mol. Biol. 91, 201-228. Musci, G., and Berliner, L. J. (1985).Biochemistry 24,3852-3856. Neidhart, D. J., and Petsko, G. A. (1988). Protein Eng. 2,271-276. Olafson, R. W., and Smillie, L. B. (1975). Biochemistry 14, 1161-1167. Olaitan, S. A,, Delange, R. J., and Smith, E. L. (1968).J.B i d . Chem. 243,5296-5301. Ofner, C., and Suck, D. (1986).J.Mol. Biol. 192,605-632. Olsson, G., Andersen, L., Lindqvist, O., Sjolin, L., Magnusson, S., Petersen, T., and SottrupJensen, L. (1982). FEBS Lett. 145,317-322. Pantoliano, M. W., Whitlow, M., Wood, J. F., Rollence, M. L., Finzel, B. C., Gilliland, G. L., Poulos, T. L., and Bryan, P. N. (1988). Biochemistry 27,8311-8317. Permyakov, E. A., Yarmolenko, V. V., Kalinichencko, L. P., Morozova, L. A., and Burnstein, E. A. (1981).Biochem. Biophys. Res. Commun. 100, 191-197. Permyakov, E. A., Murakami, K., and Berliner, L. J. (1987).J.Biol. Chem. 262,3196-3198. Pietrobon, D., Di Virgilio, F., and Pozzan, T. (1990).Eur. J. Biochem. 193,599-622. Read, R. J., and James, M. N. G. (1988).J.Mol. Biol. 200,523-55 1 . Reeke, G. N., Jr., and Becker, J. W. (1986). Science 234, 1108-1111. Rees, D. C., Lewis, M., and Lipscomb, W. N. (1983).J.Mol. Biol. 168 367-387. Richardson, J. S. (1981). Adu. Protein. Chem. 34 167-339. Richardson, J. S., and Richardson, D. C. (1988). Proteins Struct., Funct. Genet. 4,229-239. Rose, B., and Loewenstein, W. R. (1975). Science 190, 1204-1206. Satyshur, K. A,, Rao, S. T., Pyzalska, D., Drendel, W., Greaser, M., and Sundaralingam, M. (19SS).J.Biol. Chem. 263, 1628-1647. Sekharudu, Y. C., and Sundaralingam, M. (1988). Protein Eng. 2, 139-146. Shelling, J. G., and Sykes, B. D. (1985).J.Biol. Chem. 260,8342-8347. Shoham, M., Yonath, A., Sussman, J. L., Moult, J., Traub, W., and Kalb (Gilboa), A. J. (1979).J.Mol. Bi01. 131, 137-155.

144

CATHERINE A. MCPHALEN ET AL.

Silva, A. M., and Rossmann, M. G. (1985). Acta Crystallogr., Sect. B 41, 147-157. Skelton, N. J., Forsen, S., and Chazin, W. J. (1990). Biochemist? 29,5752-5761. Snyder, E. E., Buoscio, B. W., and Falke, J. J. (1990). Biochemistry 29, 3937-3943. Strynadka, N. C. J., and James, M. N. G. (1988). Proteins: Struct., Funct. Genet. 3, 1-17. Strynadka, N. C. J., and James, M. N. G. (1989). Annu. Rev. Biochem. 58,951-998. Suguna, K., Bott, R. R., Padlan, E. A., Subramanian, E., Sheriff, S., Cohen, G. H., and Davies, D. R. (1987).J. Mol. B i d . 196,877-900. Sumner, J. B., and Howell, S. F. (1936).J. Bacteriol. 32,227-237. Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985). Science 227,945-948. Swain, A. L., Kretsinger, R. H., and Amma, E. L. (1989).J. B i d . Chem. 264, 16620-16628. Szebenyi, D. M. E., and Moffat, K. (1986).J. Biol. Chem. 261,8761-8777. Szebenyi, D. M. E., and Moffat, K. (1987). In “Methods in Enzymology” (A. R. Means and P. M. Conn eds.). Vol. 139, pp. 585-610. Academic Press, Orlando. Taylor, A. N. (1983). In “Calcium Binding Proteins” (B. d e Bernard, G. L. Sottocasa, G. Sandri, E. Carafoli, and A. W. Taylor, eds.), pp. 207-213. Elsevier, Amsterdam. Teplyakov, A. V., Kuranova, I. P., Haratyunyan, E. H., Vainshtein, B. K., Frommel, C., Hohne, W. E., and Wilson, K. (1990).J. Mol. Biol. 214,261-279. Thanki, N., Thornton, J . M., and Goodfellow, J. M. (1988).J. Mol. B i d . 202,637-657. Thunnissen, M. M. G. M., Kalk, K. H., Drenth, J., and Dijkstra, B. W. (1990a).J. Mol. B i d . 216,425-439. Thunnissen, M. M. G. M., Ab, E., Kalk, K. H., Drenth, J., Dijkstra, B. W., Kuipers, 0. P., Dijkman, R., De Haas, G. H., and Verheij, H. M. (1990b). Nature (London) 347, 689-69 1. Tuffy, R. M., and Kretsinger, R. H. (1975). Science 187, 167-169. Tulinsky, A., Park, C. H., and Rydel, T. J. (1985).J. Biol. Chem. 260, 10771-10778. Tulinsky, A., Park, C. H., and Skrzypczak-Jankun, E. (1988).J. Mol. Biol. 202,885-901. van Deenen, L. L. M., and De Haas, G. H. (1964). Adv. Lipid Res. 4, 167-234. Voordouw, G., and Roche, R. S. (1974). Biochemistry 13,5017-5022. Vyas, M. N., Jacobsen, B. L., and Quiocho, F. A. (1989).J. Biol. Chem. 264,20817-20821. Vyas, N. K., Vyas, M. N., and Quiocho, F. A. (1987). Nature (London) 327,635-638. Vyas, N. K., Vyas, M. N., and Quiocho, F. A. (1990). In “Novel Calcium-Binding Proteins” (C. Heizmann, ed.). Springer-Verlag. Berlin. I n press. Wang, J. L., Cunningham, B. A., and Edelman, G. M. (1971).Proc. Natl. Acad. Sci. U.S.A. 68, 1 130- 1 134. Wasserman, R. H., and Fullmer, C. S. (1982). In “Calcium and Cell Function” (W. Y. Cheung, ed.), Vol. 2, pp. 175-216. Academic Press, New York. Wasserman, R. H., Shimura, F., Meyer, S. A,, and Fullmer, C. S. (1983). In “Calcium Binding Proteins” (B. d e Bernard, G. L. Sottocasa, G. Sandri, E. Carafoli, and A. W. Taylor, eds.), pp. 183-205. Elsevier, Amsterdam. Wells, J. A., Ferrari, E., Henner, D. J., Estell, D. A., and Chen, E. Y. (1983).NucleicAcidFRes. 11,7911-7925. Williams, R. J. P. (1986). In “Calcium and the Cell” (D. Evered and J. Whelan, eds.), pp. 145-161. Wiley, Chichester, England. Wnuk, W., Cox, J. A., and Stein, E. A. (1982). In “Calcium and Cell Function” (W. Y. Cheung, ed.), Vol. 2 pp. 243-278. Academic Press, New York. Yamashita, M. M., Wesson, L., Eisenman, G., and Eisenberg, D. (1990).Proc. Natl. Acad. Sci. U.S.A. 87,5648-5652. Yariv, J., Kalb, A. J., and Levitzki, A. (1968). Biochim. Bzophys. Acta 165, 303-305. Zell, A., Einspahr, H., and Bugg, C. E. (1985). Biochemistry 24, 533-537. Zot, A. S., and Potter, J. D. (1987). Annu. Rev. Biophys. Biophys. Chem. 16, 535-559.

COPPER PROTEIN STRUCTURES

.

By ELINOR T ADMAN Department of Blologlcel Structure. University of Washlngton. Seattle. Washington 98115

I . Introduction . . . . . . . . . . . . . . . . . . . I1. Cupredoxins: Proteins That Bind Only Type I Copper . A . Azurin . . . . . . . . . . . . . . . . . . . . B. Plastocyanin . . . . . . . . . . . . . . . . . C. Pseudoazurin . . . . . . . . . . . . . . . . . D. Cucumber Basic Blue Protein and Stellacyanin . . . E . Auracyanin and Amicyanin . . . . . . . . . . F. Summary of Cupredoxins . . . . . . . . . . . . I11. Proteins That Bind Only Type I1 Copper . . . . . . A . Superoxide Dismutase . . . . . . . . . . . . . B. Galactose Oxidase . . . . . . . . . . . . . . . IV . Proteins That Bind Only Type 111 Copper . . . . . . A . Hemocyanin . . . . . . . . . . . . . . . . . B. Tyrosinase . . . . . . . . . . . . . . . . . . V . Proteins That Bind More Than One Type of Copper . A . Ascorbate Oxidase . . . . . . . . . . . . . . . B. Ceruloplasmin . . . . . . . . . . . . . . . . C. Nitrite Reductase . . . . . . . . . . . . . . . D. Nitrous Oxide Reductase and Cytochrome Oxidase . VI Summary and Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

.

. . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145

. 148 151 157 160 . 161 . 164 165 . 168 168 171 . 172 172 177 . 178 179 184 185 . 187 190 192

I . INTRODUCTION

Copper is the third most abundant trace element in humans. after iron and zinc (Underwood. 1977). It is critical to a variety of proteins with functions ranging from electron transfer to oxygen transport to active chemistry. such as insertion of oxygen in a substrate. Table I is a selected list of copper-containing proteins . Copper proteins have been classified according to their spectroscopic properties (Malkin and Malmstrom. 1970; Fee. 1975) as type I. 11. or I11. Type I blue copper proteins are characterized by an extraordinarily intense absorption near 600 nm and by unusually small hyperfine coupling constants for the paramagnetic [oxidized Cu(II)] form of the protein . Type I1 sites have normal extinction coefficients. are found to have ADVANCES IN PROTEIN CHEMISTRY. Vol. 42

145

.

Copyright 0 1991 by Academic Press. Inc All rights of reproduction in any form reserved .

146

ELINOR T. ADMAN

TABLE I Copper Proteins" Copper-containing protein Type I copper Azurinb PIastocyaninb Pseudoazurinb Cucumber basic blue proteinb Amicyanin Auracyanin Type I1 copper Superoxide dismutaseb Lysine oxidase Galactose oxidaseb Dopamine P-monooxygenase Type 111 copper Hemocyaninb Tyrosinase Multiple types of copper Ascorbate oxidaseb

Factor V Factor VIII N 2 0 reductase Uricase

Source

Electron transfer

Bacteria Plants, algae Denitrifying bacteria Cucumber

Adman (1985) Coleman et al. (1978) Kakutani et al. (1981) Cuss et al. (1988)

Pseudomom AM1 Chloroflexus auranticus

Tobari and Harada (1981) Trost et al. (1988)

Superoxide scavenging Connective tissue biosynthesis Alcohol oxidase Catecholamine synthesis

Yeast, mammals

Fielden and Rotilio (1984)

Mammals

Knowles and Yadav (1984)

Fungi Human chromaffin gran u1es

Kosman (1984) Ljones and Skotland (1984)

O2 transport

Arthropods, mollusks Fungi, mammals

Preaux and Gielens (1984)

Plants

Mondovi and Avigliano ( 1984) Ryden (1984) Reinhammer (1984) Capaldi (1990)

Melanin synthesis

I

Ceruloplasmin Laccase Cytochrome oxidase Nitrite reductaseb Other Copper thionein

Function

02+H20

Denitrification

Reference

Vertebrates Fungi, plants Bacteria-, vertebrates Denitrifying bacteria

Robb (1984)

Liu et al. (1986)

Copper homeostasis

Vertebrates

Weser and Hartmann (1984)

Blood coagulation

Humans

Ryden (1988)

Humans Denitrifying bacteria Pigs, soybeans

Vehar et al. (1984) Riester et al. (1989) Pitts et al. (1974)

Denitrification Purine catabolism ~~

~

~~

~

~

~

Selected list of proteins in which copper is critical to function. Proteins for which three-dimensional structures are known.

COPPER PROTEIN STRUCTURES

147

larger hyperfine coupling constants, and are paramagnetic in the Cu(11) form. The original classification of type I1 sites (Fee, 1975) was intended to include only the “colorless” copper found in multi-copper oxidases. However, its properties are spectroscopically similar to those of the copper in superoxide dismutase, so it is useful to refer to these sites as type I1 as well. The type I1 copper sites are involved in chemical reactivity (as opposed to only electron transfer); for example, type I1 coppers are found in superoxide dismutase, galactose oxidase, amine oxidases such as dopamine P-monooxygenase (P-hydroxylase), and lysine oxidase. There is no other nonmetal cofactor in superoxide dismutase, but other cofactors are present in some of the amine oxidases. Type I11 copper is characterized by antiferromagnetic coupling of a pair of copper atoms and strong absorbance at 330 nm. A single type I11 pair is found in hemocyanin, in which it is involved in 0 2 transport, and in tyrosinase, in which an oxygen is inserted into substrate. A pair of copper atoms is also found in the multi-copper ascorbate oxidase, but it is coupled to the type I1 copper in a trinuclear arrangement. There are a number of excellent sources of information on copper proteins; notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS)work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. T h e new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type 111 copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. Although a great deal of work is in progress on many copper proteins, this chapter focuses on those for which there are X-ray structures, and include others inasmuch as they are known to be related to the structurally characterized ones. The future looks exciting for copper proteins-not only are there many well-characterized proteins whose structures are not yet determined, but new functions are emerging. A Cu cluster containing domain attached to a transactivating domain has been shown to turn on the gene for Cu-metallothionen (Furst and Hamer, 1989; Furst et al., 1988).

148

ELINOR T. ADMAN

11. CUPREDOXINS: PROTEINS THAT BINDONLYTYPE I COPPER

T h e structural work done in the late 1970s on type I proteins azurin and plastocyanin (Colman et al., 1977; Adman et al., 1978), coupled with insightful theoretical work by Solomon, Gray, and co-workers (Solomon et al., 1976, 1980, 1983), showed that the copper is bound in a distorted tetrahedral arrangement of two histidines, a cysteine, and a methionine, and that it was likely that the charge transfer in a copper-cysteine bond was responsible for the strength of the absorption. Physical and chemical data suggested that there might be subclasses under the type I grouping (Fee, 1975; Adman, 1985). More recently determined structures have refined this view slightly. T h e single-domain blue copper proteins are invariably involved in electron transfer; hence, it has been suggested that they might be named cupredoxin or copper redox protein, analogous to ferredoxin (Adman, 1985). Four subgroups of cupredoxins were suggested in 1985, based on spectra, amino acid composition, sequences, and X-ray structure. At that time only the structures of representatives of class I (azurin) and class 111 (plastocyanin) were known; since then, structures of classes I1 and I V have been completed. These structures lend support to the grouping, but undoubtedly the more that is learned about these proteins, the more subdivisions will be found. Table I1 summarizes a current classification, including the reaction partners where these are known. A comparison of the geometry of the copper center of three proteins for which high-resolution coordinates are available (azurin, plastocyanin, and pseudoazurin) reveals remarkable similarities, but significant differences as well. T h e distorted tetrahedron of ligand atoms, comprising a cysteine, two histidines, and a methionine in azurin is better described as a trigonal bipyramid with an axial methionine ligand and a peptide carbonyl oxygen on either side of the plane formed by the two histidine nitrogens and the cysteine sulfur. Cupredoxins differ slightly in their visible spectra, their electron paramagnetic resonance (EPR) spectra, and their redox potentials. The type I site certainly has not yet been modeled exactly by small molecules. [Since the excellent series of books edited by Karlin and Zubieta (1983, 1986) includes many articles reviewing modeling approaches to understanding the physical and chemical properties of copper proteins, these are not dealt with further here.] The question is: How does the protein structure bring about first, the inherent properties of the type I site and, second, the modest differences among them? Copper in proteins is not usually accompanied by any other cofactors such as hemes or inorganic sulfur in iron-containing proteins, so that the

TABLE I1 Subchsifcation of Type I Cupredoxins Class

Spectral characteristics and ligands

Example

1

625 nm, axial EPR, 3 Cys, H-C.H.M

Azurin"

I1

595,470, and 780 nm, axial EPR, 1 Cys,

Umecyanin? Pseudoazurin"

111

595 nm, axial EPR, 1 Cys, H..-C-H.M

Plastocyanin" Amicyanid

IV

595,480, and 780 nm, rhombic EPR, 3

Cucumber basic blue protein" Stellacyanin Mavicyanin Rusticyanin Auracvanind

H*.C.-H.M

Cys, H..C.H.M Other 3

3 cys

Source Pseudomom aeruginosa, Paracoccus denitnficans, Akaligenes faecalis Horseradish root Alcaligenesfaecalis, Achromobacter cyclochtes Plants, algae Pseudomom AM1

Electron donor

Electron acceptor

?

Cytochrome cSS1

? ?

Nitrite reductase

Cytochrome f Methylamine dehydrogenase

?

P700

Cucumber

?

?

Plant Green squash Thiobacilwferroxiduns Green Dhotosvnthetic bacteria

? ? ? ?

? ? ? ?

Structure is known. Amicyanin appears to be more homologous with the plastocyanin sequence than with the pseudoazurin sequence. Cucumber basic blue protein has been put in a class of its own in view of having a disulfide, unlike plastocyanin, although its fold is generally like that of plastocyanin. See Trost et al. (1988). a

150

ELlNOR T. ADMAN

copper environment is the protein. Much of the electronic character of the type I site is due to the fact that the copper is bound to the electronrich cysteine thiol and has tetrahedral, rather than tetragonal, geometry. The protein provides a site with exceptional affinity for both Cu(1) and Cu(II), and probably with a low-energy barrier between the two forms. The protein fold used for the cupredoxins is a Greek key p barrel. Structural variability among the proteins is limited to insertions and deletions in loop regions, while generally maintaining the topology of the protein fold, as is the case in the cytochrome c (Almassey and Dickerson, 1978) and ferredoxin protein families (Fukuyama et al., 1988). The features found to be common to all of the “blue” sites are the following (summarized in Fig. 1). (1) There is always a loop containing three of the ligands relatively close

N-terminal loop

Two hydrophobic residues sandwlchlng Met residue

Group hydrogen bondlng to N-terminal His llgand Locatlon of group which may also affect llgand His

loop containing

Adjacent drand with HIS llgand

- - _-

\

N

H dro en bonding pair of she clains

~

-1-

fis,

M \

Reorientation U of Solomon’s description of electronic structure of plastocyanin

FIG. 1. Protein factors which may affect type I copper site properties. Schematic view from the protein surface closest to the copper center.

COPPER PROTEIN STRUCTURES

151

together in sequence: Cys-X,-His-X,-Met (where n and m vary from one protein to the next). (2) There is a histidine located considerably toward the amino terminus (upstream) from the other three ligands. In the three-dimensional structure the trio of ligands occurs on a loop between the two carboxy-terminal strands of a /3 sheet, while the upstream histidine is located on the strand adjacent to these, but which, because of the Greek key topology, is considerably toward the amino terminus in sequence from these, more than 30 residues away. (3)A pair of generally conserved residues which hydrogen bond to each other next to the upstream histidine and the cysteine further stabilize the interaction between these adjacent strands of the p sheet. (4)T h e methionine is always sandwiched between two hydrophobic residues, two residues apart, provided by a loop between two p strands at the amino terminus. ( 5 ) The upstream histidine is more buried than the other, and is usually oriented further by a hydrogen bond from a residue or main-chain atom on a different strand. (6) There is one or more N H * . * Shydrogen bonds between main-chain amide nitrogens and the cysteine ligand. (7) The copper atom sites are usually not more buried than about 8 8, and each has an edge of the downstream (carboxy-terminal) histidine protruding through a more or less extensive hydrophobic face, which is likely to be at least one of the surfaces through which electron transfer occurs. Figure 1 also contains a drawing of the electronic structure of plastocyanin (Penfield et al., 1981, 1985), oriented according to the first part of Fig. 1. T h e &orbital plane appears to be normal to the peptide plane that is extended by the hydrogen-bonding pair; the T orbital of the thiolate would then also be affected, poising the cluster for electron transfer and, interestingly, apparently in the direction utilized by the multi-copper protein ascorbate oxidase (see Section V,A).

A . Azurin The refined structure of azurin from Alcaligenes denitrificans has been completed with 1.8 A resolution data (Baker, 1988), resulting in an agreement factor (R value) of 0.157 with restrained bond distance deviations at 0.016 A. (Figure 2a is the copper site; Fig. 2b, a ribbon drawing of the protein fold; and Fig. 2c, a schematic of the folding topology.) The estimated coordinate errors are -0.1 A; Cu-X bond lengths are believed to be more reliable, or -0.04 A. Because there are two molecules per asymmetric unit in this form, the maximum expected difference in bond length could be estimated to be 0.27 A. Agreement between the two molecules among Cu-X bond lengths and angles is quite good (mean values cited in Table 111).The coordination is best described as trigonal

152

ELINOR T.A D M A N

a

b

d

FIG. 2. Copper site in azurin. In this and subsequent figures the following conventions have been used. (a) The copper site is generally an enlargement of (b).The copper site is a dotted sphere, the ligand residues are represented by bonds connecting atoms in the side chain, and, where possible, the atoms bonded to the copper atom are identified by atom type. Ribbons represent portions of the backbone structure near the copper. In (b) of each figure, the main-chain polypeptide is represented by a ribbon fit to the main-chain coordinates, and the amino and carboxy termini are indicated by N and C, respectively. (c) A schematic version drawn from (b). Solid arrows represent main-chain regions participating in the p sheet roughly above the plane of the paper, while dotted or light arrows are the 0

COPPER PROTEIN STRUCTURES

153

bipyramidal, with the methionine sulfur and the carbonyl oxygen occupying axial ligand positions. The latter distance to a copper atom is rather long for a bond; nevertheless, it appears to be a significant interaction. Baker (1988) pointed out that the carbonyl actually is in a hydrophobic environment with no opportunity to bond to anything else, and that the angle at the oxygen atom is indicative of favorable interaction of lone pair electrons with the copper. The near-tetrahedral arrangement of atoms bonded to the ligand cysteine sulphur, including the hydrogen bonds from amide nitrogens, indicate that the cysteine is likely to be thiolate. As pointed out earlier (Adman, 1985; Baker, 1988), the NH*..Sbonds provide a reasonable, if simplistic, explanation for the difference in absorption maxima between azurin and plastocyanin. Increased negative charge on the sulfur could be stabilized by the extra NH...S bond so that there would be less electron density on the copper and a lower energy for the Cu-Sy charge transfer (or longer wave length). On the other hand, other factors such as longer Cu-Met S6 bonds may also alter this charge density (Solomon et al., 1983). A good discussion of the correlations of the hyperfine coupling constants, redox potentials, and distortion from tetragonal to tetrahedral symmetry of C U - N ~ Scompounds ~ has been given by Dorfman et al. (1983). These authors concluded both that steric strain is not necessary to form tetrahedrally coordinated Cu(I1) complexes and that the narrow hyperfine parameter is primarily a consequence of strong covalent character between copper and its ligands, here reflected in the short Cu-Sy bond length. Shepard et al. (1990) reported the refined structure of reduced azurin from A. denitrifcans. The reduced protein was formed at p H 6.0 in the native (oxidized) crystals using ascorbate. The refined model shows that the most significant structural differences occur at the copper. Its geometry remains much the same (already quite trigonal), and the distances from copper to the axial methionine and the carbonyl oxygen each increase by about 0.1 A. This is in contrast to results reported by

sheet below the plane. (d) Made by imagining a cylinder with its axis coincident with that of the p barrel of (c) and with the schematic picture of the structure inscribed on the outside surface of that cylinder. The cylinder is then “cut” and unrolled and flattened such that the p sheet containing the ligand residues is on the left, and the other sheet is on the right. H, Histidine; C, cysteine; M, methionine. These residues are also marked by spheres; the fact that they usually lie “under” the arrows emphasizes that these residues are inside the barrel. The striped sphere is the copper atom, and the two diamond-shaped residues are two conserved residues which hydrogen bond to each other. (a) Copper site in azurin. (b) Ribbon drawing of the azurin backbone. (c and d) Schematic of azurin topology.

TABLE 111 Cu-Ligand Geometryfor Cupredoxinf' PCY

Parameter Distance (A) cu-sy CU-NI CU-N~ cu-SG cu-0 Angle (") NL-CU-N~ Sy-Cu-Nl N2-cu-S~ sy-cu-SG SG-CU-N 1 SG-CU-N~ Reference

Az-Pde

Algal

2.12 2.08 2.01 3.12 3.17

2.12 1.89 2.17 2.92

100 135 121 109 78 93 Baker (1988)

104 125 120 108 90 102 Collyer et al. (1990)

Ox (pH 6.0) 2.13 2.04 2.10 2.90 4.00 97 132 123 108 85 103 Cuss and Freeman (1983)

Red (pH 7.8)

Pseudo-Az

*g

2.11 2.12 2.25 2.90 NA

2.38 2.34 2.36 3.02 NA

92 141 112 114 90 102 Cuss et al. ( 1986)

100 133 119 112 81 101 Church et al. ( 1986)

N I , N*'of the downstream histidine ligand; N2, N*' of the upstream histidine ligand; NA, not applicable.

2.07 2.10 2.21 2.69 4.0

2.16 2.13 2.16 2.76 3.8

99 138 121 107 86 108 Adman et al. ( 1989)

100 136 112 108 87 112 Petratos et al. ( 1988a)

COPPER PROTEIN STRUCTURES

155

Groeneveld et al. (1986) from an EXAFS study of Pseudomonus aeruginosa azurin, in which, on reduction, indications for a significantly shorter Cu-S bond were observed. Studies of apoazurin from A. denitrijicans are in progress in E. N. Baker’s laboratory (Massey Univ., Palmerston North, NZ). T h e structure of apoazurin from P . aerugznosa has been partially completed (S. S. Hasnain and E. T. Adman, unpublished observations, 1987). Apoazurin, a gift from H. R. Engeseth and D. R. McMillin (Purdue Univ.), crystallized in space group P212121 with cell dimensions of 57.26,81.11, and 110.61 8,.A difference of 2 8, in one axis and 1.5 8, in another was sufficiently nonisomorphous with the holoprotein cell dimensions that refinement of the model was begun with rigid body rotation at low resolution. Although the refinement is not yet complete, the agreement (R factor) between observed and calculated structure factors now stands at 0.26 (using PROFFT). Superposition of the Ca atoms of holo- and apoazurin shows that the four molecules in the asymmetric unit have pivoted about the hydrophobic face through which they interact. T h e resolution and status of the refinement are such that we cannot yet tell whether the more exposed histidine has rotated around its CP-Cy bond, as it apparently does when copper is removed from plastocyanin (see below). Azurin has proved suitable for a number of studies examining its physical and dynamic properties. The secondary structure predicted by Fourier transform infrared spectroscopy (Surewicz et al., 1987) on Pseudomonasfiuorescensazurin (Az-Pfe) is consistent with the known structures of the proteins from P . aeruginosa (Az-Pae) and A . denitrijicans (Az-Ade) azurins. Furthermore, the secondary structure of the apoproteins and metal-containing proteins were very similar, and also stable to -77°C before a cooperative unfolding of the P sheet begins. An early step (detected calorimetrically) in unfolding of the apoazurin and azurin reconstituted with less well-bound metals (Engeseth and McMillin, 1986) could be attributed to the a helix and/or turns. Inasmuch as the loop holding the metal ligands contains a number of bifurcated hydrogen bonds, it is likely that, in the absence of the metal, these may be destabilized at higher temperatures and unfold first. A study by Petrich et al. (1987) on the fluorescence lifetimes of excited tryptophans in azurin has proved exceptionally interesting, especially in light of the studies to be reviewed below on ascorbate oxidase. By comparing the lifetimes of tryptophan fluorescence of three azurins- AzPae (only one tryptophan, Trp-48), A. faecalis [Az-Afe (one tryptophan, Trp-1 IS)] and A. denitnjicam [Az-Ade (two tryptophans, Trp-48), and Trp-1 IS)] in both holo and apo forms-the authors found that (1) there is virtually no fluorescence quenching in the apo forms; (2) the decay of

156

ELINOR T. ADMAN

fluorescence in the holo form is doubly exponential with lifetimes of 1 and 3 nsec for Az-Afe with the more exposed tryptophan (Trp-118), while that of Az-Pae (Trp-48) is a single exponential with a lifetime of -6 nsec. It is hypothesized that the quenching of fluorescence when copper is present is due not to an energy transfer, but to an electron transfer from the excited tryptophan to the electrophilic copper center. Petrich et al. previously showed this in simpler systems of copper and tryptophanyl compounds. Arguing that the nonradiative process is electron transfer, these authors made calculations of the expected ratio of electron transfer rates for the Trp- 118/Trp-48 proteins. Their theoretical calculations suggested that the Trp-I18 rate is expected to be 10 times that of Trp-48, while the experimental ratio shows that it is -0.5 that of Trp-48, even though the tryptophan is modestly closer to the copper in the Trp-118 protein. The possibility was raised that an intervening aromatic amino acid might make the difference (i.e., facilitating electron transfer even though the distance was longer). Another notable conclusion from this paper is that these internal rates of electron transfer are extremely fast ( 10g/sec)and that the anisotropy decay of the apoprotein is not' consistent with any considerable motion of the internal tryptophan, contrary to the conclusion of a study some 8 years earlier by Munro et al. (1979). The gene for Az-Pae has recently become available (Canters, 1987; Arvidsson et al., 1989). The structures of two mutants H35N and H35L are being carried out in R. Huber and A. Messerschmidt's laboratory (Max-Planck-Institut, Martinsreid, Germany) (Nar et al., 1991). This mutant was of particular interest because His-35 (a buried histidine adjacent to the internal upstream ligand histidine) was suggested to be important in a pH-sensitive inactive form of azurin. Other mutants with ligands (Karlsson et al., 1989) and nonligands being replaced are being characterized (Pascher et al., 1989). Three mutants have been made: H35K, E91Q, and F114A and the redox potential, spectra, and rate of electron transfer with cytochrome c5s1 have been measured. It was found that H35K did not affect the rate of electron transfer, nor did E91Q, but F114A did, confirming the role of the hydrophobic surface in this process. It would be interesting to undertake the structure of the H35K mutant, to see how the charged and larger side chain is accommodated in an internal position. While the exact role of His-35 remains puzzling, its study has led to some interesting observations. Groeneveld and Canters ( 1988) carefully measured the rate of self-exchange of Az-Pae (the transfer of electrons between oxidized and reduced azurin) and found it to be both unusually high (lO'/mol/sec) and insensitive to pH. T h e high rate is suggested to be a consequence of transfer through the hydrophobic face (via His-1 17),

COPPER PROTEIN STRUCTURES

157

and, if the correlation of rate with distance is correct, the short distance implied by such a high rate indicates that the transfer is actually between these exposed histidine edges. This likely interaction is actually seen in the packing of the azurin molecules in both the crystals of the P. aerugnosa and A . denitrijicam proteins, even though these are different space groups (see Baker, 1988). In each histidine edges are -7.5 8,apart. In the A. denitrijicans protein each histidine is bridged directly to the other molecule by a solvent molecule. The lack of pH effect for self-exchange suggests that there must be an alternate path for those reactions which are pH sensitive. Unfortunately, there may be an inconsistency here. It is the path of transfer from azurin to cytochrome c551 that is p H sensitive; recently, R. Korzun (personal communication, 1989) proposed from model building that the reaction with cytochrome c551 is via the hydrophobic interface as well. This remains intriguing. A careful nuclear magnetic resonance (NMR) study of the titration behavior of His-35 in Az-Ade by Canters and co-workers (Groeneveld et al., 1988) confirmed the prediction that His-35 would not titrate-or, at best, was found to have an unusually low pK (4.6). Interestingly, when His-35 is protonated there seems to be no effect on the copper center, unlike the case with Az-Pae.

B . Plastocyanin The extremely careful and well-done structural studies of plastocyanin over the years have been quite revealing. The architecture of plastocyanin is, like that of azurin, a /3 barrel, but it is more compact and lacks the “back flap” of azurin (cf. Fig. 3). It displays a copper site much like that of azurin, except with a more remote carbonyl oxygen, one less N H . - . S bond, and a Cu-Met-SG distance slightly shorter than that of azurin. (Figure 3a is the copper site; Fig. 3b, a ribbon drawing of plastocyanin; and Fig. 3c, a schematic of the topology.) The difference in Met-SG to Cu bond length has been suggested to be one of the factors responsible for the higher redox potential of plastocyanin and plastocyanin-like blue proteins (Ainscough et al., 1987). The carbonyl oxygen is apparently forced to be more distant, or is prevented from being closer, because it is part of the penultimate residue just before the upstream histidine, and is part of the loop spanning the amino-and carboxy-terminal p sheets. This loop is longer in most azurins than it is in plastocyanin and other blue proteins. Control over the Met SG-Cu distance could be exercised either by the spacing between the histidine and methionine ligands (Xm; see Fig. 1) or to some degree by the hydrophobic packing from the aminoterminal loop (see Fig. 1).

158

ELINOR T. ADMAN

a

b

C

d

FIG.3. (a) Copper site in plastocyanin. (b) Ribbon drawing of the plastocyanin backbone. (c and d) Schematic of plastocyanin topology.

In contrast to azurin, the plant plastocyanins have a conserved negative patch of residues adjacent to a putative redox partner-binding site. Plastocyanin has, in addition, the hydrophobic face into which the edge of the second histidine ligand is inserted. Freeman and co-workers (Guss et al., 1986) have characterized the

.

COPPER PROTEIN STRUCTURES

159

reduced structure of plastocyanin as a function of pH and showed that, at low pH, the exposed histidine becomes protonated, and no longer bound to the copper, thus irreversibly forming a Cu(1) form. The same histidine is rotated 180” about CP-Cy from its liganded form in apoplastocyanin (Garrett et al., 1984), suggesting a “revolving door” two-step binding process of copper to apoplastocyanin. The other ligand groups move slightly to fill in the vacant site, although Pro-36 moves away, possibly as a consequence of the histidine flip. It is not known whether apoplastocyanin can crystallize: The apo form actually was made in the crystal by first reducing the copper at a slightly lower pH and extracting the copper with CN- in phosphate buffer. It is quite remarkable that the apo form can be made in the crystal, although the hydrophobic surface which appears to flex in forming apoazurin is not involved in packing interactions in plastocyanin. Pro-36 is also seen to undergo a conformational change when the copper of plastocyanin is replaced by mercury, this time placing it closer to the metal than it is in copper plastocyanin (Church et al., 1986). Interestingly, the mercury density could best be modeled with anisotropic thermal parameters with the major axis of anisotropy along the Hg-Met S6 bond, suggesting some disorder of the mercury. Mercury is slightly larger than copper, and the Hg-X bonds are consistently longer, with the histidine ligands differing the most (cf. Table 111). In ongoing efforts to establish the error limits on reported bond lengths and angles of refined structures of plastocyanin, Freeman and co-workers (Fields et al., 1990) have collected low-temperature data by several different methods and refined the models with three different programs. Preliminary results suggest that the Cu-ligand bond lengths agree with room-temperature measurements to within 0.05 A; complete results will be forthcoming. The structure of a Scenedesmus obliquus (algae) plastocyanin has been determined by two-dimensional NMR (Moore et al., 1988), using the poplar plastocyanin structure as a starting point for the distance geometry calculations. This plastocyanin differs from poplar plastocyanin by deletion of two residues near the conserved acidic patch, felt to be important in the interaction of plastocyanin with its redox partner P700. While the details of the structure available from NMR are less definitive than those from X-ray work, they provide a useful complementary viewpoint. In the NMR work some of the regions appear ill defined, not because the structure therein is particularly flexible, but because data in the form of appropriately close proton-proton contacts are missing. For example, in the region of the ligand-binding loop His-85 to Met-90, the root mean square difference between the poplar structure and the Scenedesmus

160

ELINOR T. ADMAN

structure is reported to be 2.5 A. This is unlikely to be a real difference, in view of the consistency of geometry seen between many kinds of blue copper proteins. Two features of the NMR structure present relevant new information. Phe-82 and Phe-29 are tyrosine and tryptophan, respectively, in the algal protein, and are apparently now, according to NMR, hydrogen bonded (internally) to each other. T h e deletion of the two residues 57 and 58 leads to an alteration of the shape of the acidic patch, a step back to the absence of the patch in evolutionarily more primitive plastocyanin. The high-resolution X-ray diffraction study (Collyer et al., 1990) of a plastocyanin from another green algae, Enteromorpha prolifera, supports the constancy of the active-site geometry within estimates of standard errors (-0.07 8, for Cu-ligand distances) and confirms that the tworesidue deletion occurs as was described for the previously cited NMR work. Although there is only 54% sequence identity between the algal and poplar plastocyanins, there appears to be complementary replacement of internal amino acids so that there is no offset of one /3 sheet to the other, as there is for azurin relative to plastocyanin. T h e effect of the two-residue deletion is to diminish the size of the negatively charged protrusion believed to play a role in interacting with electron transfer partners. The overall charge of this plastocyanin is -6, while that of poplar plastocyanin is -8. The molecules crystallize in different unit cells: Careful analysis of the packing interaction shows that hydrophobic and van der Waals packing interactions have little effect on conformation, whereas direct hydrogen-bonding contacts do have some effect, albeit a small one. It is noteworthy in view of subsequent discussion, that one “port-of-entry/exit” for electron transfer in plastocyanin is believed to be Tyr-83. This residue is on the molecular surface, one residue prior to the ligand cysteine, and protrudes from the surface next to the acidic patch. C . Pseudoazurin

The blue protein from A . faecal& strain S-6, which was isolated as a requirement for transferring electrons to a copper-containing nitrite reductase, has since been shown to have sequence homology with proteins arbitrarily designated “pseudoazurin” by Ambler and Tobari (1985), from Achromobacter cycloclastes and from Pseudomonas A M l . [Pseudomonas AM1 also produces amicyanin, which is the recipient of electrons from methylamine dehydrogenase, (see below)]. In A. cycloclastes reduced pseudoazurin donates electrons to a copper nitrite reductase (Liu et al., 1986), as it does in A . faecalis. Ambler and Tobari (1985)

COPPER PROTEIN STRUCTURES

161

suggested that a protein from P. denitnjicans previously called an azurin (Martinkus et al., 1980) may also be a pseudoazurin. T h e spectra of pseudoazurin and amicyanin are distinctly different in that there is an increased ratio of absorbance at 480 nm relative to the 600-nm band in pseudoazurin, while the visible spectrum of amicyanin is more like plastocyanin, with a reduced absorption at 480 nm. The structure of pseudoazurin from A. faecalis strain S-6 was determined in two laboratories (in part, because it crystallizes so readily; excellent crystals are formed in a matter of hours!) (Petratos et al., 1987, 1988a; Adman et al., 1989). The crystals, space group P65, have the interesting property that they are intensely blue when viewed along the sixfold axis, but are nearly colorless when viewed normal to this axis. This is assumed to be due to the fact that the plane of the Cu-Sy-Cp atoms is perpendicular to the sixfold axis, consistent with the fact that the copperthiolate bond is responsible for the blue color of the protein. T h e salient features of the A. faecalis pseudoazurin are that: (1) it has a Cu-Met bond length shorter than that of either plastocyanin o r azurin (see Table 111);(2) it has only one NH..-Sbond, as does plastocyanin; and (3) its overall architecture resembles plastocyanin (see Fig. 4), with an extended carboxy terminus folded into two a helices [a preliminary sequence comparison suggested that the folding would resemble plastocyanin (Adman, 1985)l. It retains the exposed hydrophobic face found in azurin and plastocyanin. Just how it interacts with nitrite reductase is still a subject of investigation. It is intriguing that the carboxy-terminal portion folds up onto the face of the molecule where the unique portions of other blue proteins are: the “flap” in azurin, and, as we see below in the multi-copper oxidase, entire domains. The gene for this protein has also been isolated from A. faecalis and sequenced and expressed in Escherichza coli cells (Yamamoto et al., 1987). Like azurin, it contains an amino-terminal signal sequence, suggesting that it is secreted into the periplasm in A. faecalis, although not all of the blue protein is found in this fraction. Mutants of this protein are being made and characterized in T. Beppu’s laboratory (Univ. of Tokyo) (personal communication, 1989). D . Cucumber Basic Blue Protein and Stellacyanin

Cucumber basic blue protein (Cbp) is a protein without known function, also known as cusacyanin or plantacyanin. Its structure (Guss et al., 1988) completes the repertoire of cupredoxins with known structures. The topology of its folding is similar (Fig. 5 ) to those of plastocyanin and azurin, as might have been expected from sequence similarities and

162

ELINOR T. ADMAN

a

b

FIG.4. (a) Copper site in pseudoazurin. (b) Ribbon drawing of the pseudoazurin backbone. (c and d) Schematic of pseudoazurin topology.

spectra, but, in general, leaves the copper site the most exposed of the four cupredoxins. The sequence of Cbp is quite similar to that of stellacyanin. Stellacyanin is a plant protein, also of unknown function, having visible spectra characteristic of type I copper, but lacking the methionine ligand found in all other type I proteins. A disulfide bond has been suggested as a potential copper ligand in stellacyanin; the Cbp has both a methionine and the disulfide, so that prior to the structure determina-

COPPER PROTEIN STRUCTURES

163

a

b

N

FIG. 5. (a) Copper site in cucumber basic blue protein (Cbp). (b) Ribbon drawing of the Cbp backbone. (c and d) Schematic of Cbp topology.

tion, it was thought that Cbp might provide a model for the disulfide ligand in stellacyanin. Cbp clearly does not have a disulfide ligand, but rather a methionine, as usual. An earlier observation that a disulfide bond existed between Cys-87 and Cys-93 in the apoprotein (which led to hypothesizing the pair as a putative copper ligand) is explained by the three-dimensional proximity of all three cysteines (59, 87, and 93). The disulfide bond is between 59 and 93 in the holoprotein and is too far from the copper to be liganded. Using the known sequence alignment, the

164

ELINOR T. ADMAN

spatial counterpart of the methionine ligand in stellacyanin would be Gln-97. Guss et al. (1988) suggested that this change of ligand type could explain the markedly lower redox potential (184 mV), as well as the rhombic g values seen in EPR studies of stellacyanin. Indeed, using a new triple-resonance ENDOR technique, Thomann et al. (1991) demonstrated that the fourth ligand is a nitrogen, and thus is consistent with a glutamine. Quenching of tryptophan fluorescence in stellacyanin resembles that in Cbp, suggesting that the tryptophan residues are in similar locations. The high-electron transfer exchange rate is consistent with the vastly more exposed copper and more exposed histidines that are found in azurin, plastocyanin, and pseudoazurin. Unfortunately, bond lengths have not been reported for the copper center of Cbp. Its spectrum (like that of plastocyanin) is much more like that of the A . faecalis pseudoazurin than azurin. Since little variability of the Cu-Sy bond has been seen in the three structures described above, and since the major difference between pseudoazurin and plastocyanin (or azurin) is the length of the Cu-Met S6 bond, this would suggest that the Cu-Met bond is short in this protein, as well. Its EPR is also rhombic, again, like that of pseudoazurin. E . Auracyanin and Amicyanin

A recently characterized single-domain copper protein, auracyanin (Trost et al., 1988), is a dimeric protein which has a visible spectrum more like that of the A . faecalis cupredoxin (pseudoazurin, subgroup 11; see Table 11) than that of either azurin or plastocyanin, but, because of its cysteine content and rhombic EPR, it has been put in the “other” class in Table 11. Amicyanin (Husain and Davidson, 1986; Groeneveld et al., 1988) spectroscopically resembles plastocyanin more than pseudoazurin and has about the same number of amino acids, so that its classification has been changed from subgroup I1 to I11 (the plastocyanin group; see Table 11). However, its sequence is distinctly different than the plastocyanins, and the new function may indicate yet another class. Although crystals have been reported for two amicyanins (Petratos et al., 1988b; Lim et al., 1986), the type I blue protein, which is an electron acceptor for methylamine dehydrogenase (Tobari and Harada, 1981; van Houweligen et al., 1989), neither study has yet been completed. T h e structure of methylamine dehydrogenase from Thiobacillus versutus (not a copper protein) has recently been reported (Vellieux et al., 1989). The amicyanin from P . denitnjicam has actually been cocrystallized with methylamine dehydrogenase (F. S. Mathews, personal communication,

165

COPPER PROTEIN STRUCTURES

1990), and X-ray data have been collected, so very interesting results should be forthcoming. Sequence comparisons, NMR, and Raman spectroscopy (Sharma et al., 1988) have all suggested the same sort of ligands for amicyanin. From D 2 0 resonance Raman spectroscopy it is likely that amicyanin will exhibit NH*-.Sbonds, as well. Like plastocyanin, but unlike azurin, amicyanin has a ligand histidine which is titratable. F. Summary of Cupredoxins

In summary, then, comparison of the four known cupredoxin structures helps reveal the "essence" of this kind of protein. Schematically (see Fig. 6), it can be seen that the P-barrel structure is composed of three parts: an amino-terminal portion, a middle portion, and a carboxyterminal portion (a functional grouping which probably is not relevant to the folding of the polypeptide). One part of the amino-terminal loop

Loop containing three ligands: C...H...M

Adjacent strand with "upstream" His ligand

Loop with two hydrophobic residues sandwiching Met ligand

\ Pair of side chains

--

- --

-

C-terminal sheet

"dog leg"crossover between N- and C-terminal sheets

\

N-terminal sheet

"variable"strand

- "flap" in azurin - contains "acid patch" in plastocyanin - shortened in pseudoazurin FIG. 6. Anatomy of a cupredoxin fold.

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ELINOR T. ADMAN

binds to one p sheet, one part to the other, resulting in two successive bends as the chain crosses from one sheet to the other. This loop also contains the two hydrophobic residues which always sandwich the methionine ligand, as well as atoms which may orient the amino-terminal (upstream) histidine ligand. T h e middle portion contains the upstream histidine ligand, just following a spacer which extends from the second strand of the first p sheet to the carboxy-terminal p sheet. This loop may contain a residue (His-35 in azurin, Asn-31 in plastocyanin, and water in pseudoazurin) that is adjacent to the ligand histidine and may modify its orientation. Following the placement of the ligand, the remaining residues in the middle loop provide the most variability in these structures, before adding another strand to the amino-terminal /3 sheet. In azurin the middle loop also contains the flap; in plastocyanin it contains an irregular strand and the “acid patch.” The main function of this loop, then, in addition to providing the ligand, is to provide at least two strands of p sheet: one to the amino-terminal sheet and one to the carboxyterminal sheet. The third, most constant, loop is that containing three copper ligands, Cys-X-X-X-His-X-X-X-Met. The number of residues between the ligand residues can vary. The cysteine torsional angles are invariant, but the different number of residues between cysteine and histidine may influence properties of the center, such as resonance Raman spectra (Han et al., 1991). There can be additional residues following the carboxy terminus of the loop, as in the A. faecalis pseudoazurin. If, indeed, stellacyanin folds as Cbp does, the third ligand may vary. It is intriguing that it is the His-Cys-His configuration around the copper that is threedimensionally most similar, not that of the cysteine, histidine, and methionine residues within the carboxy-terminal loop. For comparison with the cupredoxin fold illustrated in Figs. 2-6, the topology of a variable domain of an immunoglobulin is shown in Fig. 7 with the same orientation, with the hypervariable regions L1, L2, and L3 indicated. Interestingly, the face of the light-chain domain that combines with the heavy chain involves the carboxy-terminal pair of strands, that containing L3. This sort of interaction is similar to that found in ascorbate oxidase and nitrite reductase (described in Section V,A and C, respectively). The similarity of the Greek key folds of superoxide dismutase and immunoglobulins was first described in 1976, and the possibility of evolutionary relatedness was raised therein (Richardson et al., 1976). As was pointed out then, the similarity of the folds appears to be greater than expected by chance, but may only represent a preferred folding pattern. A sequence relationship to a non-copper-containing protein, a ragweed allergen, has been reported (Ryden, 1988; Hunt et al., I985), in particular, relating the so-called “plantacyanins” stellacyanin and Cbp. T h e

COPPER PROTEIN STRUCTURES

167

a

fN

‘C FIG. 7. (a) Ribbon drawing of immunoglobulin domain. (b and c) Schematic of the folding topology of the immunoglobulin domain.

folding pattern, in conjunction with the regularity of the location of histidine ligands, does suggest that most of the copper-binding proteins are evolutionarily related, and that the proteins which have a similar fold but do not bind copper are also related. Finally, there is the issue of which, if any, surface(s) of the protein(s) is used in electron transfer to specific electron transfer partners. Adman (1985) summarized the knowledge about the sites of interaction at that time. T h e hydrophobic surface surrounding the more exposed ligand histidine was one such surface (the point of view in Fig. l), and the new

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ELINOR T. ADMAN

structures are still consistent with this. The acidic patch in plastocyanin is plausible as a binding site for P700, with the adjacent Tyr-83 possibly involved as an actual conduit. We have suggested that the region corresponding to the middle loop, as described in Fig. 6, represents the most variable portion of each of these structures and perhaps will ultimately represent the portion most involved in specificity. The locations of the acidic patch in plastocyanin and the back flap in azurin are consistent with this; in pseudoazurin the carboxy-terminal helices actually occupy a similar region with respect to the copper-thiolate bond. It is quite intriguing that the electronic description by Solomon (1988) suggested that this direction of exit is important; also, that the direction of electron transfer from type I sites to the trinuclear cluster in ascorbate oxidase (Section V,A) follows this path as well. 111. PROTEINS THAT BINDONLYTYPE I1 COPPER

A . Superoxide Dismutase Although the details of the structure of superoxide dismutase (SOD), one form of which is a Cu,Zn-enzyme (Cu,Zn-SOD), have been well described previously (Getzoff et al., 1983), a brief description is repeated here, for the geometry of the copper site as well as the topology of its fold is relevant to all copper enzymes. SOD is the protein for which the term “Greek key” was coined. T h e term comes from the similarity of the symbolic representation of its folding topology to a pattern seen on Greek urns (Getzoff et al., 1983). Figure 8a is a drawing of the metal sites, Fig. 8b is a ribbon drawing of the fold of a monomer, and Figs. 8c and d are schematic representations of the fold. The protein functions as a dimer, but has independent active sites >32 A apart. The 2 A structure refinement of bovine SOD was reported in 1982 (Tainer et al., 1982); the structure of the human enzyme is being studied, as are several site-directed mutant forms (Getzoff et al., 1989). The copper and zinc are 6.3 A apart and bridged by a histidine to both. The copper environment is described as a tetrahedrally distorted square plane, with the more open side facing solvent. The ligands are four histidines: three using NE’Sand one N6 bonded to the copper. T h e zinc is tetrahedrally coordinated, with three histidine ligands (all using N6) and an aspartic acid ligand. A water molecule is positioned 3 A from the copper (see Fig. 8a). As is the case with the blue proteins, all of the metal-ligand side-chain orientations are stabilized by hydrogen bonding. Tainer et al. (1982) pointed out the significance of the buried aspartic

COPPER PROTEIN STRUCTURES

169

a

FIG. 8. (a) Copper and zinc sites in superoxide dismutase (SOD). The small sphere is waterbound to the copper site. (b) Ribbon drawing of the SOD backbone. (c and d) Schematic of SOD topology.

acid, which is hydrogen bonded to ligands for both the copper and zinc sites and which effectively reduces partial positive charge on the histidines, in turn affecting the metal environments. Asp-124 is conserved throughout the known sequences, and, when mutated to glycine, activity is completely abolished (Getzoff et al., 1989). It is intriguing that all other “orienting” interactions are with main-chain atoms and thus do not re-

170

ELINOR T. ADMAN

quire conservation of specific side chains directly, but are dependent on the fold of the protein. T h e location of the copper with respect to the Greek key fold is interesting when compared to that of the cupredoxins. While the copper in the cupredoxins lies in the interior of the /3 barrel bound by three interior-facing residues of the carboxy-terminal loop in the /3 barrel, and by a histidine in an adjacent strand, the copper in SOD lies on the outside of its /3 barrel, bound by one residue from the carboxy-terminal loop and three from the adjacent strand (cf. Figs. 2c-5c with Fig. 8c.) A structural comparison of plastocyanin and SOD, coupled with sequence alignment of plastocyanin and ceruloplasmin (Ryden, 1988), showed that three of the SOD ligands correspond to putative copper ligands in ceruloplasmin. Why this is so will become more evident after the description of the ascorbate oxidase structure and its relationship to ceruloplasmin. Getzoff et al. (1989) evaluated the function of 23 conserved residues in SOD. Fifteen are directly involved in the active site (seven ligands, the ligand-orienting asparic acid, one arginine critical to the 0; binding pocket, five glycines, and one proline deemed critical to maintaining the geometry of the active site), four at the dimer interface, and four in the /3 barrel. Additional residues are persuasively argued to be functionally equivalent in preserving the overall fold of the molecule (e.g., providing “cork” residues at one end of the barrel or another). Of all the sequences compared for the functional equivalence, only one is from a bacterium: Photobacterium leignathi, a symbiont bacteria whose host is a fish. From the latter sequence and the absence of Cu,Zn-SOD in many other prokaryotes, it has been suggested that a gene transfer has occurred from the fish to the bacterium (Lewin, 1985). Apparently, only one other bacterium has been found to have a Cu,Zn-SOD (Bannister and Parker, 1985; Steinman, 1982). In a report dealing with the sequence of cabbage SOD, Steffens et al. (1986) argued that the photobacterium sequence diverges roughly equally from the sequences of all other species and suggested that gene transfer is unlikely since, if gene transfer had occurred, the sequence should be more similar to its proposed host than it is. T h e photobacterium sequence truly differs from the eukaryote sequences, having a 12-residue insertion in the S-S subloop. Nevertheless, there are fewer differences among SODS than in the various subgroups of cupredoxins. Although the real biological function of SOD continues to be a subject of debate (Fee, 1982), the high degree of conservation of many of these residues, particularly of those in the “electrostatic loop,” argues for dismutation of 0; as a significant function (Tainer et al., 1983; Getzoff et al., 1983).

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171

The copper in this protein lies -4 8, from a smoothed surface of the protein; however, the surface is highly invaginated at the copper, providing a pocket into which not only can a superoxide ion fit, but also into which it can be actively guided by an electrostaticfield gradient (Getzoff et al., 1983). Indeed, Brownian dynamics simulations of the ionic strength dependence of the relative association rates of SOD and 05, with either Arg-141 or lysines neutralized, were consistent with the effects being largely electrostatic (Sharp et al., 1987). Although residues 121 and 133 are conserved glutamic acids, their presences were calculated to decrease rate constants over that if they were modeled as lysines. Further results of those calculations suggest that the second-order association rate constant (10") is -5.7 times the actual turnover rate (12 x 10') and that the probability of reaction once the complex is formed is about equal to the probability of dissociation (Sharp et al., 1987),which sounds surprising if the chemical reactivity is important to the superoxide/scavenging function .

B . Galactose Oxidase Galactose oxidase (see Kosman, 1984; Ito et al., 1990) is an alcohol oxidase secreted by fungi and has found commercial use in an assay for galactose in biological fluids, as well as a means for preparing radiolabeled carbohydrates. The protein is relatively easy to purify, and apoprotein readily takes up copper. The protein apparently has an affinity for surfaces and is quite basic. It is active in 6 M urea and is quite stable to proteolysis if copper is bound. Spectroscopy of the protein suggests that it has a type I1 copper site. Electron paramagnetic resonance (EPR) and spin-echo experiments suggested at least two histidine ligands and a water that is replaceable by F-, CN-, or imidazole. Tyrosine was also hypothesized to be a ligand. Thiolate was not found to be a ligand, although there is one free sulfhydryl in the protein. Two additional residues, not ligands, were determined to have an influence on the activity of the protein: a histidine and a tryptophan. Since the galactose oxidase catalyzes a two-electron reduction, the presence of another cofactor has been suggested: either a tyrosine free radical (Whittaker et al., 1989) or a form of pyrroloquinoline (Van der Meer et al., 1989). In early 1990 it became apparent that the structure of galactose oxidase from Dactylium dendroides was about to emerge. A 2.5 8, multiple isomorphous replacement (MIR) map based on area detector data from a native and three derivative crystals yielded a polypeptide chain tracing. The refined structure at 1.9 8, (R = 0.179) (Ito et al., 1991) shows that galactose oxidase consists of three domains, each of which is predominantly p

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structure. T h e first domain has a j3-sandwich structure. The second, larger, domain contains the copper and has seven four-stranded j3 sheets, arranged as petals in a flower, with a rather open center. T h e third, small, domain lies on the opposite side of the second domain from the copper. Two j3 strands from the third domain form a loop, with a histidine at its tip, filling the open center of domain 2 and forming a 10-stranded twisted j3 sheet which spans that domain. The histidine ligands the copper. T h e other ligands for the copper are two tyrosines, a histidine, and an acetate, forming a square pyramidal array, the axial ligand being a tyrosine. A surprising finding is that the sulfur atom of the “free” cysteine is covalently bound ortho to the hydroxyl of the nonapical tyrosine ligand to the copper, forming a large planar group, which includes the Cj3 of the cysteine. A tryptophan is stacked on this “pseudo-side chain,” and hence may correspond to the tryptophan detected by chemical modification as being important to the function of the protein. Although the general impression of this remarkable structure is of a topology which has nothing to do with the Greek key fold so far seen in all of the copper-binding proteins, in fact, domain 3 can be described with that topology. In this domain the 4-5 loop normally containing the amino-terminal (upstream) type I histidine is now considerably elongated and is the loop with a histidine ligand which penetrates the second domain and binds copper. There is no other histidine in this domain. T h e folding of domain 1 resembles the “jellyroll” fold found in viral proteins. N o doubt some fascinating details of the mechanism of this protein will soon be forthcoming.

THAT BINDONLYTYPE I11 COPPER IV. PROTEINS A . Hemocyanin Hemocyanins are a class of multimeric copper-containing proteins which bind dioxygen and serve as the oxygen transport portein in many species of arthropods and in mollusks. The molluscan hemocyanins generally form tubular structures of 2 10 protomers ( a j 3 ) 5 , while the arthropodan hemocyanins are generally multiples of hexamers. There can be heterogeneity within the hexameric units as well; for example, Limulus polyphemus (horseshoe crab) hemocyanin is a 48-subunit ensemble of eight immunologically distinct protomers (Lamy et al., 1983). In addition to the fascinating and complex properties of the subunit architecture of hemocyanins [described, for example, by Preaux and Gielens (1984) and Herskovits (1988)], the nature of the copper site has

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been studied extensively. The protein contains type I11 pairs of antiferromagnetically coupled copper atoms (one pair per subunit), which are diamagnetic in the reduced form. By analogy with coupled iron states in hemerythrin, several oxidation states are accessible to this copper pair. There is the deoxy form [Cu(I)-Cu(I)], both coppers reduced and deoxygenated; the blue oxy form [Cu(II)-Cu(II)], both coppers oxidized and the pair oxygenated; the colorless met [deoxygenated, but both Cu(II)]; and various semi- o r half-met forms, achievable with various exogenous ligands which are interpreted as bridging the two copper atoms (Solomon et al., 1983). One class of these ligands (NO;, F-, C1-, Br-, I-, and ascorbate-) binds tightly, apparently only one per copper dimer, whereas a second group (N;, SCN-, and CN-) apparently binds to a second site, in addition to the main binding site. Binding at the main site forces a separation greater than 5 8, between the copper atoms, breaking the superexchange pathway between the coppers and leading to an EPR-detectable copper. Because the copper atoms are not close enough for antiferromagnetic coupling to occur via a direct metal-metal interaction, it is inferred that an endogenous bridging ligand must exist. At one point is was suggested that tyrosine would be a likely bridging ligand (Solomon et al., 1983). Nitrite can react with a molluscan deoxy-hemocyanin (Astacus peptodactylus), yielding an EPR-detectable methemocyanin. T h e pH dependence of this reaction was interpreted as replacement of a p-aquo bridge (by nitrite) which becomes an unreplaceable p-hydroxy bridge at a higher pH. T h e reaction product was shown to be NO (Tahon et al., 1988). However, the binding constant for NO; is small enough to make it unlikely to be tightly bound at all, in contrast to the studies cited above. In a study of octopus hemocyanin (Salvato et al., 1989), a green halfmethernocyanin could be formed in the presence of slight molar excess of sodium nitrite and ascorbate. This form was believed to consist of cuprous and cupric metals, but the amoung of NO directly bound was less than 0.1 mol. NO; was believed to be the oxidizing species; its relationship to the endogenous ligand was not addressed. Apparently, hemocyanins are most likely to be found in deoxy, o r reduced, forms, and it is difficult to oxidize them. Several hemocyanins have been investigated by X-ray diffraction methods, in spite of the potential difficulties with heterogenous subunits. Crystals of functional unit d (an active fragment of a larger polypeptide) of the molluscan hemocyanin from Helix pomatia have been reported (Preaux and Gielens, 1984) and belong to space group P62, with six molecules in the asymmetric unit. (This seems surprising, in that the

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intact protein assembles in multimers of pentamers, but with no mention of cell dimensions, etc., it is difficult to evaluate the report.) The arthropod hemocyanins have yielded the most to diffraction studies. Studies of Limulus hemocyanin by Magnus and Love (1983, cited by Preaux and Gielens, 1984) showed a kidney bean-shaped subunit which is consistent with both electron micrographs and the structure of hemocyanin from Panuliris interruptus (spiny lobster). An attempt is being made to find crystals of an arthropod hemocyanin which diffract to high resolution and have identical subunits in the asymmetric unit. To that end Buisson et al. (1989) crystallized subunit Aa6 of Androctonus australie hemocyanin in two forms: one which diffracts only to 8 A and one which diffracts to 3 A. A high-resolution diffraction data set has been collected from crystals of the mulluscan hemocyanin active fragment Od 1 from Octopus doflieini. This is a 47-kDa tryptic fragment that binds 0 2 reversibly. The crystals diffract to 1.9 A, so there is hope that high-resolution study of oxygen binding will be forthcoming (Cuff and Hendrickson, 1990). Diffraction from crystals of intact hemocyanin from this organism have shown 10fold symmetry in two different crystal forms, one of which has quite long cell edges (570 A) (Royer et al., 1988). By far the most definitive study on an arthropod hemocyanin has been that by Volbeda and Hol(1989b), a crystallographic tour de force. Crystals of subunit b can be formed from solutions of native hemocyanin which contain three types of subunits (a, b, and c). Two subunits (a and b) are nearly identical (3% difference in sequence), whereas subunit c differs more. Subunits a and b are glycosylated at a single residue (Asn-167). While the Panuliris form has been shown to be deoxy (Volbeda et al., 1989), unpublished observations indicate that the horseshoe crab structure (Limulus) is in the oxygenated state (K. Magnus, personal communication, 1988, cited by Volbeda and Hol, 1988). There are six subunits in the asymmetric unit of the Panuliris structure, arranged in a particle best described as a trimer of dimers. The 3-2 symmetry was good enough to be useful in a phase extension technique commonly used in the structure determination of high1 symmetric viruses. Unfortunately, these crystals diffract only to 3.2 , so that there cannot be a high level of detail available for description of the active site. Nevertheless, because there are six independent copies of the molecule in the asymmetric unit, there is more information than might normally be available at this resolution. T h e structure has been carefully refined at this resolution, with and without the 3-2 symmetry restraint. The estimated coordinate error is -0.35 A. The overall R is 0.201 for data between 8 and 3.2 A. Surprisingly, while the overall B for subunits 1 and

P;

COPPER PROTEIN STRUCTURES

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2 was lower than that for the other subunits, there was a wider variation in ideality for these subunits, including substantial differences in the Cu-Cu distance; for example, for subunits 3-6 Cu-Cu distances average 3.54 A, whereas for subunits 1 and 2 they are 2.9 and 3.8 A, respectively. However, the authors do not claim that this is significant, and, in fact, when the coppers and ligands are rebuilt into a difference map from which those coordinates are omitted, the Cu-Cu distances are more equivalent. The protein monomer folds into three domains: two nearly all a-helical and the third all p. Domain 1 is at the outer edge of a threefold unit, is primarily helical, and provides some dimer contacts. The second domain (all helical) not only provides ligands for the copper site, but also provides major intersubunit contacts around the threefold axis of the hexamer, as well as some at the dimer interface. Domain 3 has Greek key topology similar to that of SOD, but is not known to bind any metal. Its function in this molecule is not obvious, although it forms loose trimeric contacts with domain 1 of a neighboring subunit, involving a network of charge interactions, but among nonconserved residues. It perhaps may be expected to differ most from one species to the next. Both dimeric and trimeric contacts involving domain 2 are more highly conserved, suggesting that this domain is crucial for transmitting structural change on oxygenation. A novel feature of the protein is the presence of three “cavities” or packing defect regions surrounding helix 2.1 of domain 2. It is thought that one of these might provide access for O2 to the copper. Residues lining these cavities are generally hydrophobic. Another function might be to provide structural flexibility for 0 2 binding. The geometry of the copper site in the four (out of six) subunits which are the most alike shows that the copper atoms are 3.54 A apart (3.45 A was found in EXAFS experiments (Woolery et al., 1984). Each has two tightly bound histidines and one less tightly bound. For &(A) the tightly bound histidines are on sequential turns of a helix, while the third comes from an adjacent helix (to the left in Fig. 9a and b). In the Cu(B) site the two more tightly bound histidines come from different helices. Arguably, one of the more tightly bound histidines bound to Cu(B) could be described as intermediate to the loosely and tightly bound histidines, which would provide a rationale for the observation that carbon monoxide binds preferentially to one copper over the other; that is, it binds preferentially to a tricoordinate site which would be Cu(A). Accessibility to these coppers could provide another reason for preferential binding. At a resolution of 3.2 A, the limit for these crystals, there is no evidence in the electron density map for an endogenous bridging ligand required according to spectroscopic analysis (Solomon, 1988), but it is not likely

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a

b

FIG. 9. (a) Copper sites in hemocyanin. (b) Ribbon drawing of the hemocyanin backbone.

that it could be the proposed tyrosyl group, inasmuch as the closest tyrosine is > 10 8, away. Hydroxyl is not ruled out. A recently synthesized model compound having a novel p-q2-q2 peroxo-dinuclear copper complex apparently has the physicochemical properties of oxohemocyanin (Kitajima et al., 1989) and may provide the most realistic model to date. The previously cited spectroscopic investigation of crystals of this hemocyanin (Volbeda et al., 1989)showed unequivocally that the crystals lacked any oxygen and that they are in a Cu(1) oxidation state. It is possible to obtain partially oxgenated states of the same crystal form, using higher concentrations of dimethyl sulfoxide (-9% versus 3%), or in a different (hexagonal) crystal form. As is the case for other copper-binding proteins, the ligands are further oriented by hydrogen bonding to neighboring atoms. There is a local twofold axis relating the Cu(A) and Cu(B) sites of hemocyanin;

COPPER PROTEIN STRUCTURES

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atoms related by this twofold axis include the ligands and the ligandcontaining helices. These show a general topological equivalence, even to the extent of suggesting a relationship to the metal-binding helical pairs in hemerythrin (Volbeda and Hol, 1989a). Details differ; for example, each of the more weakly binding histidines is further oriented by mainchain carbonyl oxygens (of completely conserved residues), but equivalent pairs of more strongly bound histidine ligands are oriented by either water o r a carbonyl o r carboxyl oxgen. One of the waters is closest to the putative 0 2 binding site and is hydrogen bonded to the completely conserved Glu-329 residue, as well as to His-344, one of the more tightly bound histidines of the Cu(B) site. Other interactions are largely hydrophobic. Indeed, the shortest distance from the copper to the molecular surface is on the order of 6 8, (measured by computing a solventaccessible surface around subunit 2); however, subunit 1 may occlude that surface in the hexameric molecule. The copper pairs are -42 A apart within a dimer, so that the cooperativity exhibited by this protein clearly involves indirect interactions. The fact that partially oxygenated hemocyanin can be found in the same crystal form (albeit with higher concentrations of dimethyl sulfoxide, which is a denaturant) argues that the structural changes either are not large or are within the variability already exhibited by the fact that the crystals at present cannot diffract to better than 3.2 A. B . Tyrosinuse

Tyrosinase, a protein involved in melanin pigment formation, is found in bacteria, fungi, plants, and mammals (Robb, 1984). It is involved in the browning reaction of fruit. Recently, some isolation and cloning of cDNA from mouse have shown that the structural gene for tyrosinase maps to the albino locus (Kwon et al., 1987), and that it can be constructed from alternative splicing mechanisms (Ruppert et al., 1988; Halaban et al., 1988).Apparently, association of tyrosinase activity with melanogenesis is one of the most proven biological activities of this protein. (With the advent of molecular biological tools, in addition to the wealth of chemistry known about this protein, determination of its structure would seem all the more tantalizing!) Tyrosinase has been shown to have a type I11 site similar to, but not exactly like, that of hemocyanin. It differs in that its active site is considerably more accessible to exogenous ligands (Solomon, 1988). Spectral analysis of competitive inhibitors shows that good inhibitors apparently force the Cu(I1) center toward a trigonal bipyramidal geometry that is likely to be an intermediate in the reaction pathway. Sequence similarities also argue for the fact that the type I11 sites in

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hemocyanin and tyrosinase resemble each other (Lerch, 1988). Fairly good sequence similarity can be found for the Cu(B) site when three hemocyanins and two tyrosinases are compared, but there is less similarity for the Cu(A) site. Small fragments can be seen to be equivalent. Putative homologs of one ligand to the Cu(B) site and one to the Cu(A) site in hemocyanin have been demonstrated to be ligands in tyrosinase by site-directed mutagenesis in Streptomyces glaucescenr tyrosinase (Huber and Lerch, 1988). V. PROTEINS THAT BINDMORETHAN ONETYPE OF COPPER T h e multi-copper oxidases include laccase, ceruloplasmin, and ascorbate oxidase. Laccase can be found in tree sap and in fungi; ascorbate oxidase, in cucumber and related plants; and ceruloplasmin, in vertebrate blood serum. Laccases catalyze oxidation of phenolic compounds to radicals with a concomitant 4e- reduction of 0 2 to water, and it is thought that this process may be important in the breakdown of lignin. Ceruloplasmin, whose real biological function is either quite varied or unknown, also catalyzes oxidation of a variety of substrates, again via a 4ereduction of 0 2 to water. Ferroxidase activity has been demonstrated for it, as has SOD activity. Ascorbate oxidase catalyzes the oxidation of ascorbate, again via a 4e- reduction of O2 to water. Excellent reviews of these three systems can be found in Volume 111 of Copper Proteins and Copper Enzymes (Lontie, 1984). Each of these proteins is blue and appears to have a minimum of four copper atoms per molecule: one type I, one type 11, and two type 111. Laccase is not known to be multimeric, nor is ceruloplasmin, but ascorbate oxidase is apparently a dimer. When the complete amino acid sequence of ceruloplasmin was determined, an internal threefold repeat suggested gene triplication (Takahashi et d.,1983). Moreover, sequence similarity to the small blue copper domains suggested that there were at least two domains with blue copper-binding sites. Analysis of fragments of laccase sequence indicated that there might be a relationship of this to small blue proteins and ceruloplasmin as well (Ryden, 1988). Nitrite reductases and nitrous oxide reductases are relatively newly found copper-containing proteins involved in bacterial denitrification. N 2 0 reductase may bear a relationship to cytochrome oxidase and, indeed, parallels it somewhat in function, being the terminal electron acceptor in its pathway.

COPPER PROTEIN STRUCTURES

179

A . Ascorbate Oxidase The relationship of the blue oxidases has been enormously clarified by the recent determination of the structure of ascorbate oxidase from zucchini by Messerschmidt,Bolognesi, and co-workers (Messerschmidtet al., 1989). This determination also represented a crystallographic tour de force, because, while two crystal forms existed, neither was quite good enough on its own for solving the structure. One crystal form included the dimer in the asymmetric unit, while the other contained two dimers. The best resolution available was 2.5 A, although derivatives were not useful beyond 3.5 A. The interested reader is referred to the original report for this intriguing story. The cDNA sequence of the cucumberderived gene has been completed (Ohkawa et al., 1989),and the similarity to the squash protein sequence was sufficient to aid in interpretation of the electron density. The monomer of ascorbate oxidase has three domains, each of which can be described as a p barrel with Greek key topology (see Figs. 10-12). Domain 2 is not involved in copper binding at all. A type I copper is bound inside domain 3, while a novel trimeric arrangement of copper atoms is bound at an interface between domains 1 and 3 by four histidines from one domain and four from the other. The histidines from domain 3,which bind the copper trinuclear cluster, bear the same relationship to the P-barrel topology as do those on domain 1 to its p barrel; that is, each cluster is on the outside of the barrel and, in fact, in a location spatially quite analogous to the copper and its ligands in CuZn-SOD, as illustrated in Figs. 8, 10, and 12. The type I copper is clearly identifiable because of the characteristic His,-Cys-Met ligands. Of the trinuclear coppers two of the coppers are -3.4 A apart, and each is bound by three histidines. By analogy to hemocyanin, then, these are more likely to be the type I11 pair, while the remaining copper would be type 11, bound by two histidines and a third small ligand such as OH- or H20, which is visible in a higher-resolution map. However, efforts to make type 11-depleted ascorbate oxidase in the crystal succeeded only in removing yet another metal ion (probably copper) at a site on the twofold axis between halves of the dimer, at a surface site unrelated to the active site. The putative type I1 site is the most accessible to incoming ligands, and inhibitors such as F-, CN-, or azide could easily bind to it, either replacing the OH- or in addition to it. At present no evidence is reported for ligands bridging the type 111 pair, but the 1.9 refinement is in progress (A. Messerschmidt, personal communication, 1990) and will be more definitive on this question. Adjacent residues in the sequence bind the type I and type I11 coppers.

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ELINOR T. ADMAN

a

A b

I

FIG. 10. (a) Copper site in domain 1 of ascorbate oxidase. (b) Ribbon drawing of the ascorbate oxidase domain 1 backbone. (c and d) Schematic of ascorbate oxidase domain 1 topology.

COPPER PROTEIN STRUCTURES

181

a

FIG. 1 1 . (a) Ribbon drawing of the ascorbate oxidase domain 2 backbone. (b and c) Schematic of ascorbate oxidase domain 2 topology.

The sequence His-Cys-His has an extended chain conformation. In this sequence, the cysteine binds type I and the two histidines, two separate copper atoms of the trinuclear cluster, thus making the shortest distance from the type I copper to the trinuclear cluster 13 A. Within the cluster, the Cu-Cu distances are 3.4,3.9, and 4 A. It should be noted that none of the histidines bridges the copper atoms, unlike SOD, in which a histidine bridges the copper and zinc. T h e pattern of histidine binding to the trinuclear cluster is rather clever: two His-X-His pairs from separate domains, His-Cys-His (507-509) and His-Gly-His (106- 108), bind the

-

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ELINOR T. ADMAN

a

d

FIG. 12. (a) Copper sites in domain 3 of ascorbate oxidase. (b) Ribbon drawing of backbone of domain 3 of ascorbate oxidase. (c and d) Schematic of ascorbate oxidase domain 3 topology.

COPPER PROTEIN STRUCTURES

183

FIG. 13. Trinuclear copper site of ascorbate oxidase.

type I11 pair, while two additional pairs, again from separate domains, His-X-His (447-450) and His-Trp-His (62-64), bind the type I1 copper and one of the type I11 coppers, with the higher sequence residue in the His-X-His binding the type 111 copper. All coppers in the trinuclear cluster are bound by NC2of the histidines (see Fig. 13). With the structure of ascorbate oxidase in hand, a new structurally based alignment of the sequences of ascorbate oxidase, laccase, and ceruloplasmin has been performed (Messerschmidt and Huber, 1990). In brief, while gene triplication for ceruloplasmin is still revelant, its sequence can be further subdivided into two domains per unit of triplicated sequence, or six domains in total. Each of these sequences bears some resemblance to each of the three domains of ascorbate oxidase, as does each of the two domains in laccase. The coppers of the trinuclear site of ceruloplasmin then are predicted to be bound between domains 1 and 6, with a type I site also lying in both domains 6 and 4 (see Huber, 1990). The relative orientation of each of these domains is not predicted by this alignment, but it turns out that the structure of nitrite reductase may shed some light on this (see Section V,C). It is noteworthy that the proximity of the copper sites in ceruloplasmin, and, indeed, the involvement of most of the correct ligand histidines, were predicted some time ago by Ryden (1982, 1984) strictly on the basis of sequence homologies to plastocyanin. A similar prediction was made for laccase based on sequence similarities around the cysteine regions (Briving et al., 1980). Proximity of the type I1 site to the type 111 site (e.g., a trinuclear site) was also predicted by Solomon and co-workers (Allendorf et al., 1985; Spira-Solomonet al., 1986) on the basis of spectroscopic analysis of azide binding to laccase. What could not have been foreseen

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was the interdomain nature of the trinuclear site. It is very satisfying to see that the structures confirm these predictions.

B . Ceruloplasmin Ceruloplasmin, the multi-copper protein found in the serum of mammals, has been the object of study for some time. Structural studies were courageously begun before the advanced technology available today. T h e early crystallographic work was reviewed in the excellent article by Ryden (1984). The most promising crystal work was on the asialo form of the material (Moshkow et al., 1977). In 1980 (Zaitsev et al., 1980) 10 8, diffraction data from the 14 crystal form of human ceruloplasmin suggested that there were noncrystallographic twofold axes in the asymmetric unit, one between the two molecules in the asymmetric unit and one internal twofold axis. No mention was made of a search for threefold axes that might be consistent with the sequence triplication. Structural work is continuing (B. K. Vainstain, personal communication, 1990). Recent publications signal the continued interest in the function of this protein. It has been called a stress enzyme, involved in influenza virus infection (Tomas and Toparceanu, 1986). An explanation for Wilson’s disease in terms of a genetic defect resulting in failure to convert from a neonatal (i.e., low) level of ceruloplasmin and copper to a normal adult level has been reported (Srai et al., 1986). Tissue specificity for the binding of ceruloplasmin to membranes was demonstrated in a study investigating the possible role of ceruloplasmin-specific receptors in the transfer of copper from ceruloplasmin to other copper-containing proteins (Orena et al., 1986). Ceruloplasmin has been shown to be effective in transferring copper to Cu,Zn-SOD in culture (Dameron and Harris, 1987), as has copper albumin. In view of the variable content of copper in this protein, it is not clear which copper is transferred. Studies of other sources of ceruloplasmin may eventually prove useful in structure elucidation, but have already clarified some of the copper chemistry. Ceruloplasmin from goose serum has been isolated, purified, and characterized. This ceruloplasmin has less carbohydrate attached, but two forms may be isolated under some conditions. It is clear that these are not products of proteolytic degradation, but perhaps they might have a different carbohydrate attached. The two type I sites have higher extinction coefficients than type I sites in other ceruloplasmins, reflecting a modestly different environment (Hilewicz-Grabska et al., 1988). The protein has been isolated from sheep plasma and purified in a rapid single-step procedure, in an effort to minimize the proteolytic degradation that frequently accompanies its purification (Calabrese et al.,

COPPER PROTEIN STRUCTURES

185

1988a). The nonequivalence of copper sites of this ceruloplasmin was characterized by EPR. In these studies it was found that H202 is effective at oxidizing both type I sites, while O2 was only effective at the “slowly reducible” species. A sequence of events was identified in which a type I copper is reduced, accompanied by a “symmetry change” of the type I1 copper (a shift of the EPR line), followed by reduction of the second type I copper (Calabrese and Carbonaro, 1986). Isolation of ceruloplasmin from chicken plasma revealed a form of the protein that apparently, as isolated, had an EPR-undetectable form of the type I1 copper (Calabrese et al., 1988b), suggestive of a more tightly coupled interaction of the three coppers in the trinuclear cluster. I n light of this work, sheep ceruloplasmin was studied further, particularly the form isolated in a single-step procedure, and it was also found to start with no detectable type I1 EPR signal. T h e fresh protein is also completely reoxidizable, in contrast to the earlier work (Calabrese et al., 1989). The mechanism for interaction between these coppers and just what form of ceruloplasmin is needed for optimal activity will make for fascinating study in the future. C . Nitrite Reductuse

Copper-containing nitrite reductases (NIR) (in contrast to the more usual heme-containing proteins) have been found in a number of denitrifying bacteria, including a particularly potent denitrifying strain of A . faecalis (S-6) (Kakutani et al., 1981) from Alcaligenes species (Masuko et al., 1984) and A . cycloclastes (Liu et al., 1986). These proteins participate in a denitrification pathway, and probably reduce NO; to NO (Kashem et al., 1987; Zumft et al., 1987), but possibly can reduce NO further to N20, requiring a second molecule of NO; (Hulse et al., 1989). There is a range of molecular weights for these proteins, and they are generally multimeric. T h e nature of the copper in these proteins is not totally clear. Dooley et al. (1988) reported that the Achromobacter protein may have two kinds of type I sites in a total of three copper sites per dimeric protein, while the A . faecalis protein was reported to be a tetrameric protein with both type I and type I1 coppers (Kakutani et al., 1981). Interestingly, the Achromobacter protein is green. Both of these nitrite reductases accept electrons from a cupredoxin. It is currently believed that NO reacts with the type I copper. Hulse et al. (1989) showed that Cu+(NO)+is an intermediate in the enzyme reaction. Suzuki et al. (1989) examined the reaction of NO (unfortunately, not reporting the relative concentrations of materials used) with the Alcaligenes and Achromobacter NIR and found that the type I EPR signal vanishes on reaction with NO and is restored when NO is removed. At

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the same time the visible absorption is substantially reduced. These results were interpreted as formation of a specific complex of NO with the type I copper. This result may need to be reinterpreted in light of structural results described below. Each of these two nitrite reductases has been crystallized. Petratos et al. (1986) reported obtaining monoclinic crystals of the Alcaligenes NIR, with one tetramer in the asymmetric unit. Turley et al. (1988) reported that the Achromobacter NIR crystallizes in a cubic unit cell (P213),with a monomer in the asymmetric unit. It was recognized at this time that the monomers could not be related by a twofold axis, and, hence, the protein could not be a dimer, as reported. It also crystallizes in an orthorhombic form with sufficient volume for either two or three monomers in the asymmetric unit. T h e 2.3 8, structure of the achromobacter NIR (Godden et al., 1991) has been determined from an MIR map based on native and two derivative data sets collected on an area detector. In the crystal the tightly packed arrangement of monomers around the crystallographic threefold axis suggests that the molecule must be a trimer, rather than a dimer, as originally determined from gel filtration studies. The chain tracing substantiates this. T h e protein folds into two domains, each of which possesses the Greek key /3-sheet folding topology (see Figs. 14 and 15). The amino-terminal domain binds a type I copper, while the carboxy-terminal domain does not bind a type I copper. A second metal site is found between domain 1 of one molecule in the trimer and domain 2 of a second molecule, so that, in all, six metals are bound by the trimer. T h e second metal is bound by two histidines from domain 1 and one histidine from domain 2. Density for a fourth ligand suggests a hydroxyl or water as that ligand. The location of the second metal site is quite interesting with respect to the type I copper: Adjacent residues in an extended conformation bind the two metals, so that they are 12.5 8, apart. This is highly reminiscent of the ascorbate oxidase structure, and, in fact, it is possible to take domain 1 of nitrite reductase and superpose it on domain 3 of ascorbate oxidase, and find that the second metal of NIR superposes on the trinuclear cluster in ascorbate oxidase. Moreover, the relationship of these two domains, although from neighboring molecules, is the same as the threedimensional relationship of domains 1 and 3 of ascorbate oxidase, within a molecule; namely, they are related by a local pseudo-twofold. Sequence similarities are being investigated to determine whether one can establish evolutionary relatedness. Figure 16 illustrates the similarity of the copper sites in nitrite reductase to those in ascorbate oxidase (see Fig. 13). The trimeric arrangement in NIR, and the putative homology with

-

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187

a

I

C-

FIG. 14. (a) Ribbon drawing of nitrite reductase domain 1 . (band c) Schematic of nitrite reductase domain 1 topology.

ascorbate oxidase and thus with ceruloplasmin, lead us to suggest a possible arrangement of the ceruloplasmin domains as shown in Figs. 16 and 17 (Fenderson et al., 1991). This arrangement is consistent with Messerschmidt and Huber’s ( 1990) suggestion that the trinuclear copper cluster is bound between domains 1 and 6 of ceruloplasmin.

D . Nitrous Oxide Reductme and Cytochrome Oxidase Nitrous oxide (N20) reductases carry out the terminal reduction step in the denitrification pathway. One has been isolated from Pseudomonas

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a

b C

C-

FIG.15. (a) Ribbon drawing of nitrite reductase domain 2 backbone. (band c) Schematic of nitrite reductase domain 2 topology.

stutzerei (Coyle et al., 1985) [formerly Pseudomonas perfectomaria (Riester et al., 1989)]and from three other organisms as well (see references cited by Riester et al., 1989). The protein is a dimer with a total molecular weight of 148K and approximately eight copper atoms per dimer, and is somewhat unstable. A pink form is isolated when purified aerobically; a purple form, when purified anaerobically. When reduced with dithionite in the absence of oxygen, a blue form is produced from either the pink or purple form. Protein which is reconstituted with stoichiometricamounts of copper from apoprotein is pink and is inactive, although the as-

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FIG.16. Cu-I and Cu-I1 sites of nitrite reductase.

FIG. 17. (a) Schematic of the trimeric arrangement of NIR. (b) Hypothetical domain arrangement in ceruloplasmin. L, Lysine.

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isolated pink form has about 50% of the activity of the purple form. T h e EPR signal from the copper is like that from an unusual type I copper, although the visible spectrum is not. T h e blue species does not have the intensity characteristic of type I sites either. Extensive analysis of the EPR and redox behavior of this unusual copper protein led to the hypothesis that the protein might contain a Cu(A) site similar to that in cytochrome oxidase (Riester et d.,1989) and that the unusual seven-line EPR is due to the Cu(A)-type site. An alternative interpretation of this EPR is based on electron spin-echo spectroscopy as well, and that is that the seven-line EPR is due to a half-met Cu-Cu pair and to unusual type I sites (Jin et al., 1989). Three sets of spin-echo peaks can be attributed to nitrogens on imidazole ligands to a CuA-type site and to another imidazole on the half-met site. The electron spin-echo spectra of cytochrome oxidase are similar, although there is not enough copper in cytochrome oxidase for a half-met site. Conceivably, the property of delocalization of the paramagnetic electron could be effected by the proposed bridging between CUBand heme a3 (nomenclature summarized by Capaldi, 1990),which are proposed to be 3-4 8, apart. Subsequent EXAFS and magnetic circular dichroism studies support the similarity of a CUA site in N 2 0 reductase and cytochrome oxidase (Scott et al., 1989). Two different Cu-S (or -CI) distances and two Cu-N (or -0) interactions at the same distance fit the EXAFS data. However, the numerology of just how many such sites there are is confusing. T h e EXAFS requires a minimum of eight sulfur distances per eight coppers, although the proposed CUAsite geometry would require two sulfurs per copper, hence leaving four coppers with no sulfur coordination. The reported sequence (Viebrock and Zumft, 1988) shows sequence similarity to cytochrome oxidase in only one site per monomer, yielding a maximum of two CUAsites per dimer and six coppers with other than CuA geometry. T h e N 2 0 reductase sequence shows some similarity, at least in the vicinity of the CUAsite, to the nitrite reductase sequence (Fenderson et al., 1991), so that it is likely that at least one domain of this protein will resemble a Greek key fold. Clearly, there will be more to come on this fascinating enzyme.

AND CONCLUSIONS VI. SUMMARY

The structural comparison of copper-containing proteins has provided a new dimension to the relationships suggested by sequence simi-

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larities. Ryden (1988) summarized the putative relationships, suggesting that a primordial single-domain cupredoxin evolved into the multidomain copper oxidases. The structures have revealed the fact that the differences reside primarily in insertions and deletions at junctions between secondary-structure elements. The mechanism of evolution (e.g., integration of new sequences into regions not essential to the Greek key fold) remains unknown. Which of the properties of a cupredoxin fold are necessary for function is the subject of site-directed mutagenesis studies. Can two of the ligands be interchanged (e.g., the upstream histidine and the methionine), effectively changing the orientation of the electron-rich orbitals important in electron transfer and still have the protein function? Can the hydrogen-bonding pair be modified? This latter point has been partially answered by the multidomain copper oxidase structure. The Tyr-Cys-Thr sequence in plastocyanin (in which threonine is a member of the hydrogen-bonding pair) is homologous with the His-Cys-His sequence in ascorbate oxidase. In the latter electron transfer is believed to flow from the type I copper (bound by the cysteine) to the trinuclear cluster, probably via these histidine residues. Hence, one might infer that the tyrosine and threonine have some role in electron transfer. Tyr-83 has been previously implicated in NMR studies as a primary site of electron transfer. The multi-copper protein structures have revealed interesting new features. T h e extra coppers are bound at domain interfaces, and can be single metals or the novel trinuclear cluster, depending on the availability of liganding histidines. A structural model of ceruloplasmin suggests that it will have at least two type I sites and, possibly, a third type I site such as stellacyanin (no methionine ligand), as well as a binding site for a trinuclear cluster. The similarity of the sequences of N 2 0 reductases and a domain of cytochrome oxidase to the sequences of proteins with known structures suggests that these, too, will have Greek key domains. Galactose oxidase and hemocyanin do not have Greek key folds in their functional domains, although each does have a Greek key domain. The need for a Greek key fold remains obscure. The apoproteins are clearly stable without metals; there are examples other than immunoglobulins of Greek key folds. So far copper seems to be found in a very limited subset of structures; other chapters in this volume show that zinc, for example, has a much wider variety of environments in proteins, as does iron. It may be that the copper-containing Greek key proteins represent a very small evolutionary niche. Structures of other copper proteins will undoubtedly reveal new surprises and help to clarify the essential role of copper in biological systems.

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ACKNOWLEDGMENTS I am grateful to Albrecht Messerschmidt, Wim Hol, and Nobutoshi Ito for providing coordinates before they were deposited. I am especially indebted to Jeff Godden’s persevering work on the nitrite reductase chain tracing. Discussions with Joann Sanders-Loehr on the manuscript were most helpful. Support from National Institutes of Health grant GM 3 1770 is also gratefully acknowledged.

REFERENCES Adman, E. T. (1985). In “Topics in Molecular and Structural Biology: Metalloproteins” (P. Harrison, ed.), Vol. I, pp. 1-42. Macmillan, New York. Adman, E. T., Stenkamp, R. E., Sieker, L. C., and Jensen, L. H. (1978).J. Mol. Biol. 123, 35-47. Adman, E. T., Turley, S., Bramson, R., Petratos, K., Banner, D., Tsernoglou, D., Beppu, T., and Watanabe, H. (1989).J. Biol. Chem. 264, 87-99. Ainscough, E. W., Bingham, A. G., Brodie, A. M., Ellis, W. R., Gray, H. B., Loehr, T. M., Plowman, J. E., Norris, G. E., and Baker, E. N. (1987). Biochemistry 26, 71-82. Allendorf, M. D., Spira, D. J., and Solomon, E. I. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 3063-3067. Almassey, R. J., and Dickerson, R. E. (1978). Proc. Natl. Acad. Sci. U.S.A. 75,2674-2678. Ambler, R. P., and Tobari, J . (1985). Biochem. J . 232,451-457. Arvidsson, R. H. A., Nordling, M., and Lundberg, L. G. (1989). Eur. J. Biochem. 179, 195-200. Baker, E. N. (1988).J. Mol. Biol. 203, 1071-1095. Bannister, J. W., and Parker, M. W. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 149-152. Briving, C., Gandvik, E.-K., and Nyman, P.-0. (1980). Biochem. Biophys. Res. Commun. 93, 454-461. Buisson, G., Duee, E., Gouet, P., Lamy, J.-N., and Lamy, J. (1990).Invertebr. Oxygen Carriers 5,183-184. Calabrese, L., and Carbonaro, M. (1986). Biochem.J. 238, 291-295. Calabrese. L., Mateescu, M. A., Carbonaro, M., and Mondovi, B. (1988a). Biochemistry Int. 16,199-208. Calabrese, L., Carbonaro, M., and Musci, G. (1988b).J. Biol. Chem. 263,6480-6483. Calabrese, L., Carbonaro, M., and Musci, G. (1989).J. B i d . Chem. 264,6183-6187. Canters, G. W. (1987). FEBS Lett. 212, 168-172 Capaldi, R. A. (1990). Annu. Rev. Biochem. 59, 569-596. Church, W. B., Cuss, J. M., Potter, J. J., and Freeman, H. C. (1986).J. Biol. Chem. 261, 234-237. Collyer, C. A., Cuss, J . M., Sugimura, Y., Yoshizaki, F., and Freeman, H. C. (199O).J. Mol. Biol. 211,617-632. Colman, P. M., Freeman, H. C., Cuss, J. M., Murata, M., Norris, V. A., Ramshaw,J. A. M., Venkatappa, M. P., and Vickery, L. E. (1977).J. Mol. Biol. 112, 649-650. Colman, P. M., Freeman, H. C., Cuss, J . M., Murata, M., Norris, V. A., Ramshaw,J. A. M., and Venkatappa, M. P. (1978). Nature (London) 272,319-324. Coyle, C. L., Zumft, W. G., Kroneck, D. M. H., Korner, H., and Jakob, W. (1985). Eur. J . Biochem. 153, 459-467.

COPPER PROTEIN STRUCTURES

193

Cuff, M. E., and Hendrickson, W. A. (1990).Abstr. Annu. Am. Crystallogr. Assoc. Meet. p. 70. Dameron, C. T., and Harris, E. D. (1987).Biochem. J. 248,669-675. Dooley, D. M., Moog, R. S., Liu, M.-Y., Payne, W. J., and LeGall,J. (1988).J.Biol. Chem. 263, 14625-14628. Dorfman, J. R., Bereman, R. D., and Whangbo, W.-H. (1983).I n “Copper Coordination Chemistry: Biochemical and Inorganic Perspectives” (K. D., Karlin and J. Zubieta, eds.), pp. 75-96. Adenine, Guilderland, New York. Engeseth, H. R., and McMillin, D. R. (1986). Biochemistry 25,2448-2455. Fee, J. A. (1975). Struct. Bonding 23, 1-60. Fee, J. A. (1982). Trends Biochem. Sci. 7,84-86. Fenderson, F. F., Kumar, S., Adman, E. T., Liu, M.-Y., Payne, W. J., and LeGall,J. (1991). Biochaistry 30,7 180-7 185. Fielden, E. M., and Rotilio, G. (1984). In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 11, pp. 27-61. CRC Press, Boca Raton, Florida. Fields, B. A., Cuss,J. M., Freeman, H. C., Bartsch, H. H., Cordes, F., and Bartunik, H. D. (1990). Abstr. Congr. Int. Union Crystallogr., 16th p. C-83. Fukuyama, K., Nagahara, Y., Tsukihara, T., Katsube, Y., Hase, T., and Matsubara, H. (1988).J. Mol. Biol. 199, 183-199. Furst, P., and Hamer, D. (1989).Proc. Natl. Acud. Sci. U.S.A. 86,5267-5271. [Cited inJ . NIH Res. 1,66-68 (1989).] Furst, P., Hu, S., Hackert, R., and Hamer, D. (1988). Cell 55,705-717. Garrett, T. P. J., Clingeleffer, D. J., Cuss, J. M., Rogers, S. J., and Freeman, H. C. (1984). J . Biol. Chem. 259,2822-2825. Getzoff, E. D., Tainer, J. A., Weiner, P. K., Kollrnan, P. A., Richardson, J. S., and Richardson, D. C. (1983). Nature (London) 306,287-290. Getzoff, E. D., Tainer, J. A., Stempien, M. A., Bell, G. I., and Hallewell, R. A. (1989). Proteins 5, 322-336. Godden, J. W., Turley, S., Teller, D. C., Adman, E. T., Liu, M.-Y., Payne, W. J., and LeGall, J. (1991).Science, in press. Groeneveld, C. M., andcanters, G. W. (1988).J.Biol. Chem. 263, 167-173. Groeneveld, C. M., Feiters, M. C., Hasnain, S. S., van Rijn, J., Reedijk, J., and Canters, G . W. (1986). Biochim. Biophys. Acia 873,214-227. Groeneveld, C. M., Oewerling, M. C., Erkelens, C., and Canters, G. W. (1988).J.Mol. Biol. 200, 189- 199. Cuss, J. M., and Freeman, H. C. (1983).J. Mol. Biol. 169,521-563. Cuss, J. M., Harrowell, P. R., Murata, M., Norris, V. A., and Freeman, H. C. (1986).J.Mol. Biol., 192, 361-387. Cuss, J. M., Merritt, E. A., Phizackerley, R. P., Hedman, B., Murata, M., Hodgson, K. O., and Freeman, H. C. (1988).Science 241,806-81 1. Halaban, R., Moellmann, G., Tamura, A., Kwon, B. S., Kuklinska, E., Pomerantz, S. H., and Lerner, A. B. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 7241-7245. Han, J., Loehr, T. M., Freeman, H. C., Codd, R., Hug, L., Adman, E. T., Beppu, T., and Sanders-Loehr, J. (1991). Submitted. Hasnain, S. S., and Garner, C. D. (1987). Prog. Biophys. Mol. Biol. 50,47-65. Herskovits, T. (1988). Comp. Biochem. Physiol. B 91B, 597-611. Hilewicz-Grabska, M., Zgirski, A., Krajewski, T., and Plonka, A. (1988). Arch. Biochem. Biophys. 260, 18-27. Huber, R. (1990). Eur. J. Biochem. 187,283-305. Huber, M., and Lerch, K. (1988).Biochemistry 27,5610-5615.

194

ELINOR T. ADMAN

Hulse, C. L., Averill, B. A., and Tiedje, J. M. (1989).J.Am. Chem. Soc. 111,2322-2323. Hunt, L. T., George, D. G., and Yeh, L. S. (1985).J.Mol. E d . 21, 126-132. Husain, M., and Davidson, V. L. (1986). Biochemistry 25,2431-2436. Ito, N., Keen, J. N., Knowles, P. F. McPherson, M. J., Stevens, C., Phillips, S. E. V., and Yadav, K. D. S. (1990). Abstr. Congr. Int. Union Crystallogr., 16th p. C-89. Ito, N., Phillips, S. E. V., Stevens, C., Oqel, Z. B., McPherson, M. J., Keen, J. N., Yadov, K. D. S., and Knowles, P. F. (1991). Nature (London) 350,87-90. Jin, H., Thomann, H., Coyle, C. L., and Zumft, W. G. (1989). J . Am. Chem. Soc. 111, 4262-4269. Kakutani, T., Watanabe, H., Arima, K., and Beppu, T . (1981).J . Biochem. (Tokyo) 89, 463-472. Karlin, K. D., and Zubieta, J., eds. (1983).“Copper Coordination Chemistry: Biochemical and Inorganic Perspectives.” Adenine, Guilderland, New York. Karlin, K. D., and Zubieta, J. (1986). “Biologicaland Inorganic Copper Chemistry,” Vols. I and 11. Adenine, Guilderland, New York. Karlsson, B. G., Aasa, R., Malmstrom, B. G., and Lundberg, L. G. (1989).FEBS Lett. 253, 99-101. Kashem, M. A., Dunford, H. B., Liu, M.-Y., Payne, W. J., and LeGall, J. (1987). Biochem. Biophys. Res. Commun. 145, 563-568. Kitajima, N., Fujisawa, K., and Moro-oka, Y. (1989).J.Am. Chem. Soc. 111,8975-8976. Knowles, P. F., and Yadav, K. D. S . (1984).In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 11, pp. 103-130. CRC Press, Boca Raton, Florida. Kosman, D. J. (1984) In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 11, pp. 1-26. CRC Press, Boca Raton, Florida. Kwon, B. S., Kaq, A. K., Pomerantz, S. H., and Halaban, R. (1987).Proc. Natl. Acad. Sci. U.S.A. 84,7473-7477. Lamy, J., Lamy, J., Sizaret, P. Y., Billiald, P., Jollts, P., Joll&s,J., Feldmann, R. J., and Bonaventura, J. (1983).Biochemistly 22,5573-5583. Lerch, K. (1988). Prog. Clin. Biol. Res. 256, 85-98. Lewin, R. (1985). Science 227, 1020. Lim, L. W. G., Mathews, F. S., Husain, M., and Davidson, V. L. (1986).J.Mol. B i d . 189, 257-258. Liu, M.-Y., Liu, M.-C., Payne, W. J., and LeGall, J. (1986).J.Bacteriol. 166,604-608. Ljones, T., and Skotland, T. (1984). In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 11, pp. 131-158. CRC Press, Lontie, R., ed. (1984. “Copper Proteins and Copper Enzymes,” Vols. 1-111. CRC Press, Boca Raton, Florida. Malkin, R., and Malmstrom, B. G. (1970).Adv. Enrymol. 33, 177-244. Martinkus, K., Kennelly, P. J., Rea, T., and Timkovitch, R. (1980).Arch. Biochem. Biophys. 199,465-472. Masuko, M., Iwasaki, H., Sakurai, T., Suzuki, S., and Nakahara, A. (1984). J . Biochem. (Tokyo) 96,447-454. Messerschmidt, A., and Huber, F. (1990).Eur.J. Bzochem. 187,341-352. Messerschmidt, A., Rossi, A., Ladenstein, R., Huber, R., Bolognesi, M., Gatti, G., Marchesini, A., Petruzzeli, R., and Finazzi-Agro, A. (1989).J. Mol. Biol. 206, 513-529. Mondovi, B., and Avigliano, L. (1984). In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 111, pp. 101-118. CRC Press, Boca Raton, Florida. Moore, J. M., Case, D. A., Chazin, W. J., Gippert, G. P., Havel, T. F., Powls, R., and Wright, P. E. (1988).Science 240,314-317.

COPPER PROTEIN STRUCTURES

195

Moshkov, K. A., Shavlovski, M. M., Saitsev, V. N., and Neifakh, S. A. (1977). Int. J . P e p . Protein Res. 9, 187-192. Munro, I., Pecht, I., and Stryer, L. (1979). Proc. Natl. Acad. Sci. U.S.A. 76, 56-60. Nar, H. Messerschmidt, A., Huber, R., van de Kamp, M., and Canters, G. W. (1991).J.Mol. Biol. 218,427-447. Ohkawa, J., Okada, N., Shinmyo, A., and Takano, M. (1989).Proc. Natl. Acad. Sci. U.S.A. 86, 1239-1243. Orena, S. J., Goode, C. A., and Linder, M. C. (1986). Biochem. Bioflhys. Res. Commun. 139, 822-829. Pascher, T., Bergstrom, J., Malmstrom, B. G., Vannglrd, T., and Lundberg, L. G. (1989). FEBS Lett. 258,266-268. Penfield, K. W., Gay, R. R., Himmelwright, R. S., Eickman. N. C., Norris, V. A., Freeman, H. C., and Solomon, E. I. (1981).J.Am. Chem. Soc. 103,4382-4388. Penfield, K. W., Gewirth, A. A., and Solomon, E. I. (1985).J. Am. Chern. Soc. 107,45194529. Petratos, K., Beppu, T., Banner, D. W., and Tsernoglous, D. (1986).J.Mol. Biol. 190, 135. Petratos, K., Banner, D. W., Beppu, T., Wilson, K. S., and Tsernoglou, D. (1987).FEBS Lett. 218,209-214. Petratos, K., Dauter, Z., and Wilson, K. S. (1988a).Acta Crystallop-. Sect. B 44,628-636. Petratos, K., Dauter, Z., Wilson, K. S., Lommen, A., von Beemen, J., and Canters, G. W. (1988b).J . Mol. Biol. 199, 545-546. Petrich, J. W., Longworth, J. W., and Fleming, G. R. (1987).Biochemistry 26,2711-2722. Pitts, 0. M., Priest, D. G., and Fish, W. W. (1974).Biochemistry 13,888-892. Preaux, G., and Gielens, C. (1984). In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 11, pp. 159-206. CRC Press, Boca Raton, Florida. Reinhammer, B. (1984). I n “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 111, pp. 1-36. CRC Press, Boca Raton, Florida. Richardson, J. S., Richardson, D. C., Thomas, K. A., Silverton, E. W., and Davies, D. R. (1976).J.Mol. B i d . 102, 221-235. Riester, J., Zumft, W. G., and Kroneck, P. M. H. (1989).Eur.J. Biochem. 178, 751-762. Robb, D. A. (1984).In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 11, pp. 207-240. CRC Press, Boca Raton, Florida. Royer, W. E., Hendrickson, W. A., Cuff, M. E., Miller, K. I., and van Holde, K. E. (1988). Abstr. Annu. Am. Crystallop-. Assoc. Meet. p. 104. Ruppert, S., Muller, G., Kwon, B., and Schutz, G. (1988). EMBO J . 7,2715-2722. Ryden, L. (1982).Proc. Natl. Acad. Sci. U.S.A. 79,6767-6771. Ryden, L. (1984). In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 111, pp. 37-100. CRC Press, Boca Raton, Florida. Ryden, L. (1988). Prog. Clin. B i d . Res. 274,349-366. Salvato, B., Giocometti, G. M., Beltramini, M., Zilio, F., Giocometti, G., Magliozzo, R. S., and Peisach, J. (1989). Biochemistry 28,680-684. Scott, R. A., Zumft, W. G., Coyle, C. L., and Dooley, D. M. (1989).Proc. Natl. Acad. Sci. U.S.A. 86,4082-4086. Sharma, K. D., Loehr, T. M., Sanders-Loehr, J., Husain, M., and Davidson, V. L. (1988). J . Biol. Chern. 263,3303-3306. Sharp, K., Fine, R., and Honig, B. (1987). Science 236, 1460-1463. Shepard, W., Anderson, B. F., Baker, E. N., and Norris, G. E. (1990).Abstr. Congr. Int. Union Crystallop-., 16th p. C-85. Solomon, E. I. (1988). Prog. Clin. Biol. Res. 274, 309-330.

196

ELINOR T. ADMAN

Solomon, E. I., Hare, J. W., and Gray, H. B. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 1389-1393. Solomon, E. I., Hare, J. W., Dooley, D. M., Dawson, J. H., Stephens, P. J., and Gray, H. B. (1980).J.Am. Chem. SOL. 102, 168-178. Solomon, E. I., Penfield, K. W., and Wilcox, D. E. (1983). Struct. Bonding53, 1-57. Spira-Solomon, D. J., Allendorf, M. D., and Solomon, E. I. (1986).J. Am. Chem. SOC. 108, 53 18-5328. Srai, S. K., Burroughs, A. K., Word, B., and Epstein, 0. (1986). Hepatology 6,427-432. Steffens, G. J . , Michelson, A. M., Otting, F., Puget, K., Strassburger, W., and Flohe, L. (1986). Biol. Chem. Hoppe-Seyler 367, 1007-1016. Steinman, H. M. (1982).J. Biol. Chem. 257, 10283-10293. Surewicz, W. K., Szabo, A. G., and Mantsch, H. H. (1987).Eur.J. Biochem. 167,519-523. Suzuki, S., Yoshimura, T., Kohzuma, T., Shidara, S., Masuko, M., Sakurei, T., and Iwasaki, H. (1989). Biochem. Biophys. Res. Commun. 164, 1366-1372. Tahon, J. P., Van Hoof, D., Vinckier, C., Witters, R. B., d e Ley, M., and Lontie, R. (1988). Bi0chem.J. 249,891-896. Tainer, J. A., Getzoff, E. D., Beern, K. M., Richardson, J. S., and Richardson, D. C. (1982). J. Mol. Bi01. 160, 181-217. Tainer, J. A., Getzoff, E. D., Richardson, J. S., and Richardson, D. C. (1983).Nature (London) 306,284-287. Takahashi, N., Bauman, R. A., Ortel, T . L., Dwulet, F. E., Wang, C.-C., and Putnam, F. W. (1983).Proc.Natl. Acad. Sci. U.S.A. 80, 115-119. Thomann, H., Bernardo, M., Baldwin, M., Lowry, M., and Solomon, E. I. (1991).J. Am. Chem. SOC.113,5911-5913. Tobari, J., and Harada, Y. (1981). Biochem. Biophys. Res. Commun. 101,502-508. Thomas, E., and Toparceanu, F. (1986). Virologie 37,279-287. Trost, J. T., McManus, J. D., Freeman, J. C., Ramakrishna, B. L., and Blankenship, R. E. (1988). Biochemistry 27, 7858-7863. Turley, S., Adman, E. T., Sieker, L. C., Liu, M.-Y., Payne, W. J., and LeGall, J. (1988). J. Mol. Biol. 200,4 17-4 19. Underwood, E. J. (1977). “Trace Elements in Human and Animal Nutrition,” 14th Ed. Academic Press, New York. [Quoted in Ettinger, M. J. (1984). In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 111, pp. 175-229. CRC Press, Boca Raton, Florida.] Van der Meer, R. A., Jongejan, J. A., and Duine, J. A. (1989).J.Biol. Chem. 264,7792-7794. van Houweligen, T., Canters, G. W., Stobbelaar, G., Duine, J. A., Frank, Jzn, J., and Tsugita, A. (1989). Eur.J. Biochem. 153, 75-88. Vehar, G. A., Keyt, B., Eaton, D., Rodriguez, H., O’Brian, D. P., Rotblat, F., Oppermann, H., Keck, R., Wood, W. I., Harkins, R. N., Tuddenham, E. G. D., Lawn, R. M., and Capon, D. J. (1984). Nature (London) 312,337-342. Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, Jzn, J., Jongejan, J. A,, Duine, J. A., Petratos, K., Drenth, J., and Hol, W. G. J. (1989).EMBOJ. 8,2171-2178. Viebrock, A., and Zumft, W. G. (1988).J. Bacteriol. 170,4658-4668. Volbeda, A., and Hol, W. G. J. (1988). Proc. Clin. Biol. Res. 274,291-307. Volbeda, A,, and Hol, W. G. J. (1989a). J.Mol. Bwl. 206,531-546, Volbeda, A., and Hol, W. G. J. (1989b).J. Mol. Biol. 209,249-279. Volbeda, A., Feiters, M. C., Vincent, M. G., Bouwman, E., Dobson, B., Kalk, K. H., Reedijk, J., and Hol, W. C .J. (1989). Eur.J. Biochem. 181,669-673. Weser, U., and Hartmann, H. J. (1984). In “Copper Proteins and Copper Enzymes” (R. Lontie, ed.), Vol. 111, pp. 151-173. CRC Press, Boca Raton, Florida.

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Whittaker, M. M., DeVito, V. L., Asher, S. A., and Whittaker,J. W. (1989).J. Biol. Chem. 264,7104-7106. Woolery, G. L., Powers, L., Winkler, M., Solomon, E. I., and Spiro, T. G. (1984).J . Am. Chem. SOC. 106,86-92. Yarnarnoto, K., Uozuni, T., and Beppu, T. (1987).J. Bucteriol. 169,5648-5652. Zaitsev, V. N., Naumov, A. S., and Moshkov, K. A. (1980). Sou. Phys. Crystullogr. 25, 100- 102. Zumft, W. G., Gotzrnann, D. J., and Kroneck, P. M. H. (1987). Eur. J . Biochem. 168, 301-307.

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PERSPECTIVES ON NON-HEME IRON PROTEIN CHEMISTRY

.

.

By JAMES B HOWARD* and DOUGLAS C REESt Department of Biochemistry. University 01 Minnesota School 01 Medicine. Minneapolis. Minnesota 55455 Division of Chemistry and Chemical Engineering. California institute of Technology. Pasadena. California 91125

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Background and Scope of Review . . . . . . . . . . . . . . . . B . Structures of Non-heme Iron Centers . . . . . . . . . . . . . . C . Magnetic and Redox Properties of Iron Centers . . . . . . . . . . D . Structural Overview of Non-heme Iron Proteins . . . . . . . . . . E . Problems in Identification and Characterization of Iron Center . . . . I1. Structuresand Functionsof Mononuclear Iron Proteins . . . . . . . . A. Oxygen-Utilizing Enzymes . . . . . . . . . . . . . . . . . . . . B . Bacterial Photosynthetic Reaction Center . . . . . . . . . . . . . C. Transferrins . . . . . . . . . . . . . . . . . . . . . . . . . I11. Structures and Mechanisms of Binuclear Octahedral Iron Proteins . . . . A . Hemerythrin . . . . . . . . . . . . . . . . . . . . . . . . . B. Ribonucleotide Reductase . . . . . . . . . . . . . . . . . . . . C. Methane Monooxygenase . . . . . . . . . . . . . . . . . . . . D . Oxygen Activation by Binuclear Octahedral Iron Centers . . . . . . IV . Tetrahedral Iron: Fe : S Proteins . . . . . . . . . . . . . . . . . . A . Ferredoxins . . . . . . . . . . . . . . . . . . . . . . . . . B . Nitrogenase . . . . . . . . . . . . . . . . . . . . . . . . . C. Aconitase: A Nonredox Fe : S-Containing Enzyme . . . . . . . . . D . Fe :S Proteins with Other Prosthetic Groups . . . . . . . . . . . E . Fe : S Proteins with Non-thiolate Ligands: Rieske-Type Fe :S Proteins . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

. . . . . . . .

. .

. . .

199 199 201 206 209 218 227 230 235 237 239 240 244 248 249 251 251 256 262 265 269 271 272

I . INTRODUCTION A . Backpound and Scope of Review To the broader biochemical community the term “non-heme iron proteins” has frequently suggested a limited group of low-molecularweight proteins confined to electron transfer between enzymes in a limited number of reactions. such as nitrogen fixation and photosynthesis.’

’

The usage of “iron” and “sulfur” in this chapter is generic and implies iron atoms and sulfur atoms . “Fe : S” implies a cluster composed of iron and sulfur. Our usage is unique; “Fe protein” is the name of a specific enzyme. a component of the nitrogenase complex (see Section 1V.B). “p-0x0” refers to an oxygen atom bridge between metals where the oxygen is formally Oz- . ADVANCES IN PROTEIN CHEMISTRY. Vol. 42

199

Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

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JAMES B. HOWARD AND DOUGLAS C. REES

This view is a relic from 25 years ago, when >95% of the known ironcontaining proteins had heme as a prosthetic group. This humble beginning did not portend the explosive discovery of enzymes and proteins with non-heme iron centers. We now know that non-heme iron proteins are ubiquitous to all living cells. Furthermore, the active-site iron participates in reactions as diverse as electron transfer, substrate oxidationreduction, nonredox group transfer, oxygen insertion, and regulation of enzymatic activity. These proteins are essential enzymes in biological processes from the Krebs cycle to nucleotide synthesis to the transfer and processing of inorganic nutrients. Although heme and non-heme iron structures are significantly different, there are some similarities in biochemical function. There are examples from both categories of proteins that serve as electron transfer agents, 0 2 carriers, oxidases, or oxygenases. In other cases non-heme iron proteins have no counterpart in the heme protein category; for example, aconitase, nitrogenase, and hydrogenase are found only as non-heme iron enzymes. Because the non-heme iron prosthetic groups have simpler structures and in many cases can form spontaneously, it is likely that enzymes with non-heme iron appeared earlier in the development of primordial life forms. Nevertheless, both types of iron centers have persisted even when serving chemically similar functions. In addition to their varied biological roles, non-heme iron proteins contain a magnificent assortment of iron sites having a multitude of chemical and structural properties. Indeed, the catalog of iron centers is a bit like the taxonomy of insects-a seemingly limitless variation of a few structural themes, yet each new form sufficiently different to define a new species. It is beyond the scope of any review of non-heme iron proteins to be inclusive, and there are excellent recent reviews which detail selected topics. Rather, it is our intention to provide in one chapter an overview of the major classes with an emphasis on proteins for which a crystal structure is available. This review begins with a survey of the types of protein iron structures and a discussion of some methods and problems associated with establishing the iron center type. This should provide an introduction to readers less familiar with the area. Sections I1 to IV include the current status and recent developments for a limited number of proteins from the major iron classes. These have been chosen in the subjective vein of a limited review; the omission of a topic does not indicate its relative importance or interest, only the limitation of space. The purpose of this section is to emphasize the diversity of iron center structures and functions.

NON-HEME IRON PROTEIN CHEMISTRY

Mononuclear Iron

1Fe

20 1

Binuclear Iron

2Fe:2S 3Fe:4S

4Fe:4S

Tetrahedral Iron FIG. 1. Iron center types found in non-heme iron proteins.

B . Structures of Non-heme Iron Centers The six established categories of non-heme iron centers are shown in Fig. 1. Protein structures and model compounds representing all six types have been determined by X-ray diffraction crystallography.' A summary of the currently available protein crystallographic structures of non-heme iron centers and ligands is provided in Table I. As discussed in more detail later, these structures have been adapted to perform diverse functional roles. Our objective is to understand how the protein structure modifies the iron center such that the unique activities are expressed. Although a number of iron-containing enzyme systems have been identified, unfortunately an insufficient number have been studied in adequate detail such that a comprehensive theory can be developed. However, there are some patterns that can be inferred, and these may be useful in considering new iron proteins or enzymes. Non-heme iron is defined as iron coordinated by ligands other than Because the number of protein three-dimensional structures completed is relatively small, much of our understanding of the metal center structures comes from studies on inorganic model compounds. Although it is beyond the scope of this chapter to discuss these compounds separately, we have integrated some of the results in our discussion of the relevant protein structures.

TABLE I Iron Ligands in Non-heme Iron Proteins A. Mononuclear octahedral iron centersD Protein Fe-superoxide dismutase

Lactoferrin Amino-terminal lobe Carboxy-terminal lobe

N 0 N

3,4-Protocatechuate dioxygenase Photosynthetic reaction center Rhodopseudamonas vindis

Rhodobacter sphueroldes

X

Y

2

ASP-156 06

His-73 Nc2

H20

His-253 N E ~ His-597 N E ~ His-BlGO N E ~

Tyr-92

Tyr-PI08

Asp-60 06 Asp-395 06 Tyr-Bl47

His-L19O N E ~ His-L19O N E ~

His-M264 N.52 His-M266 Nc2

His-M2 17 N E ~ His-M2 19 N E ~

Tyr-435

-Y

-X

Reference

His-26 N E ~

His- 160 Nc2

Azide

Stallings et al. (1983), Carlioz et al. ( 1988), Ringe et al. (1983). Stoddard et al. (1990)

Tyr-192

Solvent

Anderson et al. (1987, 1989) Bailey et al. (1988)

-2

Tyr-528

Solvent

Solvent ? Solvent

?

?

His-Pl62 N E ~

H20

His-L230 Nc2 His-L230 N E ~

Glu-M232

Glu-M232

OE 1

0.52

Gly-M234

Glu-M234 082

?

O E1

Ohlendorf et al. (1988)

Deisenhofer et al. (1984) Allen et al. (1987, 1988)

B. Binuclear iron centersD Myohemerythrin-Fe1

X Glu-58

Y Asp-111

Myohemerythrin-Fe2

Asp- 111

Glu-58

Ribonucleotide reductase-Fe 1

His-118 N6 1 His-241 N6 1

Hz0

Z His-73 N E ~ His-25 N ~2 ASP-84

Glu-115

Glu-204

Ribonucleotidereductase-Fe 2

02-

Glu-115

-X His- 106 N E ~ His-54 N ~2 Asp-84

02-

Glu-238

H20

-2 0202-

-Y His-77 N E ~ Azide

Sheriff et al. ( 1987) Stenkamp et al. (1984)

Nordlund et al. (1990)

C. Iron-Sulfur Proteins*

A

Rubredoxin Clostridium prtteurianum Desulphovibrio gigas Desulphovibrio vulgaris 2Fe : 2s Ferredoxin Spirulinu platenssis Apharothece sacrum Halobacterium murisrnortii 3Fe : 4Si4Fe ;4s Ferredoxin Peptococcus aerogenes 4Fe :4s-1 4Fe : 4s-2 Arotobacter vinelundii 4Fe :4.5 3Fe : 4s Bacillus thennoproteolyticw 4Fe :4s Desulphovibrio gagas 3Fe :4 s High-potential iron protein 4Fe : 4 s Aconitase 3Fe : 4s (inactive) 4Fe :4 s (active) Nitrogenase iron protein (A. vinelandii) Trimethylamine dehydrogenase I

-

B

D

C

CYS-6

cys-9

cys-39

cys-42

Watenpaugh et al. (1980) Frey et al. (1987) Adman et al. (1977)

cys-4 1 cys-4 1 CYS-63

CYS-46 CYS-46 CYS-68

cys-49 cys-49 cys-7 1

cys-79 cys-79 cys-102

Fukuyarna et al. (1981) Tsutsui et al. (1983), Tsukihara et al. (1990) Sussman et al. (1989)

CYS-8 cys-35

cys- 11 CYS-38

cys- 14 cys-4 1

cys-45 CYS-18

Adman et al. (1973, 1976)

cys-39 CYS-8

cys-42 CYS-16

cys-45 cys-49

cys-20

-

Stout et al. (1988) Stout (1988, 1989)

cys- 11

cys- 14

cys- 17

CYS-6 1

Fukuyarna et al. (1988, 1989)

CYS-8

cys- 14

cys-50

-

Kissinger et al. (1988, 1989)

cys-43

CYS-46

CYS-63

cys-77

Carter et al. (1974)

CYS-358 CYS-358 Cys-97A

cys-424 cys-424 Cys-132A

cys-42 1 cys-42 1 Cys-97B

-

Robbins and Stout (1989a,b)

CYS132B

Georgiadis et al. (1990)

cys-347

cys-350

cys-353

CYS-366

Lim et al. (1986)

Hz0

Ligand positions about the iron are designated X,Y, Z, -X, -Y, and -2, where X, Y, and 2, define the directions of an idealized right-handed orthogonal coordinate system centered about the iron site. Ligand positions A, B, C, and D are defined with respect to the idealized geometry illustrated in Fig. 4a for rubredoxins and the 3Fe :4s and 4Fe :4.5 proteins. For the 2Fe : 2s proteins ligands A, B, C, and D are assigned in order of increasing residue number of the liganding groups.

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JAMES B. HOWARD AND DOUGLAS C. REES

polydentate tetrapyrroles such as protoporphyrin. Coordination can be either tetrahedral or octahedralltrigonal bipyramidal (although not formally correct, five coordination is frequently considered a subset of six). Protein amino acid side chains, solvent, substrates, and inhibitors are the monodentate ligands. However, as discussed in Section IV,D,2, even this distinction between heme and non-heme iron centers is blurred; recently, a family of enzymes has been found which contain a heme iron structurally and electronically coupled to a 4Fe:4S cluster by a common bridging ligand (Ostrowski et al., 1989). Other references for much of the general discussion of non-heme iron properties can be found in reviews such as those by Lovenberg (1973a,b, 1977), Que and True (1991), Spiro (1982), and Beinert (1990). 1 . OctahedralITrigonal Bipyramidal Iron Centers

T w o types of iron prosthetic groups have been classified by spectroscopic methods as having nominal octahedral coordination. From the crystal structures of mononuclear iron proteins [Table I(A)], the coordination was found to be better described as trigonal bipyramidal in the absence of substrate or inhibitors; during catalysis or in the presence of substrates and inhibitors, the coordination may be expanded to octahedral. Protein histidyl, aspartyl, glutamyl, and tyrosyl residues have been unambiguously identified as ligands. In addition, water, azide, and carbonate have been found in some specific proteins. Typical metal-ligand distances for coordination of an imidazole nitrogen or a carboxylate oxygen to octahedral ferrous iron are about 2.2 and 2.1 A, respectively (Lehnert and Seel, 1978; Carrel1 et al., 1988). In model compounds these distances are sensitive to both oxidation state and coordination number of iron; decreases of 0.05-0.15 A are associated with a decrease in coordination number from octahedral to tetrahedral geometry, or an increase in oxidation state to ferric iron. For the binuclear iron cluster [Table I(B)] the two irons share either a common face o r edge of their respective octahedrons; that is, there are either two or three bridging ligands. To date, all of the characterized protein clusters have a p-0x0 (or the protonated p-0x0, p-hydroxo) and one or two carboxylate bridge^.^ In addition, each iron has one histidyl ligand, with the remaining ligands mainly as carboxylic acids and histidines; water and tyrosines are ligands in selected cases. The dominant

’

For the purposes of this chapter, we limit the discussion to p-0x0 bridged clusters. Model compounds of binuclear octahedral iron without 0x0 bridges have electromagnetic properties different from the related clusters with p-0x0 bridges (Sureruset al. (1989);only the p-0x0 bridged clusters have been found in proteins so far.

NON-HEME IRON PROTEIN CHEMISTRY

205

feature of the cluster is the short 0x0-iron bonds (i.e., 1.7- 1.9A), which, in combination with other bridging ligands, result in an Fe-Fe distance of 3.2-3.3 A and Fe-0-Fe angles of 125"-135". trans to the short 0x0 ligand is the apical o r longest bonded ligand, which is generally the least basic in model compounds. In some cases one of the irons may be pentacoordinate. However, it remains nominally octahedral, rather than trigonal bipyramidal, as found in the mononuclear centers; at this vacant site exogenous ligands bind. 2. Tetrahedral Iron Centers Ligands for tetrahedral iron are principally cysteinyl thiols [Table I(C)]. I n addition the multinuclear tetrahedral iron clusters have inorganic sulfur as bridging ligands. For this reason tetrahedral iron proteins are frequently referred to as iron-sulfur, or Fe : S, proteins. Recently, the Fe :S proteins have been found to have more variety in the terminal ligands; for example, there is either crystallographic or spectroscopic evidence for histidyl, carboxyl, and water ligands (see Section IV for examples). Clusters containing multiple iron atoms can be considered higher-order derivatives of the simplest mononuclear iron center. For example, the binuclear iron cluster can be viewed as the fusion of two mononuclear Fe(SR)4 centers, with two of the four thiolate ligands replaced by bridging inorganic sulfur atoms. T h e tetranuclear iron cluster can be regarded as two fused binuclear iron clusters, and, again, two terminal ligands are replaced with bridging inorganic sulfur atoms. Trinuclear iron clusters appear to be derived from 4Fe : 4s clusters by the removal of one iron and are sufficiently unstable without a protein scaffolding that models of the cubane 3Fe:4S cluster have not yet been successfully prepared. This building of higher-order clusters from smaller units is likely to be the spontaneous synthetic route (Berg and Holm, 1982). Likewise, many of the spectroscopic properties for individual iron atoms in the multinuclear iron clusters can be related to the simplest mononuclear iron protein, rubredoxin (Palmer, 1973). The homogeneous sulfur coordination of the tetrahedral iron is reflected in the remarkable similarity of the bond angles and distances among the iron-sulfur structures, with only small differences originating from either the oxidation state of the cluster o r protein-induced distortion. [See Berg and Holm (1982)for an extensive summary of representative Fe : S models and proteins.] Although the bridging Fe-S bond is longer (-2.3 A) than the binuclear Fe-0 (-1.8 A), the Fe-Fe bond distance (-2.7 A) is considerably shorter than in the octahedral clusters, due to the tetrahedral bond angles (-109") for S-Fe-S. This probably introduces more Fe-Fe bonding than in the octahedral clusters.

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JAMES B. HOWARD A N D DOUGLAS C. REES

C . Magnetic and Redox Properties of Iron Centers Unlike heme iron, all non-heme irons are high spin (i.e., they have the maximum number of unpaired d electrons) in both formal oxidation states (Fez+ or Fe3+).4p5 All classes of iron centers shown in Fig. 1 have been found with enormous variation of the redox potential E6. For example, the -2/-3 redox couple for 2Fe : 2s proteins ranges from 100 to -450 mV. Based on extensive model and protein studies, Eb is controlled by polarity of the pocket (solvent) surrounding the center, hydrogen bonding from the protein (solvent) to both the terminal and bridging ligands of the center, the ligand strength (e.g., the amount of covalency between the ligand and the iron), and basicity of the terminal ligands (Berg and Holm, 1982; Rees and Farrelly, 1990; Que and True, 1991). Of obvious interest to the biochemist is the possibility that the protein or substrate can modulate the ligand field strength and thereby affect the chemistry carried out. The mechanisms by which such changes occur may be equivalent to understanding the origin of the diverse chemical activities exhibited by iron centers. Mononuclear octahedral/ trigonal bipyramidal iron centers are found in either the ferric or the ferrous oxidation state (Whittaker et al., 1984; Arciero et al., 1983).Because the iron may participate directly in catalysis as either a Lewis acid or base, only one state is the active form for a given enzyme. Transient redox changes may occur during turnover, but the enzyme returns to its initial condition. In contrast the tetrahedral mononuclear iron proteins appear to function primarily as electron transfer agents and therefore change oxidation state with a single turnover. T h e irons in binuclear clusters are strongly antiferromagnetically coupled, at least in some oxidation states. In the formalism of the Heisenberg superexchange coupling model, the spin Hamiltonian, X , equals - 2 j ( S & ) , wherej, the coupling constant, is large and negative for antiferromagnetic coupling. In antiferromagnetic coupling the d electrons can be thought of as spin-paired between irons, while in ferromag-

+

This generalization has some qualifications. Recently, nitrile hydratase was reported to be a low-spin Fez+ enzyme. Demonstration of the spin state is incomplete at this time and the low-spin claim for the enzyme may be revised. Also, the binding of NO to high-spin ferrous iron (S = 2) results in a one-electron reduction and intermediate spin (S = 8) ferric iron. Finally, low-spin ferric iron has been observed for cyano 3,4-protochatechuate dioxygenase (Whittaker and Lipscomb, 1984). The oxidation state of a cluster is generally designated by the net charge on the cluster; for example, [2Fe : 2S]'+ is the oxidation state at which formally there is one Fe3+,one Fez+, and two S2-. The oxidation state can also be identified for the cluster with its ligands: for the example above, [2Fe : 2s : 4RS-I5-. The three- and four-iron clusters are designated similarly.

NON-HEME IRON PROTEIN CHEMISTRY

207

netic coupling the electrons are spin-aligned and additive. T h e magnetic and spectral properties of the cluster are a function of the coupling. For example, binuclear clusters in the diferric oxidation state are antiferromagnetically coupled such that the two high-spin irons ( S = # result in an S = 0, electron paramagnetic resonance (EPR)-silent system (Gibson et al., 1966). The addition of one electron converts the cluster to an S = 4, low-spin EPR-active system, although the iron atoms remain high spin (S = 1 and S = 2). When coupled iron atoms are in different oxidation states, yet retain their individual formal charges, the cluster is designated a “trapped” or “mixed” valence state.6 This is a property of all protein and related model binuclear iron clusters and may be an intrinsic property of these systems (cf. multinuclear clusters, discussed below). Three oxidations states are potentially available in a binuclear iron center. Enzymes with octahedral p-0x0 bridged iron clusters can be isolated in each of the three states: the diferric and diferrous states appear to be the functional terminal oxidation states for most of the enzymes, while the mixed valence state may be an important intermediate o r transition state for some reactions (Que and True, 1991). In these enzymes the cluster participates primarily as a two-electron partner in the redox of substrates, perhaps using sequential one-electron steps. Without additional coupled redox steps the enzyme is in a new oxidation state after one turnover. In contrast only the diferric and mixed valence oxidation states have been found for 2Fe : 2s clusters. T h e diferrous state may not be obtainable because of the high negative charge on [2Fe : 2S(4RS)I4- versus - 1 or 0 net charge for the diferrous octahedral (i.e., non-Fe : S) clusters. The 2Fe : 2 s proteins either are one-electron donor/acceptors or serve as transient electron transfer intermediates. The multinuclear tetrahedral iron clusters have the potential for additional formal oxidation states. Because not all of these states have been found in proteins or model compounds, it is possible that some oxidation states may be unstable. For a given Fe : S protein only one redox couple is used; the other possible states appear to be excluded by restrictions of the protein structure. This selection rule is illustrated with two 4Fe :4 s lowmolecular-weight electron transfer proteins : ferredoxin and highpotential iron protein (HiPIP). T h e 4Fe:4S clusters in both proteins were shown by X-ray crystallography to be virtually identical. However, the redox potential and oxidation states for the two proteins are vastly The term “mixed valence state” has been used in two connotations: (1) to mean two coupled irons with different formal charge (e.g.,Fez+: Fe3+)and (2) to mean two (or more) irons coupled and electronically delocalized to have fractional formal charge (e.g.,2Fe2.5+ from delocalized Fez + Fe3+).We use “mixed valence” only in the former meaning.

208

JAMES B. HOWARD AND DOUGLAS C. REES

-400 m V [4Fe:4S:4Cys]3- H Ferredoxin

+350 m V [4Fe:4S:4Cys]2-

t)

[4Fe:4S:4Cys]l-

HiPIP

FIG.2. Three-state model of the oxidation states available to 4Fe : 4s clusters.

different, as illustrated in Fig. 2. On this basis Carter et al. (1972) proposed three allowed states, one in common and one unique to each of the two proteins. T h e larger number of amide hydrogen bonds between the protein and the inorganic sulfurs in the ferredoxin presumably stabilizes the more electron-dense -3 state (Adman et al., 1975). Whatever the specific origin of the restriction, it must be due to the protein structure and not inherent in the cluster, because all three oxidation states can be achieved once the proteins are denatured (Cammack, 1973). On refolding of the protein, the original restricted redox states are reestablished. Spectral properties indicate that the electronic coupling in the multinuclear clusters is more complex than in the binuclear clusters. For example, an electron may be fully shared (i.e., delocalized) by two irons in a cluster such that each iron is formally in a fractional valence state. This iron pair is then coupled to the second pair in a four-iron cluster. One iron or more may retain formal valency as a trapped valence. A good example of a cluster with a combination of delocalized and trapped valence iron is the one-electron reduced 3Fe : 4 s cluster (Moura et al., 1982). In the oxidized state all three irons are Fe3+. As shown by Mossbauer spectroscopy, on reduction, the added electron is delocalized over two of the three irons, resulting in two fractional valence Fe2.5+and one trapped valence Fe3+. All three irons remain coupled as a composite S = 2 spin system. The theoretical treatment for these clusters is not fully developed; however, a model eliciting ferromagnetic and antiferromagnetic coupling (i.e., double-exchange coupling) seems promising (Papaefthymiou et al., 1987). For some [4Fe : 4 s : 4RSI3- and [4Fe : 4Se : 4RSI3- clusters in both proteins and model compounds, the cluster has been observed in more than one spin state, for example, as a combination of S = f, 8, up to 8 (Lindahl et al., 1985; Watt and MacDonald, 1985; Dunham et al., 1985; Moulis et al., 1984; Carney et al., 1988). Using the double-exchange model, the multiple spin states have been rationalized as a consequence of the degree of interaction between the ferromagnetic and antiferromagnetic components (Noodleman, 1988). If the interaction is small, then small changes in the individual ferromagnetic and antiferromagnetic

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couplings can lead to the population of additional spin states. Because changes in the protein buffer (solvent)can shift the ratio of the observed spin states, it is tempting to suggest that perturbation of the ligand strength (through protein conformational distortion) might alter the coupling constants. Likewise, the spin state for model compounds is sensitive to solvent and crystal lattice packing. On the other hand, there is no clear connection between the multiple spin states and any enzymological or chemical property of the cluster (e.g., the redox potential or level of enzyme activity). It should be kept in mind that the multiple spin states are due to multiple low-lying energy levels. At biochemically relevant temperatures different higher-lying levels are increasingly populated. That is, the spin states problem may turn out to be of interest mainly to physicists, not to enzymologists.

D. Structural Overview of Non-heme Iron Proteins A representative sampling of non-heme iron proteins is presented in Fig. 3. Evident from this atlas is the diversity of structural folds exhibited by non-heme iron proteins; it may be safely concluded that there is no unique structural motif associated with non-heme iron proteins in general, or even for specific types of non-heme iron centers. Protein folds may be generally classified into several categories (i.e., all a,parallel a/& or antiparallel p) on the basis of the types and interactions of secondary structures (ahelix and p sheet) present (Richardson, 1981). Non-heme iron proteins are found in all three classes (all a:myohemerythrin, ribonucleotide reductase, and photosynthetic reaction center; parallel alp: iron superoxide dismutase, lactoferrin, and aconitase; antiparallel p: protocatechuate dioxygenase, rubredoxins, and ferredoxins).This structural diversity is another reflection of the wide variety of functional roles exhibited by non-heme iron centers. Despite the lack of structural similarities among non-heme iron proteins, there are several themes common to the iron center environments in different proteins. These themes should be taken as rough generalizations present in at least several of the characterized non-heme iron proteins, but they certainly are not fundamental laws of nature that are always obeyed. 1 . Environment of Non-heme Iron Center

Although the iron centers are coordinated by polar groups (aspartate, glutamate, histidine, tyrosinate, and thiolate side chains), the overall environment created by residues surrounding the iron-ligand center often has a hydrophobic character. Amino acids located in this “second

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a

b

U

U

FIG. 3. Stereo views of the polypeptide fold and metal centers in non-heme iron proteins. The data set identifier in parentheses indicates the coordinate set from the Brookhaven Protein Data Bank (Bernstein etal., 1977) used to generate the figure. Relevant references are provided in Table I. (a) Escherichia coli Fe superoxide dismutase (courtesy of M. Ludwig), (b) 3,4-protocatechuate dioxygenase (courtesy of D. H. Ohlendorf), (c) R. sphaeroides photosynthetic reaction center (IRCR), (d) lactoferrin (reproduced with permission from Anderson et al., 1987),(e) myohemerythrin (BMHR),(f) ribonucleotide reductase (courtesy of H. Eklund), (g) C. pasteurianum rubredoxin (BRXN), (h) S . platensis 2Fe :2s ferredoxin (SFXC), (i) P. aerogenes ferredoxin (lFDX), (j) A. vinelandii ferredoxin (4FD1). (k) B. thennoproteolyticus ferredoxin (lFXB), (1) high-potential iron protein (lHIP), (m) A.

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d

e

f

uznelandzz nitrogenase iron protein (D. C. Rees, unpublished coordinates), (n) aconitase, 3Fe : 4 s form (5ACN), and (0)trimethylamine dehydrogenase, 4Fe :4 s and flavin-binding domain (reproduced with permission from Lim et aL, 1986).

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g

C

h

1

FIG. 3. (continued)

C

213

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k

1

C

FIG. 3. (continued)

C

2 14

JAMES B. HOWARD AND DOUGLAS C. REES

n

U

0

FIG. 3. (continued)

shell” region of various non-heme iron proteins include: Escherichia coli iron superioxide dismutase (Tyr-34, Tyr-76, Trp-77, Trp-122, Trp-158, and Tyr-173),Rhodobacter sphaeroides photosynthetic reaction center (ValL194, Ile-L229, Phe-M216, Ile-M223, and Met-M262),myohemerythrin (Phe-55, Met-62, and Tyr-l14), and Peptococcus aerogenes ferredoxin clus-

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ter 1 (Ile-4, Ile-9, Ala-13, Tyr-28, Pro-46, Val-47, and Pro-50) and cluster 2 (Tyr-2, Pro-19, Ile-22, Ile-30, and Ile-36). As noted by Yamashita et al. (1990), this hydrophobicity pattern about metal centers (polar ligands and apolar second-shell residues) is not restricted to iron sites, but includes many types of metal-binding sites. The polarity of the protein environment will influence functionally important properties of the iron, such as redox potential and pKa values of liganded groups, including bound solvent species (Rees and Farrelly, 1990).Qualitatively,the more apolar the environment, the more favored will be states of the iron center (including ligands) with lower absolute charge. In addition to general hydrophobicity effects, residues in the second shell may also influence oxidation and ionization states in the iron center by more specific types of hydrogen-bonding interactions. A classic example described previously is provided by Fe :S proteins ferredoxin and HiPIP. By donating more hydrogen bonds (with a partial positively charged hydrogen) to the cluster, ferredoxin can apparently stabilize the more electron-rich fully reduced form of the cluster, compared to the cluster in HiPIP (Adman et al., 1975).Likewise, ribonucleotide reductase has hydrogen-bonding interactions of potential functional significance. Metal ligands His- 1 18 and His-241, each of which coordinates a different iron atom, are linked by a hydrogen-bonding network involving Asp-237 and Gln-43. In the photosynthetic reaction center the secondary quinone acceptor, Q B , is hydrogen bonded to a histidine ligand of the iron. In addition to influencing the polarity of the iron environment, surrounding side chains may also exert steric control over the ability of the iron to accommodate conformational changes. Events of this type may range from fairly large-scale changes associated with substrate or ligand binding to more subtle effects associated with expansion or contraction of the center on change in oxidation state. While the interior of proteins tends to be efficiently packed, packing densities in the active site were observed by Richards (1974) to be lower than average. This suggests that a loose atomic packing may be necessary in the active site to permit and facilitate conformational adjustments required for enzyme function. Adman (1982) presented a preliminary study of packing interactions around the 4Fe : 4s clusters in ferredoxin and HiPIP to determine how the expansion/contraction of the cluster during reduction/oxidation (Laskowski et al., 1979) could be accommodated by the protein. While problems in defining the packing density of surface residues precluded a detailed analysis of redox-linked structural changes, this intriguing approach should be extended as a way of understanding how non-heme iron proteins modulate the properties of the metal center.

2 16

JAMES B. HOWARD AND DOUGLAS C. REES

2. Nonlocal Nature of Iron Ligands There are two classes for the distribution of ligand residues within the primary protein sequence: (1) residues grouped within short segments of sequence, and (2)those that are diversely spaced. The average difference between the lowest and highest residue numbers for the iron ligands in single-subunit proteins given in Table 1 is 79, with a range of 24 (P. aerogenes ferredoxin cluster 2) to 202 (lactoferrin, carboxy-terminal lobe) There are also several examples, such as the photosynthetic reaction center or the nitrogenase iron protein, in which the ligands are contributed by residues on different subunits. The number of residues spanned by the metal ligands in non-heme iron proteins is much larger than is minimally necessary to coordinate the iron. Tetrahedral coordination of the structural zinc by four cysteines in liver alcohol dehydrogenase requires only 15 residues (Eklund et al., 1976), while octahedral calcium sites in calcium-binding protein span 12 residues (Moews and Kretsinger, 1975). These considerations suggest that other factors besides simple metal coordination (e.g., substrate binding or proximity of catalytic groups) influence the location of ligands in the protein sequence. However, clustering of the ligands does provide local structure to the protein and increases stability over the widely separated ligands. For example, the three cysteinyl ligands, 39, 44, and 47, of spinach ferredoxin are stable in 6 M guanidine, while ligand Cys-77 readily exchanges with nonligand cysteinyl-18 (J. B. Howard, unpublished observations). These effects may be important for ferredoxins, in which three of the four ligands are frequently clustered with two to four intervening residues. There is a striking tendency for the iron center in mononuclear trigonal bipyramidal iron proteins to be located at the interface between domains or subunits. The irons in iron superoxide dismutase and lactoferrin are coordinated by ligands from two domains within a single subunit and the irons in protocatechuate dioxygenase are located at the interface between two subunits (although the ligands are in only the 0 subunit), while the iron in the photosynthetic reaction center bridges two subunits. In contrast the currently characterized binuclear iron proteins and simple iron-sulfur proteins do not exhibit this tendency for the metal center to occur at a subunit or domain interface. This motif is again present, however, in more complex iron-sulfur proteins such as aconitase, trimethylamine dehydrogenase, and nitrogenase Fe protein. As previously discussed (Section I,D,l), the iron centers tend to be predominantly buried in the protein, but access to the solvent may be critical for metal binding and catalytic processes. Location of the iron center at an

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interface may provide a mechanism for altering access to the metal by varying the relative positions of domains. Several studies suggest that some variations in metal ligands may be tolerated without adverse structural or functional consequences. For example, if the ligand Cys-20 of the Azotobacter vinelandii ferredoxin 4Fe :4 s cluster is replaced by alanine, a structural adjustment allows the previously nonligand Cys-24 to coordinate the cluster (Martin et al., 1990). In this case the cluster is able to “recruit” replacement ligands from other residues in the second-shell region. Williams et al., (1991) recently replaced all five iron ligands in Rb. sphaeroides photosynthetic reaction center with Cys, Gln, and Glu. Surprisingly, many of these mutants are able to form functional reaction centers which support photosynthetic growth. While the quantitative consequences of ligand replacements on metal content and electron transfer kinetics need to be established, these preliminary results suggest that, at least in some instances, rather large changes in iron coordination may be tolerated. The sequence associated with 4Fe :4s clusters has an interesting stereochemical property. A tetrahedral center with four distinct substituents (designated A, B, C, and D) may exist in two nonsuperimposable forms, independently of whether the center is a carbon atom or a 4Fe:4S cluster. If D is assigned to the most remote ligand in the sequence, then the remaining ligands, A, B, and C, may be positioned about the cluster in the two topologically distinct arrangements illustrated in Fig. 4a and b. Only the arrangement represented in Fig. 4a, with the polypeptide chain incorporating ligands A, B, and C proceeding in a left-handed direction about the cluster D ligand direction, has been observed in 4Fe:4S proteins [Table 1(C)]. Similar observations have been reported for other tetrahedrally coordinated metal sites, such as the structural zinc sites in liver alcohol dehydrogenase (Eklund et al., 1976) and aspartate carbamoyltransferase (Honzatko et d.,1982). The origin of this preference is uncertain, but presumably it must create a favorable geometry for the side chains to ligate the metal center. An energetic analysis of this se-

(a)

(b)

FIG. 4. (a and b) Topologically distinct arrangements of protein ligands to tetrahedral iron centers.

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JAMES B. HOWARD AND DOUGLAS C. REES

quence should indicate whether the observed pattern corresponds to the only energetically favorable form, or whether alternate bonding geometries might also exist. Similar stereochemical considerations are also relevant to linear nFe :nS clusters. In this case six distinct ligand arrangements are theoretically possible, only one of which has been observed in 2Fe : 2s clusters.

E . Problems in Identijication and Characterization of Iron Center To develop a unifying view of iron center catalysis, properties of the iron center in individual enzymes must be determined. Obviously, the definitive solution for the structure is atomic resolution of the active enzyme and postulated intermediates determined by diffraction or nuclear magnetic resonance (NMR) spectroscopy. Just as obviously, these methods are limited by enormous time, effort, and instrumentation requirements as well as by practical and theoretical considerations. This point is emphasized by the paucity of available protein structures. I n addition to the strictly structural details of the iron center, chemical and physical properties are required and, in some cases, these results augment diffraction or NMR structural studies. Discussed below are a few of the more common processes by which this information is obtained. 1. Composition of Cluster Components The first step in systematically evaluating the number and types of iron centers in a protein is to establish the stoichiometry of the metal and other constituents, such as inorganic sulfur. Although this may seem obvious, the problem of stoichiometry has not always received the attention it deserves. For example, the iron-containing hydrogenases from Clostridium pasteurianum and ribonucleotide reductase from E. coli have been extensively studied for 20 years. However, it was only recently discovered that these proteins contain 35-100% more iron than their accepted values (Adams et al., 1989; Lynch et al., 1989). In both cases the spectroscopic and enzymological results had been forced to fit erroneous iron analyses and, hence, were incorrectly interpreted. Likewise, there were persistent reports that the nitrogenase Fe protein contained eight iron atoms in preparations of highest activity. Careful reevaluation showed the correct amount to be four iron atoms, the previously accepted value (Anderson and Howard, 1984). It seems likely that other metal stoichiometries will also need to be corrected. Generally, the experimental emphasis has been on the vagaries of quantifying the iron concentration in the presence of an organic matrix (i.e., the protein). An equally important aspect of establishing the metal

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stoichiometry is knowing the protein concentration by which the metal concentration is to be divided. Too often, colorimetric methods such as Lowry, Bradford, o r bicinchoninic acid are used after standardizing against bovine serum albumin. These methods are highly dependent on the individual protein and can give quantities as much as 200% over or under the true value. Even once standardized, colorimetric methods are sensitive to the buffer and other reagents. Although no single method is without qualifications, the most credible procedure is quantitative amino acid analysis after acid hydrolysis (Anderson and Howard, 1984; Adams et al., 1989). If the metal analysis is performed on a portion of the acid hydrolysate, the problem of “deproteinizing” the sample is alleviated and a direct comparison of the metal and protein concentration can be made. Corrections must be made for the amount of tryptophan and cysteine which are poorly determined without additional special procedures. If a rigorous protein concentration determination is performed, the most serious limitation to obtaining the metal stoichiometry is knowing the true molecular weight of the protein. Perhaps one of the most elegant object lessons demonstrating the importance of careful compositional analysis comes from the work by Beinert et al. (1983) on the 3Fe cluster. Even with structural analysis by numerous sophisticated spectroscopic methods and by X-ray diffraction, there was substantial controversy about whether the cluster was a modified cubane structure with four inorganic sulfurs and three ligands or a more open chair structure with three sulfurs and six ligands. T h e analysis of aconitase indicated four sulfurs and resolved the question in favor of the modified cubane. Further X-ray diffraction studies confirmed the cubane 3Fe :4 s cluster for aconitase and ferredoxins from A. vinelandii and Desulfovzbrio gigas (Stout et al., 1988; Stout 1988; Kissinger et al., 1989).

2 . Fe :S Cluster Zdentijication by Extrusion One approach for identifying the type of iron center in a protein has been to remove the center, intact, by ligand exchange between the protein and an exogenous acceptor ligand (reviewed by Berg and Holm, 1982). T h e removed, or “extruded,” center is identified by comparison of its spectral properties (usually absorption spectrum) to known model compounds. Alternatively, the extruded center can be inserted into a second apoprotein which has been previously determined to accept only one type of iron center. The reconstructed “standard” protein is then analyzed by EPR. T h e latter method, interprotein cluster transfer, requires that acceptor apoproteins for all known classes of centers are included in the reaction mixture and that the reconstituted “reporter”

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protein can be readily analyzed. Although, in principle, the extrusion technique could be applied to any metalloprotein for which appropriate acceptor ligands and model compounds are available, in practice it has been most successful with Fe:S proteins having a limited number of types of chromophores and clusters. Unfortunately, the utility of this method for many of the more interesting enzymes is restricted by the complexity of the protein. If more than one type of cluster is present, the multiple component analysis on the extruded mixture may lead to ambiguous conclusions. In addition nonmetallochromophores may interfere. Holm and co-workers (Wong et ad., 1979) circumvented some of these problems for Fe :S proteins by their choice of spectral analysis and exogenous thiolate ligand. Namely, they used '"F NMR spectroscopy to analyze the products of thiolate extrusion with p-trifluoromethylbenzenethiol. Contact shifts for the fluorine resonances are considerably different for 2Fe and 4Fe clusters. Important restrictions on the use of the '"F NMR detection are the quantity of protein needed, the synthesis of the ligand, and access to the spectrometer. Three additional qualifications should be considered when using extrusion studies. First, clusters can interconvert during the ligand exchange reaction. In the extreme, cluster resynthesis is possible and is likely if less stable forms of oxidation states are present. Second, nonstandard clusters such as the 3Fe clusters or mixed metal clusters may not have appropriate models for spectral comparison and may be overlooked o r incorrectly assigned. Third, quantification of the extruded cluster may be low because protein unfolding was inadequate for complete thiol exchange. This is of concern particularly for larger proteins, which may have limited solubility in the mixed organic-aqueous solvents required for the extrusion of stable clusters. Not withstanding these caveats, cluster extrusion can be a useful tool for the Evaluation of Fe : S proteins. 3. Identification of Metal Ligands

Spectroscopic methods can indicate the presence of sulfur, oxygen, or nitrogen ligands and, in some cases, even the type of amino acid side chain or extra protein ligand. However, these methods are not able to distinguish between individual residues, for example, which tyrosines, histidines, carboxylic acids, or cysteines in the protein sequence are the ligands. In lieu of a definitive three-dimensional structure, solution chemical approaches have been used to ascertain which specific amino acid residues are ligands and how the centers are distributed within the protein stucture. The results of chemical modification studies can be verified by site-specific mutagenesis, although both methods have the

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22 1

potential for misleading interpretation; namely, primary effects must be distinguished from nonspecific general effects on the protein structure. Chemical modification methods, in conjunction with spectroscopic observation, have been useful in the study of ligand exchange and cluster reorganization and in assigning specific metal centers to their spectroscopic signature. The amino acid sequence of the protein may give a good preliminary indication of possible ligands. T h e rapid advances in both protein and nucleotide sequencing methods make it possible that a suitable data base can be accumulated to make this approach reasonable. Not only are ligands likely to be conserved, but also other residues involved in the protein folding around the metal center. A comparison of sequences from divergent species may reveal residues as potential ligands. For example, only 16 of the approximately 360 residues in the B2 subunit of ribonucleotide reductase are invariant among nine sequences compared (Nordlund et al., 1990). All of these residues are ligands, critical residues in the binuclear cluster organization, or part of the tyrosyl radical pocket. If the ligands for one member of a general class are known, sequences similar to those around the ligand may be found in a new class of metalloprotein. For example, the characteristic distribution of cysteines found in ferredoxins (i.e., two to four residues between grouped cysteine ligands) has been found in larger enzymes which indicate both potential ligands and the types of iron clusters. This approach has been useful in a number of more complex proteins, such as beef heart succinate dehydrogenase (Yao et al., 1986) and E. coli fumarate reductase (Johnson et al., 1988). However, this distribution of cysteines is only indicative of potential ligands, but is not required. In addition, all of the ligands need not be grouped; at least one ligand may be located outside the grouped ligands. A comparison of sequences has indicated that many iron-containing proteins may have arisen by gene duplication. This generates a protein with a second set of potential ligands and associated “active-site” residues, some of which may be deleted by subsequent mutations. T h e first protein class to be identified as having gene duplication was 4Fe and 8Fe ferredoxins, in which, in some cases, -70% of the residues in the first half are identical to residues in the second half. Curiously, the ligands for each cluster, as determined by crystal structure, come from both sets of duplicated residues. For these proteins the gene duplication must have occurred prior to the division of the prokaryotes and archaebacteria (Hausinger et al., 1982). Sequence comparisons may also suggest new ligands. Recently, the complete amino acid sequence for a ferredoxin from the thermophilic organism Pyrococcus furzosus was determined (Eccleston et al., 199 1). All

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five cysteines were conserved with the cysteines in the D. gzgm ferredoxin. Three of the conserved cysteines correspond to three thiol ligands found in the D. gigas ferredoxin crystal structure (Kissinger et al., 1989); the other two conserved cysteines form a disulfide. In the P.furiosus sequence the missing cysteinyl residue was replaced by an aspartyl residue, which was postulated as a potential fourth ligand. I n many cases the protein sequence is of little help in predicting the ligands, because there is no discernible pattern of potential ligands o r there is an excess of candidates. For example, in the nitrogenase Fe protein there are 10 conserved cysteines, yet only four are needed for the single 4Fe :4s cluster. The probable ligands in several proteins have been deduced, using one of the variations on a general scheme, as outlined in Fig. 5 for cysteinyl ligands (Hausinger and Howard, 1983; Cupp and Vickery, 1988; Plank et al., 1989). The idea is to react specifically the ligand residues after metal center chelation (path 1, Fig. 5); alternatively, the ligand residues are protected by the cluster, while all similar nonligand residues are reacted under denaturing conditions (path 2, Fig. 5 ) . In the latter version the ligands are labeled in a subsequent step after removal of the iron center. Thiolate ligands are particularly well suited for this type of analysis, because cysteines usually are present in relatively small numbers and are highly reactive to a broad spectrum of alkylating reagents, many of which can be obtained radiolabeled. In the example in Fig. 5 , RX would be a radiolabeled alkylating reagent such as iodoacetic

Reduce R X T X Alkylation Exposed -S _

_

f

M

-\\\

Identify Rlabeled Cysteines

Reduce TX RX M Alkylation Exposed -S Fe, s

7

FIG.5. Labeling schemes for identification of cysteine ligands to Fe :S clusters. RX and TX are alkylating agents as described in text.

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acid, while T X would be the same reagent without label. Because these reactions are not wholly specific or quantitative, identification of the ligands requires knowledge about the fraction of each individual residue modified at each step in the procedure. As an internal calibration the protein having its cysteines uniformly labeled with a second isotope can be added. The internal standard protein also provides markers for identifying the individual peptides in a map following protease digestion. Additional information can be obtained by analyzing the results at various times of reaction. For example, residues exposed on the surface (S1) may be distinguished from ligands (SL)by the requirement for the chelator and the relative rates of alkylation. Variation [ l] in Fig. 5 requires a chelation procedure that does not unduly disrupt the protein structure. A complication in the interpretation of the data can occur if the protein has a nonligand cysteinyl residue exposed to alkylation when the cluster is removed. Choice of chelator and oxidation state of the cluster is also important. If the cluster contains Fe3+ and a Fe2+ chelator is used without exogenous reducing agent (e.g., dithionite), then cluster destruction can proceed with partial di- and polysulfide formation (Plank et al., 1989). The driving force for the reaction is the preferential binding of Fe2+. Also, chelation must be anaerobic, to limit oxidative destruction and polysulfide formation (Petering et al., 1971). Although this is usually an undesirable side effect, the reaction can be used to advantage in some cases. Because the ligands are involved in the oxidative formation of di- and polysulfides, they can be identified as the newly blocked (i.e., protected by sulfide formation) cysteines. This method was recently used to correctly identify the aconitase ligands (Plank et al., 1989). Variation [2] in Fig. 5 also has limitations that should be noted. First, the cluster-protected residues must be distinguished from protein disulfides. Although Fe:S proteins were generally thought not to have disulfides, the D. gigas (Kissinger et al., 1989) and P. furzosus (Eccleston et al., 199 1) ferredoxins were reported to have disulfides. Second, the cluster must be stable under conditions that fully denature the protein. Finally, the nonligands must be modified rapidly to prevent exchange with the ligands. In some cases this is a formidable obstacle even if high concentrations of the alkylating reagent are present when the denaturant is added. For example, the 10 nonligand cysteines in the nitrogenase Fe protein fully exchange with the cluster ligands before the denaturation is sufficient to allow alkylation of buried cysteines (Hausinger and Howard, 1983). In contrast, no exchange between ligands and nonligands was found for one form of aconitase in 6 M guanidine (Plank et al., 1989).

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4 . C?ystallographic Methods of Structure Determination

Crystallography remains the only technique with the potential to determine the structure of all atoms (proteins plus cofactors) in a macromolecule of essentially unlimited molecular weight, provided that suitable (i.e., “diffraction-quality”) crystals can be prepared. While the technical details are beyond the scope of this chapter (see, e.g., Blundell and Johnson, 1976), several general aspects of a diffraction experiment are relevant to the interpretation and critical assessment of a macromolecular structure. The ultimate objective of a crystallographic analysis is a list of coordinates describing the atomic positions in a structure. These coordinates cannot as yet be directly determined experimentally, however. The experimentally measurable quantities are the intensities of the diffraction maxima of the crystals, which are related to the structure by the amplitudes squared of the Fourier transform of the atomic coordinates. Lost in the process of data collection is the critical second component of the Fourier transform: the phases. Both amplitudes and phases are required to reconstruct the electron density of the structure from the diffraction pattern, and most of the effort in a crystallographic analysis is in deriving the missing phase information by various indirect methods. Once the phases are determined, they can be combined with the amplitudes and Fourier transformed to generate an electron density map of the structure. Various computer graphics and refinement methods can be then used to obtain a set of atomic coordinates for the molecule. While the theoretical foundations of crystallography are solid, the practical details are not always straightforward. The critical steps of phase determination and model building, in particular, often require subjective interpretations. In ideal cases the crystallographic analysis of a structure is both unambiguous and exhilarating. If there are errors or problems at any stage of the analysis, however, the resulting structure (should it ever be obtained) may be suspect. In extreme cases the structure can be completely incorrect; in other cases perhaps only a small part of the structure is in error. The recent commentary by Branden and Jones (1990) provides a useful discussion of the various warning signs in a structure analysis and possible approaches to overcoming these problems. With care, the structure determination will be successfully completed. It is important to recognize, however, that even in these cases there are fundamental limitations on the accuracy of the derived atomic coordinates. The two principal reasons for this (not entirely independent) are the limited resolution of the diffraction pattern and structural disorder.

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Even the highest-resolution macromolecular structure determinations (<1.5 A resolution) are rarely at “atomic” resolution, in the sense that the

atoms are resolved from each another. The width of the electron density distribution about an atom depends strongly on the resolution of the diffraction analysis and is, in general, on the order of several angstroms. Clearly, the width of this distribution limits the accuracy to which the atomic coordinates may be determined. Least-squares refinement methods can reduce the average error in coordinates to perhaps several tenths of the limiting resolution, but this is still orders of magnitude higher than the coordinate errors (-0.00 1 A) routinely obtained in smallmolecule structure determinations. The comparatively limited resolution of a macromolecular structure determination implies that the ratio of the number of experimental observables (intensity measurements) to the number of refined parameters (coordinates) is much smaller for macromolecular structures than for small-molecule structures. T o increase the robustness of a macromolecular least-squares refinement, it is now conventional to supplement the least-squares equations with various restraints, to maintain “proper” geometry. By this process the geometry of the final coordinates will be biased toward the imposed restraints, making it difficult to obtain an objective estimate of a given geometrical parameter. These considerations are of special relevance for determining metal-ligand distances in metalloprotein structures: If a certain distance restraint is imposed on the structure, then the resulting distance cannot be taken to provide an unbiased estimate of that parameter, while, if the distance is unrestrained, it will quite likely adopt a chemically unreasonable value. In most favorable cases metal-ligand bond distances can probably be determined with an error of less than a few tenths of an angstrom, but this requires an accurate well-refined structure at resolutions below 1.5 8, [as illustrated by the myohemerythrin structure (Sheriff et al., 1987), for example]. At lower resolutions correspondingly less quantitative detail may be obtained. T h e identity of the metal ligands should be discernible at resolutions sufficient to trace the polypeptide chain (3-3.5 A), but in these lower-resolution studies it is impossible to determine accurately metal-ligand distances, or even to answer such basic questions as whether a carboxyl group is coordinated to the metal in a mono- or bidentate fashion. Changes in structure associated with ligand binding or metal oxidation state can be subtle, and may require very high-resolution data to describe quantitatively. Difference Fourier methods can provide a sensitive approach to visualizing conformational changes between families of related structures. In addition to specifying where atoms are located, the electron density

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map also contains useful information on the number of electrons occupying a particular volume of space. All other factors being equal, the electron density at an atomic position is proportional to the atomic number. Consequently, crystallography can be used to identify atom types, as is routinely done in small-molecule crystallography. Once again, however, the situation is not nearly so favorable in macromolecular work. Thermal motion, static disorder, phasing errors, etc., complicate the relationship between atomic number and electron density. While the qualitative relationship remains valid in macromolecular structures (e.g., metal atoms will have larger peaks than carbon atoms), it is not possible to distinguish more subtle atomic differences. Iron and manganese would be very difficult to distinguish solely on the basis of a macromolecular structure determination (although it might be possible using anomalous scattering techniques). Likewise, the oxidation state of iron cannot be assigned from a structure determination. The resolution of macromolecular structures is almost always insufficient to directly locate hydrogen atoms (except with the use of neutron diffraction techniques). Consequently, hydrogens must be positioned by indirect inference. While this may be relatively straightforward, in many important cases it is difficult to do this unambiguously. An important situation often arises in distinguishing between hydroxide ion or water coordinated to a metal in a protein; this cannot be decided solely on the basis of an X-ray structure determination. Finally, protein structures are dynamic, with all the constituent atoms in constant motion. If the position of a given atom is close to the timeaveraged value, then it can be reliably located in the electron density map. If a region of the protein is conformationally flexible, however, then the time-averaged electron density of that region is rather diffuse, and atoms cannot be described accurately by a single position. T h e degree of diffuseness of the electron density is modeled during structure refinement by atomic temperature factors; the temperature factor is proportional to the mean square displacement of an atom from the average position. Many other factors besides dynamic motion also influence atomic temperature factors, however, so that they cannot always be interpreted literally. T h e relative magnitudes of the atomic temperature factors do provide a measure of the reliability of the atomic position (the lower the temperature factor, the more reliable the atomic position), and so they can be used to provide an indication of the quality of particular regions of a structure.

5 . Spectroscopic Methods for Identijication of Iron Centers A wealth of spectroscopic methods are available for the study of metalloproteins. Most require some level of sophistication in physics for rigor-

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ous interpretation and have provided a common area of interest for the interaction of spectroscopists, inorganic chemists, and biochemists. Protein samples present problems that require experience beyond studying simpler inorganic or organic molecules. For example, it is generally not possible to obtain molar concentrations as high with proteins as with smaller compounds. Likewise, proteins are severely restricted in terms of what solvents and temperature ranges can be used. Thus, state-of-the-art instruments with the highest sensitivity and stability are needed; these are frequently a one-of-a-kind modification of commercial instruments. For many of the spectroscopies, the availability of instrumentation and expertise to study proteins is limited to a few laboratories. It is beyond the scope of this review to discuss these spectroscopies. Indeed, the reviews and texts are so numerous, it is not reasonable even to present a reference list. Readers who want an introduction to the biological application of a method should begin with appropriate volumes of Methods in Enzymology. The properties of some of the methods mentioned in this chapter are given in Table 11.

11. STRUCTURES AND FUNCTIONS OF MONONUCLEAR IRONPROTEINS The diversity of functional roles in mononuclear iron proteins is exemplified by the list in Table 111. These include proteins involved in several types of oxygen reactions, iron transport, and water insertion. Although protein structures are available that represent different functional catagories, there are insufficient numbers of structures to permit a generalization correlating enzyme activity to properties of the iron environment. For example, it is not yet clear what specific interactions between the protein and the iron establish the oxidation state, the redox potential, or the Lewis acidity of the iron. However, some patterns can be inferred based on crystal structures and spectroscopic studies. Namely, in the resting state (active) form of the enzyme, the iron is pentavalent (usually trigonal bipyramidal), frequently has nonprotein ligands (usually H 2 0 or OH-), and is high spin. These properties allow the ready exchange of iron ligands with substrates, including expansion of the coordination sphere. For most classes of enzymes, the iron only undergoes a transient change in oxidation state; the exceptions are the mixed-function monooxygenases, in which the iron is reduced to the starting oxidation state by coupling to a second cofactor-linked reaction. For the purposes of this review we restrict ourselves to classes for which we have a protein structure.

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TABLE I1 Spectroscopic Methods to Characterize Metal Centers in Nan-heme Iron Proteins Electronic spectra Visiblelnear-infrared Ligand charge transfer, characteristic for S, 0, N ligands and coordination number Useful to follow change in states of protein In some cases large extinction coefficient allows sensitive detection Circular dichroism (CD) spectra Coordination geometry; metal ligands Electron paramagnetic resonance (EPR) spectra Often require low temperature to observe (-2-10 K) Observe paramagnetic systems such as organic radicals and noninteger spin systems g Values indicate spin type of system Splitting of g indicates anisotropy of ligand field Due to sensitivity to impurities, should correlate signal quantity to number of iron centers Electron nuclear double resonance (ENDOR) spectra Identification of liganded residues Mossbauer spectra Require low temperature to observe in many systems (-2-70 K) Excellent for identifying all irons in sample Determine oxidation and spin state of individual irons from magnitude of isomer shift (6) Identify magnetically coupled iron and magnetic state by response to applied external field Identify number of irons in a grouping (i.e., cluster) Quadrupole splitting (AEQ)correlates to electric field gradient and, based on model compounds, can identify some ligand types Can observe changes in ligand field induced by sample perturbation Can only detect iron sites Magnetic circular dichroism (MCD) spectra Require low temperature to observe (-2-70 K) Clusters have individual spectral signatures Response to external field is used to determine magnetic susceptibility and electronic ground state of spin system Nuclear magnetic resonance (NMR) spectra Observe some atoms from protein and solvent, report on protein and, indirectly, the iron center. Observe resonances of atoms in metal environment; chemical shift, relaxation rate, and temperature dependence of shift related to paramagnetism of metal, including coupled integer systems Observe atoms not directly bonded to metal; magnetization transferred at a distance Observe at normal biological temperatures Determine bulk magnetic susceptibility Identify specific protein components associated with individual magnetic centers (irons in specific environments) Resonance Raman spectra Cluster symmetry, ligand types, and oxidation states Extended X-ray absorption fine structure (EXAFS) spectra Metal ligand distances; number and types of liganded atom

TABLE I11 Reactions Catalyzed 4 Mononudear Fe Proteins Reaction Oxidase SH4 + 0 g - f S + 2H20 Dioxygenase Intradiol S(0H)z + 0 2 + S’(C0OH)z Extradiol S(OH)2 + 0 2 --* S’(COOH)(OH)(CHO) 2-Oxoglutarate SH + 2-oxogiutarate + 0 2 + SOH + COB+ succinate Peroxidase S+OZ--*SOOH Dismutase 20; + 2Hf + H202 + 0 2 Mixed-function oxidase Monooxygenase SH+02+DH2+SOH+HgO+D D = Pteridine

D = NADH Dioxygenase S + 0 2 + NADH + S(OH)2 + NAD H ydratase RCN + H 2 0 + RCONHp Fe transport Protein structure, electron transfer

Example

Fe state

Reference

Isopenicillin N-synthetase

+2

C hen et al. ( 1989)

3,4-Protocatechuate dioxygenase 1,2-Catechol dioxygenase

+3 +3

Whittaker et al. (1984) Kent et al. (1987)

4,5-Protocatechuate dioxygenase 2,3-Protocatechuate dioxygenase

+2 +2

Arciero et al. (1983) Lipscomb et al. (1988)

Proline 4-hydroxylase Lysine hydroxylase

+2 +2

Pankalainen et al. (1970) Hausmann (1967)

Lipoxidase

+3

deCroot et al. (1975)

Escherichia coli superoxide disrnutase

+3

Yost and Fridovich (1973)

Tyrosine monooxygenase Phenylalanine monooxygenase Tryptophan monooxygenase 4-Methoxybenzoate monooxygenase

+2

Kaufman and Kaufman (1985)

+2 +2 +2

Bill et al. (198 1)

Phthalate dioxygenase Benzene dioxygenase Toluene dioxygenase

+2

Nitrile hydratase Transferrin Lactoferrin Bacterial photosynthetic reaction center

+3 +3 +3

Sugiura et al. (1987) Baker et al. (1987)

+2

Feher et al. (1989)

+2 +2

Keyser et al. (1976) Axcell and Geary (1975) Subramanian et al. (1979)

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A. Oxygen-Utilizing Enzymes

There are at least six types of reactions involving oxygen that are catalyzed by mononuclear iron enzymes. One of these, iron superoxide dismutase, is similar to a manganese-containing enzyme described elsewhere (Ludwig et al., 1991). Of the remaining classes the most extensively studied are the dioxygenases, although there is a structure only for 3,4-protocatechuate dioxygenase (3,CPCDase) (Ohlendorf et al., 1988). Th e substrates for these enzymes contain vicinal dihydroxyls, which are converted to aldehydes, or carboxylic acids with the insertion of both atoms of dioxygen. Dioxygenases can be further classified as either intradiol or extradiol, depending on whether the dioxygen is inserted between or adjacent to the diols. The range of substrates utilized by these enzymes is impressive, and an important consequence of this work is the search for specific enzymes (and bacteria harboring the enzymes) that can oxidize toxic aromatic compounds in the environment. The intradiol dioxygenase, 3,4-PCDase, is a (apFe3') 12 structure in which the protomers are packed on the faces of a truncated tetrahedron (Ohlendorf et al., 1988). T h e two subunits of a protomer have similar folding patterns, consistent with their partial sequence homology. Although they can be characterized as a p barrel, they are unusual in having both parallel and anti-parallel p sheets. T h e active site of the enzyme is at the interface of the two subunits, with the ligands provided by the p subunit (see Table I and Figs. 3 and 6). Although the iron is below the protein surface, there is a flat hydrophobic channel to the surface which could serve as a binding pocket for planar aromatic substrates. The entrance to the pocket has an arginine (residue 133) from the a subunit to stabilize the carboxylic acid on the substrate. A quasi-iron-binding site is located in the a subunit which approximates the true active site by virtue of the similarity in subunit fold and sequence homology. However, two of the ligands in th e p subunit have been substituted in the a subunit and the iron has been replaced with two hydrogen bonds. A similar conversion of iron ligands to nonligand residues, with the resulting loss of a binding site, has been described for the ferredoxin family (see Section IV,A). It is tempting to speculate that the ap dimer is derived from dioxygenases having only one type of subunit in their multimer structure. When the structures of dioxygenases with single-subunit types become available, it will be interesting to see whether the motif of shared active sites is retained [cf. thymidylate synthase (Montfort et al., 1990)l. In the crystal structure of PCDase, the iron has trigonal bipyramidal pentacoordination with one water ligand. Recent extended X-ray absorption fine structure (EXAFS) studies indicate that the bound water is more

NON-HEME IRON PROTEIN CHEMISTRY

23 1

FIG.6 . Active-site region of 3,4-protocatechuatedioxygenase.(Courtesyof 0.Ohlendorf.)

likely OH- (True et al., 1991). Based on EPR and Mossbauer spectroscopy, Lipscomb and co-workers (Whittaker et al., 1984) have shown that the active protein contains ferric iron, Furthermore, using a series of '70-labeled substrates and water, they found that water (hydroxide) binds directly to the iron, but is displaced by the two hydroxyls of poor substrates [e.g., homoprotocatechuic acid (HPCA)] (Whittaker and Lipscomb, 1984; Orville and Lipscomb, 1989). For the transition state inhibitor, 2-hydroxyisonicotinicacid N-oxide, both the N-oxy and water (hydroxide) are bound to the iron. Thus, at least two nonprotein ligands can be bound simultaneously. Because the EXAFS results for the HPCAenzyme complex are best explained by the pentavalent structure, one of the enzyme ligands must be displaced as well as the water (hydroxide) in order to accommodate the two substrate hydroxyls. However, this does not exclude expansion of the iron coordination sphere at other points in the enzyme turnover. Furthermore, the EPR spectra of the enzyme with a good substrate, protocatechuic acid, is substantially less intense, with only -20% of the iron in the bidentate form (both hydroxyls as ligands), suggesting that good substrates may bind in other modes than the pentavalent structure proposed for poor substrates. Because ferric iron does not react with triplet oxygen, intradiul dioxygenases must use a mechanism besides oxygen activation as the first step.

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-ooc

-9 F? , -

-ooc

t

O

o

C

T

I-

1’

t

FIG. 7. Model for oxygen activation by intradioi dioxygenases. PCA, the substrate protochatechuic acid.

I n this case the ferric iron site seems to activate the substrate. Our generalization of this model is shown in Fig. 7. T h e iron binds the substrate and could serve, as a Lewis acid, to facilitate ketolization of either the bidentate or monodentate substrate. The hydroxide (water) ligand may be more significant than simply a “placeholder” ligand to be displaced by substrate. Since both substrate hydroxyls must lose their

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233

protons in these initial stages of the reaction and there are no general bases in the active site (J. Lipscomb, personal communication), the displaced protein ligand and the hydroxyl likely function as general bases as well. Delocalization of an unpaired electron between the iron and substrate generates radical character on the aromatic ring, which can now react with oxygen (Cox and Que, 1988). In a subsequent rapid step the iron is reoxidized and a peroxy intermediate is formed. Alternatively, a transient ferrous iron intermediate might be formed which would react with dioxygen to give a ferric superoxide. Although no oxidation state other than ferric has been detected for any of the inhibitor-substrate complexes, Ballou and co-workers (Bull et al., 1981) found evidence for at least two other intermediates by rapid quench kinetics. One of these may be the elusive transient oxidation state. In any case it is difficult to reconcile the required “electron pushing” in the “paper chemistry” without some electron delocalization that is equivalent to short-lived redox changes. By any mechanism some reorganization around the iron must occur to accommodate the developing tetrahedral carbon at position 4. A number of other intradiol dioxygenases have been studied by NMR, EPR, Mossbauer, EXAFS, and Raman spectroscopies. By comparison to 3,4-PCDase, all intradiol oxygenases appear to have similar reaction mechanisms; most significantly, they all have high-spin ferric iron centers, with some tyrosyl and histidyl ligands. In contrast the extradiol dioxygenases all have high-spin ferrous centers, which are EPR silent. However, binding of N O by ferrous iron oxidizes the iron with a resulting EPR-active S = f system (Arciero and Lipscomb, 1986). Indeed, the N O complex is often cited as an analog of ferrous 0 2 . Using the NO complex with the extradiol dioxygenase, 4,5-PCDase, EPR results indicated that as many as three nonprotein ligand sites can be occupied by water and substrates. T h e prevalent model for these enzymes has dioxygen activation as the initial step, which is allowed for ferrous iron (see Fig. 8). In summary, the intradiol dioxygenases have ferric iron, which serves as a Lewis acid to activate the substrate. The subsequent intermediates with lower oxidation states may be able to activate oxygen, and certainly must bind oxygen intermediates. Extradiol dioxygenases are exclusively ferrous iron and initially activate dioxygen as well as bind the substrate. As a consequence many of the proposed intermediates in the two mechanisms resemble each other (cf. Fig. 7 and 8).Although these roles for iron are undoubtedly important, perhaps the most consequential role is to coordinate the specificity of the reaction. Significantly, intradiol dioxygenases never insert oxygen in the extradiol positions and vice versa. Furthermore, the site of dioxygen insertion is highly specific. For example, the aromatic ring of protocatechuic acid can be cleaved by dioxygen

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JAMES B. HOWARD AND DOUGLAS C. REES

FIG.8. Model for oxygen activation by extradiol dioxygenases.PCA, protochatechuic acid.

insertion at either the 2,3 or 4,5 position; the enzyme catalyzing the one reaction does not catalyze the other, although both enzymes probably have the same activated substrate-peroxy intermediate (Lipscomb et al., 1988).Clearly, the iron and the active-site topology must control the site of insertion. One mechanism that may allow for the specificity is analogous to the retention of configuration by pseudorotation in phosphorus chemistry. Namely, the two substrate hydroxyls are likely to be constrained to an equatorial and an axial position on the penta/ hexacoordinate Fe. Reorientation with respect to the bound peroxy group could occur by elongation of the trigonal bipyramidal short axis. This would require a considerably smaller protein conformational change than to accommodate substrate rotation in a binding site as restricted as that in the 3,4-PCDase structure. Although it is beyond the scope of this chapter to discuss the other oxygen-utilizing enzymes for which there are no crystal structures, it should be noted that mixed-function monooxygenases and oxidases ap-

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pear to follow the postulates outlined above. In particular, ferrous enzymes activate oxygen while binding the substrate and ferric enzymes activate the substrate. B . Bacterial Photosynthetic Reaction Center Reaction centers (RCs) are integral membrane protein-pigment complexes that carry out the primary photochemical events of photosynthesis. In purple photosynthetic bacteria RCs are composed of three membrane-spanning subunits (designated L, M, and H) that are associated with four bacteriochlorophylls (Bchl’s), two bacteriopheophytins (Bphe’s), two quinones (Qs), and one non-heme ferrous iron (reviewed by Feher and Okamura, 1978; Feher et al., 1989). Two of the Bchl molecules are organized into a special pair, or dimer, which is the primary donor for photosynthetic electron transfer. Light-induced electron transfer in the RC proceeds from the dimer to an intermediate acceptor (a Bphe molecule) to a primary acceptor and ultimately to the secondary Q acceptor ( Q B ) . RCs contain 1 1 transmembrane-spanning a helices from the three protein subunits that serve as a scaffold to organize the cofactors (Fig. 3). The cofactors are arranged into two connected branches: A and B (Deisenhofer et al., 1984, 1985). Along each branch cofactors appear in the sequence: dimer, monomeric Bchl, Bphe, Q, and iron. The dimer and iron groups are common to both branches. A striking feature of the RC is the approximate symmetry of the structure, in which both the cofactors and the L and M subunits are related by a twofold rotation. Both the dimer and the iron are located near this rotation axis, at opposite ends of the membrane-spanning portion of the RC. Although there are many interesting aspects of RC structure and function, it is the role of the non-heme iron that is of special relevance to this review. The Fe is coordinated at the interface between the L and M subunits by four histidine residues (L190, L230, M219, and M266 in the Rb. sphaeroides RC sequence) and by the two oxygen atoms of Glu-M234, in a distorted octahedral environment (Deisenhofer et al., 1985; Allen et al., 1987, 1988) (Fig. 9). Two of the histidine rings, from residues L190 and M219, are located between the rings of the quinones QA and QB. These histidines are part of a Q~-His-L190-Fe-His-M2 19-QA system, with close, but beyond hydrogen-bonding distance, to His-M2 19. The transfer of electrons from the semiquinone form of QAto QB is the last electron transfer step occurring within the RC. The location of the iron and associated ligands between these two groups suggest that both the iron and the histidine ligands may play a role in electron transfer. The

(a)

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JAMES B. HOWARD AND DOUGLAS C. REES

FIG.9. Iron environment in the Rb. sphaeroides photosynthetic reaction center.

precise role played by iron in this process is uncertain, however. Relevant experimental observations (Debus et al., 1986) related to the participation of iron in QA-to-QB electron transfer steps are: (1) the iron remains ferrous during this process, with no detectable change in oxidation state; (2)removal of the iron quantitatively alters, but does not eliminate, the rate of electron transfer; (3) replacement of iron by other divalent metal ions yields RCs with native electron transfer rates, both in vivo and in vitro; (4)bacteria containing RCs with the histidine ligands to iron substituted by glutamate, glutamine, or cysteine are able to grow photosynthetically (Williams et al., 1991). Consequently, it appears that (i) Fe is not essential for the electron transfer step; (ii) metal ions accelerate the electron transfer rate between QA and QB, but are not absolutely required for this step; and (iii) the detailed electronic structure of the metal-ligand complex is not critical to the electron transfer step. Rather, metal ions must participate in electron transfer in a more general fashion, perhaps by facilitating superexchange coupling between the two QS (Okamura and Feher, 1989) and/or possibly by functioning in a structural capacity to orient the quinones properly for electron transfer. The location of the iron at the L-M subunit interface suggests that the metal might play a role in structural stabilization and/or assembly of the RC. The coordination of the metal by the protein ligands provides a major polar interaction in the membrane-spanning region of the RC. These interactions help stabilize a bundle of four a helices that forms the core of the RC at the L-M subunit interface. Although the Fe can be removed from the native RC without complete loss of activity, it is possible that these interactions may play a role in stabilizing various intermediates during the assembly of the RC in the membrane. Little is known about this assembly process, which is an intriguing problem involving the association of three subunits and 10 cofactors.

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C . Trunsferrins Although the thermodynamically favored form of iron under aerobic conditions is Fe3+,the facile hydrolysis of this species to insoluble ferric hydroxides severely limits the bioavailability of iron. Consequently, a variety of biological systems have been developed to reversibly transport and store iron in a usable form. Of special interest to this review are the transferrins, a class of non-heme iron proteins that serve as the primary iron transport proteins in vertebrates (reviewed by Aisen and Listowsky, 1980). Three major types of transferrins have been characterized: lactoferrin, serum transferrin, and ovotransferrin. These proteins have molecular weights of about 80,000 and contain two iron-binding sites, each of which reversibly coordinates a single Fe3+ with an association constant of >lozo M-'. One of the characteristic features of transferrins is the stoichiometric and obligatory coupling between iron and anion (usually COi- o r HCO;) that appears to be intimately involved in a binding process that is both extremely tight and kinetically reversible. Detailed pictures of the iron-binding sites in transferrins have been provided by the crystal structures of lactoferrin (Anderson et al., 1987, 1989; Baker et ul., 1987) and serum transferrin (Bailey et al., 1988). Each structure is organized into two lobes of similar structure (the amino- and carboxy-terminal lobes) that exhibit internal sequence homology. Each lobe, in turn, is organized into two domains separated by a cleft (Fig. 3 and 10). The domains have similar folding patterns of the alp type. One iron site is present in each lobe, which occupies equivalent positions in the interdomain cleft. T h e same sets of residues serve as iron ligands to the two sites; two tyrosines, one histidine, and one aspartate. Additional extra density completes the octahedral coordination of the iron and presumably corresponds to an anion and/or bound water. The iron sites are buried about 10 8, below the protein surface and are inaccessible to solvent. T h e coupling between iron and anion binding is a critical component of transferrin function. In the absence of anion, transferrins bind Fe3+ weakly and nonspecifically. The putative anion site identified crystallographically is in an electrostatically positive region, adjacent to the side chain of an arginine and the amino terminus of an cy helix. T h e anion site does not bridge the two domains, and so does not function directly as a latch that closes the cleft around the iron. It is possible that the anion may partially compensate for the presence of several basic residues in the cleft that enhance iron binding. On release of iron, transferrin undergoes a major conformational change, characterized by a hydrodynamically less compact structure. Crystallographic analysis of apolactoferrin reveals that the amino-

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JAMES B. HOWARD AND DOUGLAS C. REES

FIG. 10. Iron environment in lactoferrin. (Reproduced with permission from Baker el al., 1987.) Revisions to the residue numbering are described in Anderson et al. (1989).

terminal lobe cleft opens up in the apoprotein, exposing the iron site to the solvent (Anderson et al., 1990). The cleft opening is created by pivoting of two helices and a bending of two antiparallel strands that connect the two domains. Curiously, the cleft of the carboxy-terminal lobe remains closed; this may reflect the influence of crystal-packing forces. On the basis of this work, a model for the coupled binding of anion and iron to lactoferrin has been proposed. The open cleft in the amino-terminal lobe exposes three basic side chains that may electrostatically attract the anion. At this stage the iron could be coordinated by the anion and the two protein ligands that would be present in the same domain. Closure of the cleft would complete the iron coordination sphere by providing the remaining two ligands. The molecular details of the physiologically critical step of iron release from serum transferrin are unclear. The anion may play an important role in this process by providing a handle for modulating the affinity of transferrin for iron. If the anion were to be removed (perhaps by protonation followed by dissociation), then the binding of iron to transferrin would be greatly weakened, facilitating dissociation of the metal. It seems

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NON-HEME IRON PROTEIN CHEMISTRY

clear that the anion is critical to the ability of transferrins to combine thermodynamic affinity with kinetic lability in their interactions with iron. 111. STRUCTURES AND MECHANISMS OF BINUCLEAR OCTAHEDRAL IRONPROTEINS There are relatively few constituents of this class of non-heme iron proteins, yet each member has its own unique set of interesting biochemical and structural properties. These proteins are listed in Table IV. The three enzymes with unambiguous physiologically defined rolesribonucleotide reductase (RNR), methane monooxygenase (MMO), and hemerythrin (Hr)-have the common property of binding oxygen. However, the activated oxygen intermediates are used to distinctly different ends. In the spirit of discussing only a few examples of a particular class, we have selected these three proteins since they reflect variations on the common theme of oxygen binding. Additional binuclear octahedral iron proteins have been identified. Of these the purple acid phosphatase has been the most vigorously studied and has a distinctly different biochemical function. Indeed, the protein may be involved with iron transport as well as a hydrolase (Doi et al., 1988; Nuttleman and Roberts, 1990). Because there are insufficient structural data to provide a basis of comparison, we have omitted it from our discussion. Likewise, rubrerythrin has only recently been identified as a member of the class, and no enzymology has been established (LeGall et al., 1988);thus, its inclusion in this discussion would be premature. There

TABLE 1V Proteins with Binuclear Octahedral Iron Centers Protein

Reaction

Ribonucleotide reductase Hemerythrin

2SH + ribose + 2-deoxyribose + -S-SHr + O2 [Hr - O,]

Methane monooxygenase Purple acid phosphatase

NAD+ + 0 2 + CH4+ NADH + H20 + CHsOH ? (ROPOs + ROH + PO4)

Rubrerythrin

? (Redox)

*

Reference Thelander and Reichard (1979), Stubbe (1989) Okamura and Klotz (1973), Kurtz (1990) Fox et al. (1989), Woodland and Dalton (1984) Chen et al. (1973), Campbell and Zerner (1973), Doi et al. ( 1988) LeCall et al. (1988)

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JAMES B. HOWARD AND DOUGLAS C. REES

are several excellent reviews for those wanting a comprehensive accounting of binuclear iron proteins with emphasis on their relationship to model compounds (e.g., Que and True, 1991; Sanders-Loehr, 1989; Lippard, 1988).

A . Hemerythrin H r was the first binuclear iron protein for which a three-dimensional structure was determined and is the most extensively studied. Thus, the protein has become the standard of comparison for other binuclear iron proteins, with the inherent hazards of defining a class on the basis of one example. Indeed, recent crystal structures for model compounds and the RNRB2 subunit indicate some “surprises” and substantial diversity in the class. Nonetheless, the structure of Hr is an appropriate place to begin. H r is an oxygen-transporting protein found in four phyla of invertebrates and occurs in several oligomeric forms (monomers to octamers of identical approximately 118 amino acid protomers) (Klotz and Kurtz, 1984). Diferrous H r (deoxy-Hr) binds one 0 2 and is simultaneously oxidized to the diferric state (oxy-Hr). The iron oxidation is readily reversible with the release of 0 2 . In contrast, if deoxy-Hr is chemically oxidized in the absence of 02,the resulting diferric protein (met-Hr) cannot bind oxygen, but can bind a number of anions, such as azide (azidomet-Hr). Thus, the oxygen binding and iron oxidation are intimately connected. However, because oxy-Hr is in the ferric oxidation state, technically oxy-Hr can be considered oxygenated met-Hr and similar to azidomet-Hr. This borne out by a variety of results, as discussed below. High-resolution crystal structures for azidomyomet-Hr (monomeric met-Hr), met-Hr, and azidomet-Hr have been determined by two groups and provide the basis for visualizing oxygen binding (Stenkamp et al., 1984; Sheriff et al., 1987). Except for a few differences (discussed below), the structures are the same. met-Hr structure is substantially a-helical (>60%),with a four-helix bundle motif having two pairs of antiparallel helices crossing at approximately 22%. The helices are connected by short sections of tight p turns. This motif was subsequently found in other proteins (e.g., cytochrome c‘ and tobacco mosaic virus coat protein) and is not unique to proteins with iron ligands. A relatively large cavity composed of hydrophobic residues is formed at the interface of the four helices distal to the iron cluster. However, there is no known function for the cavity which could be the remnant of a substrate-binding site used by an evolutionary precursor. The irons are coordinated by ligands from all four helices with three

NON-HEME IRON PROTEIN CHEMISTRY

CB..

24 1

n

FIG. 11. Environment of the binuclear irons in myohemerythrin. (Reproduced with permission from Sheriff et al., 1987.)

bridging ligands (the p-0x0 and two carboxylic acids),as shown in Fig. 1 1; the remaining terminal ligands are all histidines. Ligands derived from a single helical segment are spaced such that one ligand is above the other and staggered on the same helical face and these ligands are directed to the same iron atom (His-54 and Glu-58 to Fe2; His-73 and His-77 to Fel; and His- 106 and Asp-1 11 to Fel). The two carboxylic acids are also bridging ligands. With this arrangement the cluster axis is perpendicular to the helical axes. The short Fe-pi-0 bond (1.78-1.80 A) is identical in all three met-protein forms and model compounds, whether determined by EXAFS or crystallography [see Que and True (1991) for a compilation of known models]. In contrast the Fe-Fe bonds determined from the crystal structures of met-Hr and azidomet-Hr (3.21-3.25 A) are larger than those found in EXAFS or in model compounds (3.06-3.19 A). The myomet-Hr values appear closer to the latter. However, it is not clear whether this is a real or significant difference, given the uncertainties in determining selected bond distances by X-ray diffraction in larger structures (see Section I,E,4). In the met-Hr structure the two iron atoms differ in coordination number. Fe2 is pentacoordinate, yet has bond angles typical of octahedral iron with an open coordination site. This is in distinction to the pentavalent mononuclear iron proteins, which are better described as

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JAMES B. HOWARD AND DOUGLAS C. REES

trigonal bipyramidal (see Section II,A), However, the difference in coordination between the two iron sites is not reflected in the Mossbauer spectrum, which has only a single quadrupole doublet; namely, the electronic environment is similar for both irons (Clark and Webb, 1981). T h e isomeric shift is typical of high-spin ferric (6 = 0.46 mm/sec), while the large AEQ (1.57 mm/sec) probably reflects the asymmetry in the electric field gradient due the short p-0x0 bond. Based on this structure and numerous model compounds, these Mossbauer parameters are frequently quoted as evidence for diferric iron in octahedral coordination with a short p-0x0 bridge. Magnetic susceptibility measurements and chemical shifts in NMR spectra of met-Hr indicate very strong antiferromagnetic coupling between the irons and explain the absence of an EPR spectrum [namely, the two S = 3 Fe3+ are antiferromagnetically coupled to give a diamagnetic S = 0 ground state (Maroney et al., 1986)l. The structure of azidomet-Hr shows that the sixth coordination site on Fe2 is filled. Because Fourier difference maps for oxy-Hr and azidometH r are reported to show minimal differences, it is reasonable to use the high-resolution azidomet-Hr structure as the model of oxy-Hr (Stenkamp et al., 1985). T h e similarity of the iron environment for oxy-Hr and azidomet-Hr has been confirmed by EXAFS (Zhang et al., 1988). Likewise, the Mossbauer spectra clearly reflect the increased coordination in oxy-Hr and azidomet-Hr; the spectra now have a pair of equally intense quadrupole doublets with the same isomeric shift (Clark and Webb, 198 1 ; Okamura et al. , 1969). The binding of 0 2 at one iron induces a new electric field gradient, distinguishing it from the second iron. Although the irons remain strongly antiferromagnetically coupled in oxy-Hr, the coupling constant, J , is decreased -40% (Dawson et al., 1972). This implies that when oxygen is bound, some new hydrogen bonding to the p-0x0 bridge occurs, decreasing the exchange coupling. By the same argument the bridge 0x0 is not fully protonated because the coupling is still large ( J = -77 cm-'). Based on the resonance Raman transitions at 503 cm-' which appear when 0 2 is added, the bound form of oxygen appears to be the protonated two-electron reduced hydroperoxide (Shiemke et al., 1986). Although low-resolution difference Fourier maps for oxy-Hr and deoxy-Hr show little change in the protein structures, some of the iron center properties are significantly altered in deoxy-Hr. The differences provide a rationale for an oxygen-binding mechanism. The Mossbauer spectrum for deoxy-Hr has a single quadrupole doublet with an isomeric shift typical of high-spin ferrous iron (6 = 1.14 mm/sec; AE? = 2.76 mm/sec) (Clark and Webb, 1981). As for met-Hr the two iron environments are similar, yet differ in coordination number; for exam-

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ple, the circular dichroism spectrum in the infrared region has transitions at 5000 and 10,000 cm-', indicating both five and six coordination (Reem et al., 1989).However, the interesting changes are in the iron bond distances, as measured by EXAFS. In deoxy-Hr the Fe-Fe bond is increased by 0.33 A and Fe-p-oxo bonds by 0.16 A (Zhange et al., 1988).In addition Mossbauer spectroscopy and magnetic susceptibility indicate that the iron coupling is substantially decreased. The weak coupling (if any) is consistent with a fully protonated p-0x0 bridge (i.e., the iron are bridged by a hydroxo group). The latter is thought to disrupt the paths for superexchange coupling and greatly reduces the magnetic interaction between the irons. Recently, Que and True (1991) presented a model of 0 2 binding incorporating these results and the earlier proposals by Stenkamp et al. (1985) and Reem et al. (1989). We have further adapted the model as shown in Fig. 12. Two alternative paths for 0 2 binding can be considered. In path a oxygen binds at the pentavalent ferrous iron and is reduced to give a superoxide radical. In this mixed-valence state the superoxide abstracts the proton from the bridging hydroxyl, allowing coupling of the iron and subsequent oxidation of the second iron. Alternatively, in path b the electron transfer is concerted followed by proton transfer. Path a emphasizes that proton transfer in the redox cycle of oxygen binding has a mediating effect on the Fe-Fe interactions. The difference in coupling in the two states raises the metachemical question: Is coupling between irons a consequence of, or the driving force for, the reaction? Furthermore, because the bent Fe-0-Fe bond increases the basicity of the p-0x0 bridge, the proton transfer is facilitated by the geometry of the cluster. Hence, the role of the other bridging ligands is to ensure the bending. Evidently, changes in this bond angle in the transition state would alter the proton transfer potential and the degree of iron coupling. This is an

\

0-H

Fe/*+ deoxy-Hr Weakly Coupled

> 02

NO.

/

Fe:

0-

0-H Fe'3+

1

'0 H' Fe13+ oxy-Hr Strongly Coupled

FIG. 12. Model for oxygen binding to hemerythrin (Hr).

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JAMES B. HOWARD AND DOUGLAS C. REES

interesting point which we briefly consider in the sections on RNR and MMO. One elegant experiment should be mentioned that provides evidence for the superoxide intermediate or transition state (Nocek et al., 1988). NO, a putative 0 2 analog (cf. Section II,A), binds moderately tightly to deoxy-Hr. T h e Mossbauer spectrum of the complex has two quadrupole doublets, one which is similar to the diferric state (6 = 0.68 mm/sec, AEQ = 0.61 mm/sec) and one which is similar to the diferrous state (6 = 1.21 mm/sec, AEQ = 2.65 mm/sec). That is, the iron cluster has undergone a le- reduction to give the mixed-valence state (semimet-Hr). ( N0 )H r is EPR active, with g = 2.77 and 1.84, as expected for antiferromagnetic coupling of the S = 8 (Fe3+-NO) and S = 2 (Fe'+) sites. The resonance Raman bending modes associated with the Fe-NO bond are sensitive to D20, which indicates some hydrogen bonding to the NO. This structure is analogous to the proposed first intermediate in 0 2 binding by path a (Fig. 12).

B . Ribonucleotide Reductase RNR catalyzes the conversion of ribonucleotides to deoxyribonucleotides. Because of this central position in generating precursors for DNA synthesis, the enzyme has been the target for pharmacological modification. The reaction has been proposed to proceed through a radical mechanism involving the cyclic transfer of a preexisting radical in the enzyme to the substrate and back (Stubbe, 1989). Several classes of RNR have been identified on the basis of the type of radical and associated metallocenter. For the purposes of this chapter, only the E . coli enzyme, which has a tyrosyl radical and a binuclear iron cluster, is considered. The E . coli enzyme is a tetramer composed of two types of subunits. The larger subunit, B 1, contains the substrate-binding sites, an extensive array of allosteric effector sites, and the redox active disulfide-thiolate pair (Thelander and Reichard, 1979). T h e redox state of the protein is maintained by coupling the latter through disulfide interchange with thioredoxins. In the E . coli enzyme the smaller subunit, B2, has both the binuclear iron site and the radical located on Tyr-122 (Petersson et al., 1980). A comprehensive study using selectively deuterated tyrosine has shown that the radical resides primarily on one of the two P-CHZs, which is aligned with the ring 7r electrons (Sjoberg et al., 1978). The radical is probably neutral, rather than a cation, as proposed for other aromatic radicals (Stubbe, 1989). The iron center in the active B2 subunit has spectral properties that, in general, are similar to those of met-Hr, and therefore, by analogy, it has

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been classified as a binuclear octahedral cluster. For example, the visible spectrum has electronic transitions at 325, 390, and 600 nm, similar to met-Hr; the resonance Raman spectrum has the Fe-O-Fe stretching (496 cm-I); and EXAFS analysis found Fe-Fe of 3.22 8, and Fe-0 of 1.79 8, (Petersson et al., 1980; Scarrow et al., 1987; Sjoberg et al., 1987). Even after reaction with hydroxyurea, which quenches the tyrosine radical (met-RNRB2), these properties remain (Petersson et al., 1980). However, the Mossbauer spectrum is clearly different from that of metHr; there are two quadrupole doublets of equal size, (cf., met-Hr in the absence of exogenous ligands, which has only one) (Atkin et al., 1973; Lynch et al., 1989). Although the isomer shifts are normal for high-spin ferric iron, both AEQS are large, one especially so. Because the two doublets persist even after quenching the tyrosine radical, the two iron environments must be inherently different, without regard to the tyrosine. However, B2 is a dimer of identical protomers, and a single cluster should be shared with twofold symmetry. This conundrum was recently solved when the iron content of the protein was carefully redetermined and four irons per dimer rather than two were found (Lynch et al., 1989). That is, there is one binuclear cluster per protomer rather than one per dimer. This reinterpretation of the iron center arrangement has been confirmed by the recent crystal structure (Nordlund et al., 1990). A 2.2 8, electron density map is now available for RNRB2 (Nordlund et al., 1990) (see Fig. 3). T h e general protein structure consists of eight main strands of a helix. One helix, E, serves as a hydrophobic core about which five other helices (B, C, D, F, and G) are packed. Helix A interacts with helices C and F through a hydrogen-bonded network. T h e dimer is stabilized by symmetrical interprotomer hydrogen bonding between surface helices and four-strand antiparallel p sheet (two strands from each protomer) across the bottom. Helices B, C, E, and F provide the iron center ligands, His-I18 and -241, glu-115, -204, and -238, and Asp-84 (see Fig. 13). Two sets of ligands are nearest neighbors on the helical surface, where the residues are separated by the distance n + 3. However, this apparent similarity in the helical motif and iron center arrangement between RNRB2 and H r is illusory. Although both cluster-binding regions are formed by pairs of antiparallel a helices, the order or stereo sense of the helices differs in the two proteins. More significantly, the iron cluster axis is parallel to the helical bundle axis in RNRB2, but perpendicular in Hr. The RNRB2 iron cluster has several fascinating features that clearly distinguish it from the H r center and may provide a clue to its unique chemical function. For example, the cluster ligands are predominantly oxy functions, with only one histidine per iron. (All terminal ligands in

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JAMES B. HOWARD AND DOUGLAS C. REES

0

Tyr 122

20L

241

G l u 11s

T r p 48

I

Asp 231

I Gln L 3

FIG. 13. Environment of the binuclear iron in ribonucleotide reductase. (Reproduced with permission from Nordlund et al., 1990.)

H r are histidines.) Besides the characteristic p-0x0 bridge (protonated) and carboxylate ligands (bridge and terminal), there are two water ligands, one on each iron. An additional unusual feature is that both oxygens of Asp-84 ligand a single iron. Although both irons have octahedral coordination, the bidentate aspartic acid ligand of Fel distorts the octahedron toward a trigonal bipyramid. The distortion undoubtedly results in different electric field gradients for each iron expressed as the two quadrupole doublets seen in the Mossbauer spectrum. Finally, the cluster has only two bridges rather than the three of the H r cluster. This could result in more flexibility between irons. In what may be a compensation for the “missing” third bridge ligand of Hr, there is an elaborate hydrogen-bonding network that forces the two histidyl ligands to within 4 8, of each other. Besides being the scaffolding on which the iron cluster is arranged, the helical bundle provides the correct orientation for Tyr-122, the site of the radical. This residue is the third on a common face of helix C, composed of ligands Glu-115 and His-1 18. This positions the tyrosine 5.3 8, from the nearest iron and 3.3 8,from the nearest ligand (Asp-84).Likewise, the putative 0 2 binding pocket is formed by residues near the ligands on the surfaces of the helices. Besides Tyr-122, these include Phe-208 and Phe212 (above ligand Glu-204 on helix E) and Ile-234 (above ligand Glu-238

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on helix F). These amino acids are among the few conserved residues found on B2 from different species. Perhaps the most surprising facet of the RNRB2 structure was the location of the tyrosine radical. Because its enzymological role is to accept the electron to initiate the chemistry of ribonucleotide reduction, it was assumed the tyrosine would be located near the surface of B2 where B 1 binds. Remarkably, the tyrosine is 10 8, from the surface and buried in a hydrophobic pocket. Although being buried accounts for the stability of the radical, the direct participation of the radical in electron transfer seems unlikely without substantial structural reorganization. Nordlund et al. (1 990) proposed that the extensive hydrogen-bonding network involving the histidine ligands may provide a path for transferring the radical to the surface. This hypothesis provides a new and active role for the iron cluster in catalysis. Presumably, at least some conformational changes would be required to bring the tyrosine radical into the coordination sphere of the iron. That the cluster has only two bridging ligands may be an important structural requirement for the electron transfer through this path; changes in the hydrogen-bonding network could alter the Fe-Fe bond distance and coupling. The conformational changes would be induced only in an intermediate involving substrate and the catalytic subunit, thus protecting the radical from gratuitous reactions. At least one clear role of the iron cluster is in the formation of the tyrosyl radical (Atkin et al., 1973; Petersson et al., 1980). Because the radical can be quenched by scavengers in vivo and the radical must be introduced for de novo synthesis of the enzyme, creation of the radical is a continuing intracellular process. In uitro, the radical formation can be accomplished from several different states of the protein (Sahlin et al., 1989; Lynch et al., 1989; Atkin et al., 1973). Starting with diferric B2, the iron must be converted to the ferrous state either by metal replacement or by reduction in situ. The Mossbauer spectrum of diferrous RNRB2 has a curious feature; namely, there is only a single quadrupole doublet, indicating that both irons have essentially identical environments (Lynch et al., 1989). This is surprising, since it is the Asp-84 bidentate distortion that gives rise to a second quadrupole doublet in the diferric state. T o have equivalent sites in the diferrous state, either one iron has gained a similar distortion or one iron has lost the distortion. Either way, structural changes in the ligands apparently have occurred. When oxygen is added to the diferrous enzyme, partial activity is recovered concomitantly with radical formation and iron oxidation. The 0 2 has been proposed to bind at the site occupied by water on Fel in the diferric RNRB2 structure (Nordlund et al., 1990). T h e oxygen would extend toward the tyrosine and into a hydrophobic pocket (see above).

-

248

JAMES B. HOWARD AND DOUGLAS C. REES

The radical on the tyrosine apparently is generated in combination with the redox of the irons from intermediates similar to those in Hr 0 2 binding. Consistent with this mechanism is the observation that H202 can generate the radical starting with the diferric RNRB2 (see below for more extensive discussion of the chemistry) (Sahlin et al,, 1990). Quantitative generation of the radical and activity requires a reducing agent such as ascorbate, as well as the 0 2 . An additional electron is needed because the protein only supplies three electrons (two ferrous irons and one tyrosine) for the four-electron reduction of 02.In the absence of other reducing agents, presumably some of the diferrous protein serves as the reductant (without 0 2 bound), leading to incomplete recovery of activity. In uivo, the diferric state is reduced by an NADH-dependent reductase; the subsequent formation of the radical with O2 oxidation appears to follow the same mechanism as in vitro (Fontecave et al., 1989). Thus, RNRB2 may be considered an oxygenase with the overall reaction written as: Ared

+ -Tyr + 2Fe2+ + O2+ -Tyro + 2Fe3+ + 2H20 + Aox

Notice that two water molecules have been generated, one of which is likely to be at the site occupied by a water ligand in the crystal structure of the active protein. Indeed, it is tempting to speculate that the water ligands and/or the p-0x0 group may be the product of the activation process and not solvent derived (although they might exchange with solvent).

C. Methane Monooxygenuse MMO has been identified as a member of the binuclear octahedral iron family (Woodland et al., 1986; Fox et al., 1988).The enzyme can hydroxylate a wide variety of hydrocarbons, including some toxic waste compounds; hence, the enzyme has elicited considerable interest beyond its contribution to binuclear iron chemistry and mechanisms of oxygen activation. Although we do not have a crystal structure and have only limited spectroscopic data, the properties of the enzyme indicate that it should be considered with the other oxygen-activating binuclear iron proteins. The protein is composed of three independent components: component A, the hydroxylase; component B, a mediating protein; and component C, the reductase (Green and Dalton, 1985; Fox et al., 1989). These proteins have been isolated in high purity and activity. The hydroxylase contains iron centers which have Mossbauer parameters simi-

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lar to those for Hr and RNRB2 (Fox et al., 1988), which is the main criterion for proposing that the enzyme contains a binuclear octahedral cluster with some form of p o x 0 bridge. However, the absence of charge transfer bands of over 300 nm, and a strong g = 16 low-field EPR resonance for ferromagnetically coupled iron (Hendrich et al., 1991), indicate major differences in ligands compared to Hr and RNRB2 (Fox et al., 1988,1989). Indeed, this evidence suggests that the p-0x0 group may be either doubly protonated or a bridging amino acid. The active form of the hydroxylase is the reduced diferrous enzyme, as shown by Miissbauer spectroscopy; the other oxidation states (diferric and mixed valent) are inactive, but can be reactivated by reduction. Reduced hydroxylase is sufficient for a single turnover of 1 mol of oxygen and 1 mol of substrate. After one turnover the cluster is diferric. Thus, the enzyme supplies two of the four electrons for the reduction of oxygen. In this respect the reaction is similar to that for RNRB2, and it is likely that there are similar intermediates for both reactions (see Section 111,D). The NADH-dependent reductase, which contains a 2Fe :2 s cluster and FAD as cofactors, converts the oxidized hydroxylase binuclear cluster to a diferrous state after each catalytic cycle. It should be emphasized that the reductase does not participate directly in the hydroxylation reaction; its sole function is to regenerate the reduced enzyme in a separate reaction (Fox et al., 1988). The latter reaction is reminiscent of the NADH-linked reduction of inactive diferric RNRB2 (see Section IILB).

D. Oxygen Activation by Binuclear Octahedral Iron Centers The enzymes discussed above have a number of properties in common, including similar, although not identical, iron centers (see references to the various specific enzymes discussed in Sections II1,A-C). Furthermore, the chemistry performed by the enzymes appears to be related by a hierarchy of intermediates, as shown in Fig. 14. At some stage in each enzymatic reaction oxygen is bound at the iron center. In all three enzymes oxygen is bound only by the diferrous state, with subsequent redox leading to the partial reduction of the oxygen and the oxidation of both irons. There is good spectroscopic evidence for the diferric hydroperoxide in oxy-Hr. Likewise, since hydrogen peroxide can serve as the oxy donor with diferric RNRB2, there is chemical evidence for the intermediate in a second enzyme. The ferric peroxide intermediate could undergo either heterolytic or homolytic cleavage to give either a ferry1 or diferryl oxene. In the path shown in Fig. 14, the organic radical is generated by reaction with the Fe center after heterolytic 0-0 bond cleavage. For RNRB2 this is the

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JAMES B. HOWARD AND DOUGLAS C. REES

O(X)

--y-

O(X)

FIG.14. Intermediates in the activation of oxygen by binuclear iron centers. Hr, Hernerythrin; RNR, ribonucleotide reductase; MMO, methane monooxygenase.

metastable tyrosine radical; the final reduction of the iron oxene gives water. For MMO the organic radical is the substrate which provides the additional electron for reduction of the ferryl oxene to ferric iron. At one level these are analogous reactions, the difference being that in RNRB2 two waters are generated, while in MMO one water and one hydroxylated hydrocarbon are formed. Clearly, the mechanisms for RNRB2 and MMO could proceed by different pathways. However, the alternate mechanism of homolytic cleavage generating the highly reactive hydroxide radical seems less satisfying for a controlled and specific enzyme reaction. The later stages of the putative mechanism are similar to the ferryl oxene intermediates proposed for cytochrome P-450 enzymes and heme oxidases and peroxidases (Murray and Groves, 1986). T h e ferryl iron in cytochrome P-450 is thought to be stabilized by the electron-rich heme rings. For binuclear iron the mechanism of stabilization is less clear, although the strong coupling of the iron through the 0x0 bridge may provide a mechanism to delocalize the electron density. It is interesting to compare the oxygenase activities of binuclear and mononuclear iron enzymes. The iron in mononuclear oxygenases may serve either as a Lewis acid to activate the substrate (ferric enzymes) or as a Lewis base to activate oxygen (ferrous enzymes). It appears that in the binuclear enzymes the iron center performs both functions. The diferrous center first activates oxygen to the hydroperoxide and is converted

NON-HEME IRON PROTEIN CHEMISTRY

25 1

to a diferric cluster. With the subsequent formation of the highly reactive diferryl oxene, even deactivated chlorinated hydrocarbons can be activated as radicals for oxygenation. I n contrast the ferric mononuclear iron enzymes are only able to catalyze oxygenation of aromatic substrates already partially activated by ring substitutions.

1V. TETRAHEDRAL IRON:Fe : S PROTEINS The range and diversity of Fe:S proteins are so broad that a comprehensive discussion of this family is no longer possible. Representative categories of Fe :S proteins are listed in Table V. The following discussion is limited to a few examples which emphasize the diversity of the field and for which there are crystal structures. A . Ferredoxins Bacterial ferredoxins provide a fascinating example of how a diverse range of specific structures can be derived from a single protein family motif. Indeed, it is the only group of non-heme iron proteins in which sufficient numbers of structures are available to form a hypothesis that may have predictive value. Bacterial ferredoxins typically contain 50 to > 100 amino acids and function as electron transfer carriers in a variety of metabolic reactions. Sequence analyses (George et al., 1985; Bruschi and Guerlesquin, 1988) have demonstrated the relatedness of these ferredoxins; yet within this family considerable variability exists in both the number and type of Fe : S clusters present in the protein. Ferredoxins isolated from diverse bacteria contain the following combinations of cluster types: two 4Fe :4 s (eight Fe ferredoxins); one 4Fe : 4 s and one 3Fe :4 s (seven Fe ferredoxins); one 4Fe : 4s; o r one 3Fe :4s. A combination of sequence and structural analyses has provided an elegant framework for understanding the relationships between these different ferredoxins. T h e following discussion draws heavily on the presentation by Fukuyama et al. (1988, 1989), to which the reader is referred for additional details. Proteins containing two 4Fe : 4s clusters, typified by the P. aerogenes ferredoxin (PaFd), show striking sequence similarities between the amino- and carboxy-terminal halves of the molecule, suggesting that these proteins evolved by gene duplication of a more primitive protein (Section I,E,3). Of particular note is the pattern of four cysteines in the two halves of the molecule (Cys-8, -1 1, -14, -18 and Cys-35, -38, -41, -45 in the amino- and carboxyl-terminal halves, respectively, of the PaFd se-

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JAMES B. HOWARD AND DOUGLAS C. REES

TABLE V Reactions Performed by Fe :S Proteins Reaction Electron transfer 1Fe, Rubredoxins 2Fe, Ferredoxins 3Fe, Ferredoxins 4Fe (2 X 4Fe), Ferredoxins, high-potential iron protein Oxidoreductase Nitrogenase, MoFe-, VFe-, and Fe-only classes Hydrogenases, Fe-only and NiFe classes Sulfite and nitrite reductase, heme and Fe :S cofactors Xanthine oxidase, flavin, Fe : S, Mo-pterin cofactors Trirnethylamine dehydrogenase, flavin and Fe : S cofactors Furnarate reductase Succinate dehydrogenase NADH-Q-oxidoreductase CO dehydrogenase, NiFe clusters Forrnate dehydrogenase, W and Fe : S cofactors Hydratase and isomerase Aconitase, isopropylrnalate isornerase, and hornocitrate isornerase Dihydroxy-acid dehydratase Mannonic and altronic acid hydratases 2-Hydroxyglutaryl-CoA dehydratase Maleic acid hydratase Hydrolase Endonuclease I11 Protein stability/regulation of activity Glutarnine phosphoribosylpyrophosphate arnidotransferase

Reference Lovenberg (1973a,b, 1977) Lovenberg (1973a,b, 1977) Moura et al. (1978) Lovenberg (1973a,b, 1977) Burgess (1984), OrmeJohnson (1985) Adarns (1990) Ostrowski et al. (1989) Massey (1973) Lim et al. (1986) Morningstar et al. (1985) Lusty et al. (1965), Davis and Hatefi (1971) Hatefi (1985) Stevens et al. (1989) Yarnarnoto et al. (1983) Ernptage (1988) Flint and Ernptage (1988) Dreyer (1987) Schweiger el al. (1987) Dreyer ( 1985) Cunningham et al. (1989) Grandoni et al. (1989)

quence). This pattern of internal sequence homology is less evident in other members of the ferredoxin family, however. While most ferredoxins show sequence similarities to PaFd in the amino-terminal half (especially between Cys-8 and - 14), the degree of sequence conservation in the carboxy-terminal half varies widely among different proteins. Some ferredoxins, such as those from Bacillus thermoproteolyticus (BtFd), lack three of the four cysteines in the carboxy-terminal half (corresponding to Cys-35, -38, and -41 of PaFd). Significantly, proteins missing some or all of these cysteines are found to coordinate only one Fe :S cluster. Insertions and deletions of residues, both internally and at the termini of

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different ferredoxins, are also apparent from sequence comparisons. Based on these considerations, it is possible to arrange different ferredoxins into several groups of similar sequences and to construct a possible evolutionary tree relating these proteins. The structures of four bacterial ferredoxins have been crystallographically determined (Table I). Fortunately (and presumably not entirely coincidentally), these ferredoxins belong to different groups within the ferredoxin family. PaFd has two 4Fe : 4s clusters, the A. vinelandii ferredoxin (AvFd) has one 4Fe :4 s and one 3Fe :4 s cluster, BtFd has one 4Fe:4S cluster, and the D. @gas ferredoxin (DgFd) has one 3Fe:4S cluster. Cysteines liganding the clusters are listed in Table I, while structures of PaFd, AvFd, and BtFd are illustrated in Fig. 3. As described below, there are strong similarities in the folding of these proteins. Based on the structure of PaFd, the two cluster-binding sites are designated sites 1 and 2. Table VI summarizes the cluster types bound to these two sites in the different ferredoxin structures. T h e structure of PaFd was the first to be crystallographically determined (Adman et al., 1973). The basic fold of the protein may be described as a pair of two stranded antiparallel p sheets. The two 4Fe : 4 s clusters are sandwiched between these p strands on one side and several helical segments on the other side. The clusters are packed in a predominantly hydrophobic environment. The internal sequence homology is clearly reflected in the structure; the two clusters and much of the polypeptide chain are related by an approximate internal twofold rotation axis. T h e two clusters are ligated by the two sets of four cysteines in the two halves of the molecule. Surprisingly, each cluster is liganded by cysteines from both halves of the sequence, rather than cysteines from only one half (which are adjacent in the sequence). Cluster 1 is coordinated by Cys-8, - 11, and - 14 in the amino-terminal half and Cys-45 of the carboxy-terminal half, while cluster 2 is coordinated by Cys-35, -38, and -4 1 of the carboxy-terminal half and Cys- 18 of the amino-terminal half.

TABLE VI Contents of Cluster Sites 1 and 2 in Bacterial Ferredoxim Ferredoxin

Site 1

Site 2

P . aerogenes A. vinehndii B . thmoproteolytictu D. @gas

4Fe :4 s 3Fe : 4s 4Fe :4s 3Fe :4 s

4Fe :4 s 4Fe : 4 s a Helix a Helix

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JAMES B. HOWARD AND DOUGLAS C. REES

This distribution provides a cautionary tale to inferring ligating patterns from sequence data: Sequence proximity need not imply that the ligands bind the same center (see Section I,E,3). The redetermined structure of AvFd (Stout et al., 1988; Stout, 1988, 1989) demonstrated that the same basic folding pattern observed in PaFd was conserved among ferredoxins containing two clusters. There are two major differences between AvFd and PaFd: (1) a 3Fe : 4 s cluster occupies site 1 in AvFd, and (2) AvFd has a 52-residue extension at the carboxy terminus compared to PaFd. The extension forms an CY helix covering the side of the p sheet opposite the clusters and wraps around the part of the protein containing the 3Fe : 4 s cluster. Since the clusters are a primary determinant of the protein structure, the adaptations permitting the coordination of only one cluster are of great interest. The solution to this problem, revealed in the BtFd structure (Fukuyama et al., 1988, 1989), is quite elegant. T h e protein fold around the cluster in site 1 is similar in both the one- and two-cluster ferredoxins. Since the four cysteines that coordinate cluster 2 in PaFd are absent in BtFd, it is not possible for a second cluster to bind. Instead, in the region corresponding to cluster 2, the polypeptide chain in BtFd folds into an a helix. This helix packs in the structure so as to fill part of the volume normally occupied by the second cluster. The fourth cysteine in the carboxy-terminal half (Cys-61 in BtFd, which corresponds to Cys45 in PaFd) is retained in the sequence and coordinates cluster 1. Consequently, the four cysteines conserved in bacterial ferredoxin sequences are the first three in the amino-terminal half and the fourth cysteine in the carboxy-terminal half (Cys-8, -1 1, -14 and -45 in PaFd). It is interesting to note that no examples have been found in which either (1) the ligands for cluster 1 have been replaced, leaving only cluster 2 in the protein, or (2) a 3Fe :4 s cluster occupies site 2. The crystal structures do not reveal whether there are inherent structural restrictions to such mutations. T h e replacement of the second cluster by an CY helix has also been observed in DgFd (Kissinger et al., 1989), which contains a single 3Fe : 4 s cluster. Unlike BtFd, two of the four cysteines that would ligate the second cluster of PaFd are present in DgFd. In an interesting twist these two cysteines are linked in a disulfide bridge. Sequence comparisons suggest that this disulfide is also present in other (but not all) singlecluster ferredoxins (e.g., P. furiosus ferredoxin) (Eccleston et al., 199 1). Most of the differences between BtFd and DgFd occur as sequence insertions in surface loops and as a carboxy-terminal extension in BtFd. T h e factors leading to the accommodation of 4Fe : 4 s versus 3Fe : 4 s clusters are less clear. An obvious explanation is that the presence of

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either three or four cysteines specifies the coordination of a 3Fe :4 s or 4Fe :4 s cluster, respectively. Although this appears to be a contributing factor, the situation is apparently more complex. T o some degree all 4Fe :4 s clusters can be converted to 3Fe clusters by irreversible oxidative damage. However, for our discussion it is more germaine to consider two classes of 3Fe :4 s proteins. One type readily undergoes reversible 3Fe :4S-to-4Fe : 4 s conversion with minimal structural change (e.g., aconitase, DgFd, and P. furiosus ferredoxin). All three proteins have a unique non-thiol fourth ligand in the 4Fe : 4 s form; this is the site from which an iron is lost in going to the 3Fe form. In aconitase the fourth ligand is a water (hydroxyl) and in P. furiosus the usual cysteine ligand, equivalent to Cys-11 of PaFd, is replaced with aspartic acid. In DgFd Cys-1 1 appears to be modified (perhaps as a disulfide to methyl thiol) and is rotated away from the cluster. T h e second type of 3Fe : 4 s protein is exemplified by AvFd. In this class the 3Fe : 4 s cluster is stable. Because the cluster is more completely buried within the protein fold, it cannot be converted to the 4Fe form without full denaturation of the protein. Curiously, the AvFd has a potential fourth ligand, Cys- 11, in the immediate environment of the cluster. However, Cys-11 is forced away from the 3Fe:4S cluster due to a sequence insertion of two residues in AvFd sequence compared to PaFd, and, as a consequence, Cys-1 1 is too distant from the cluster to coordinate a fourth iron. T h e presence of Tyr-13 (structurally equivalent to Cys-11) partially blocks the fourth iron site. Thus, the 3Fe : 4 s cluster is accommodated, whereas considerable structural rearrangement would be needed for a 4Fe : 4 s cluster (Stout, 1989). The number and distribution of cysteines are not the sole discriminating factors for the cluster type. A case in point is the replacement of a liganding cysteine by a nonliganding residue. When Cys-20, a ligand to the 4Fe : 4 s cluster in AvFd, is changed to alanine, a structural rearrangement occurs that allows a previously nonliganding group, Cys-24, to coordinate the 4Fe : 4s cluster (Martin et al., 1990). These considerations indicate that a complex and subtle set of steric and chemical factors is involved in the binding of 4Fe :4 s and 3Fe :4 s clusters. Consequently, it is not yet possible to predict from the sequence alone the type of cluster that will be coordinated to a protein. The role of 3Fe clusters is open to speculation. Some proteins appear to be functional only as the 3Fe form (e.g., AvFd and fumarate reductase). For others the 3Fe form may be an unfortunate side effect of having a reactive readily displaced ligand which is required for the enzyme mechanism as in aconitase. Because the 3Fe cluster can take up other metals (e.g., nickel, cadmium, or zinc) (Moura et al., 1986; Surerus et al., 1987; Surerus, 1989), it is possible that the 3Fe form may be the precursor for

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more complex clusters with new functions (Stevens et al., 1989). In the case of ferredoxins such as DgFd and PfFd, the ready interconversion between forms allows one protein structure to perform more than one function (i.e., to have forms with different redox potential). DgFd can be isolated in either of two forms; DgFd I contains a 4Fe : 4 s cluster and couples the phosphoroclastic reaction to hydrogenase (Eb = -460 mV), while DgFd I1 contains a 3Fe : 4 s cluster and serves as the electron donor to sulfite reductase (Eb = - 130 mV) (Moura et al., 1978). T h e intriguing possibility exists that the disulfide with Cys-11 is sensitive to the redox state of the cell and controls which metabolic path is operative. Finally, the interconversion of cluster types may serve as an oxygen-sensitive regulation site to turn the enzyme on and off. This has been proposed for glutamine pyrophosphoribosylpyrophosphateamidotransferase (amidophosphoribosyltransferase) (Grandoni et al., 1989). A brief historical note on the structure of the iron-sulfur clusters in ferredoxins is relevant. After the first analytical results revealed the presence of (nearly) equimolar iron and acid-labile sulfur, it was clear that the metal center in ferredoxins did not resemble any previously characterized cofactor type. The early proposals for the Fe : S center structure were based on a linear chain of iron atoms coordinated by bridging cysteines and inorganic sulfur (Blomstrom et al., 1964; Rabinowitz, 1971). While the later crystallographic analyses of HiPIP, PaFd, and model compounds (Herskovitz et al., 1972) demonstrated the cubanetype structure of the 4Fe : 4 s cluster, the original proposals have turned out to be somewhat prophetic. Linear chains of sulfide-linked irons are observed in 2Fe : 2 s ferredoxins and in the high-pH form of aconitase. Cysteines linked to several metal atoms are present in metallothionein. Th e chemistry of iron-sulfur clusters is rich and varied, and undoubtedly many other surprises await in the future. B . Nitrogenase The nitrogenase enzyme system catalyzes the reduction of atmospheric dinitrogen to ammonia, which is one of the principal mechanisms for the assimilation of dinitrogen into the metabolic processes of living organisms (reviewed by Burgess, 1984; Orme-Johnson, 1985). The nitrogenase system consists of two proteins, the iron (Fe) protein and the molybdenum iron (MoFe) protein. T h e first step in the general scheme of dinitrogen reduction involves the reduction of Fe protein by an electron carrier (e.g., ferredoxin). Electron transfer from Fe protein to MoFe protein requires hydrolysis of MgATP. Under tightly coupled conditions t w o MgATP molecules are hydrolyzed per electron transferred. This step

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must be repeated until the MoFe protein has accumulated sufficient electrons for substrate reduction. Each electron transfer appears to involve a cycle of association/dissociationof the protein complex. Substrate reduction apparently occurs on the MoFe protein, although involvement of the Fe protein at this step is plausible, since Fe protein is the only known reductant for MoFe protein that results in catalytic activity. Important details of the reaction mechanism, including the types of partially reduced nitrogen intermediates generated, the sequence of electron flow through the various clusters, and the role of MgATP in this process, are, at best, only sketchily understood. As the names of the component proteins imply, iron-containing redox groups are essential to nitrogenase function. These clusters are of the Fe : S type, but they exhibit many unique features that are not present in “simpler” protein and model systems. A brief summary of the properties of the nitrogenase proteins, with emphasis on the metal clusters, follows. 1 . Fe Protein Fe protein is a dimer of two identical subunits, with a total molecular weight of about 60,000. The dimer contains one 4Fe :4s cluster, exists in the + 1/+2 states and has a redox potential of about -300 mV. T h e cluster is coordinated to both subunits through two cysteines, numbered 97 and 132 in the A. vinelandii protein sequence (Hausinger and Howard, 1983; Howard et al., 1989). These studies indicate that the cluster is symmetrically liganded to each subunit, presumably resulting in a dimer with an internal twofold symmetry axis. Spectroscopic, extrusion, and redox studies of Fe protein suggest that the 4Fe :4s cluster generally resembles that of ferredoxin, although it is quite likely that the cluster environment differs significantly between ferredoxins and Fe protein. The most striking indication of this is the ability to generate 2Fe: 2 s clusters in oxidized Fe protein in the presence of MgATP and chelator; such interconversions have not been observed in ferredoxins (Anderson and Howard, 1984). In addition the Fe protein cluster appears to be much more exposed to solvent than in ferredoxins, as indicated by rapid D20 exchange in pulsed EPR studies (Morgan et al., 1990). The nucleotide-binding properties of the component proteins indicate that Fe protein is critically involved in the coupling of electron transfer to ATP hydrolysis. Fe protein has two nucleotide-binding sites per dimer, with dissociation constants measured in the range of 10 to > l o 0 Detailed binding constants and cooperativity between binding sites have been difficult to establish, in part due to the extreme oxygen sensitivity of the nucleotide-protein complex. Binding of adenine nucleotides leads to pronounced changes in the redox, spectroscopic, and cluster chelation

m.

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behavior of Fe protein. The complex kinetics of cluster chelation in the presence of MgATP are consistent with the presence of two conformers of Fe protein (Diets and Howard, 1989). While chelation can occur from either conformer, interconversion of the two forms is prevented by MgATP binding. The oxidized form of Fe protein binds MgATP more tightly than the reduced form, resulting in a decrease in redox potential of the 4Fe : 4 s center from -300 to less than -400 mV in the presence of this nucleotide. ATP does not appear to bind directly to the cluster, since stimulated spin-echo EPR studies indicate that the phosphates are >6 A from the cluster (Morgan et al., 1990). T h e tertiary structure of the A. vinelundii Fe protein has been crystallographically determined (Georgiadis et al., 1990). At the current stage of the analysis, the fold of the polypeptide chain and the positions of many (but not all) side chains are available. The overall shape of the Fe protein dimer resembles a butterfly (Fig. 3). Each subunit of Fe protein is a single domain with an alp-type fold. The cluster is located near the “top” of the dimer, at the “head” of the butterfly. A cleft exists “underneath” the cluster in the middle of the dimer, at the interface between the two subunits. Unlike the ferredoxin 4Fe : 4 s clusters that are completely surrounded by protein, and hence buried from exposure to solvent, the Fe protein cluster is exposed to solvent along the top face. This solvent accessibility was observed in pulsed EPR studies. An a helix containing residues 98-1 10 extends from the cluster. Two residues, Arg-100 and Glu-112, which have been implicated in the binding of Fe protein to MoFe protein (Pope et al., 1985; Murrell et al., 1988; Lowery et ul., 1989; Willing and Howard, 1990; Wolle et al., 1991), are located in or near this helix, suggesting that, at a minimum, this part of Fe protein interacts with MoFe protein. Although the MgATP-binding sites have not been completely described crystallographically, Fe protein contains a nucleotidebinding site involving residues near the amino terminus (Robson, 1984). On the basis of this motif, the ATP phosphates will be located near the cleft region, approximately 20 8, from the cluster. It is possible that the remainder of the ATP molecule will bind in the cleft region, between the two subunits. T h e construction of Fe protein from two subunits, with the cluster substantially forming the interface between them, implies that it may be easier to couple conformational changes associated with nucleotide binding to the properties of the 4Fe:4S cluster than would be possible in proteins in which the cluster is embedded in the interior of the protein. 2 . MoFe Protein T h e MoFe protein is a tetramer containing two a and two /3 subunits, both a and p having molecular weights around 60,000. The tetramer

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contains two molybdenum and 30 2 iron atoms distributed among six clusters. These clusters are of two types: the FeMo cofactor (or M centers) and the P clusters. Two M centers are present per tetramer. The properties of the M centers are described in more detail in Section IV,B,3. Each tetramer also contains four P clusters, each having four irons. The properties of the P centers differ in several respects from more usual 4Fe :4S clusters (Zimmermann et al., 1978;Huynh et al., 1980;McLean et al., 1987; Lindahl et al., 1988): The iron atoms in each center may be assigned by Mossbauer spectroscopy to three distinct types, designated D, Fe2+,and S; oxidized P clusters exist in multiple spin states; and under many conditions oxidized P clusters are EPR silent. The four P centers may be grouped into two pairs with slightly different spectroscopic properties. The two oxidation states of P centers differ by an odd number of electrons. The general picture suggests that a total of four electrons may be removed from the set of P centers, although this view is not universally accepted (Watt et al., 1981; Hagen et al., 1987). Tantalizing glimpses of MoFe protein structure are being revealed by crystallographic analyses. At low resolution MoFe protein exhibits pseudo-222 symmetry (Yamaneet al., 1982),suggesting that the polypeptide folds of the a and p subunits are similar. While the structures of the subunits and cofactors are still not known, some intriguing observations concerning the relative locations of the cofactors in the MoFe protein structure are available. Bolin et al. (1990)has collected anomalous scattering data from crystals of the Clostridium pasteurianum MoFe protein. Based on analyses of these diffraction data, which are dominated by the iron and molybdenum atoms in the various cofactors, Bolin has evidence that the MoFe protein cofactors are organized into four discrete units at low resolution. These four units are arranged in two pairs related by the twofold molecular symmetry axis. Units within each pair are approximately 19 8, apart, while equivalent units related by the molecular twofold are approximately 70 8, apart. Since six centers have been identified in MoFe protein spectroscopically and chemically, the finding that only four units are present suggests that pairs of centers are in such close physical proximity that they appear as single objects at low resolution. The interpretation favored by Bolin is that the four units represent the two M centers and two pairs of P centers. The latter might actually consist of clusters larger than 4Fe :4s. While eight iron-containing P centers have been proposed (Hagen et al., 1987) (two per tetramer), spectroscopic evidence (Lindahl et al., 1988) has been presented discounting this possibility. Resolution of this issue will be of great interest in relating the cofactor arrangement and properties to the electron transfer reactions of nitrogenase.

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3. Cofactor

One type of the constituent metallocenters in the MoFe protein has the properties of a somewhat independent structural entity. This component, referred to as the FeMo cofactor (FeMo-co), was first identified by Shah and Brill (1977) as the stable metallocluster extracted from acid-denatured MoFe protein. The FeMo-co was able to fully activate a defective protein in the extracts of mutant strain UW45, a protein which subsequently was shown to contain the P clusters but not the EPR-active center. The isolated cofactor accounted for the total S = 3 system observed by EPR and Mossbauer spectroscopies of the holo-MoFe protein (Rawlings et al., 1978). Elemental analysis indicated a composition of M o : Fe6-8 :s6-9 for the cofactor, which, if there are two FeMo-co’s per a2&, accounts for all the molybdenum and approximately half the iron in active enzyme (Nelson et al., 1983). Although FeMo-co has been extensively studied [reviewed in Burgess (1990)l the structure remains enigmatic. T o date, all attempts to crystallize the cofactor have failed. This is possibly due to the instability and resultant heterogeneity of the cofactor when removed from the protein. Also, there is a paucity of appropriate models for spectral comparison (see Coucouvanis, 1991, for a recent discussion). Final resolution of this elusive structure may require its determination as a component of the holoprotein. T h e salient points for the structure, as determined using both the isolated FeMo-co and the holoenzyme, can be outlined as follows: (1) Cofactor is the site of substrate reduction (Rawlings et al., 1978; Hawkes et al., 1984; Imperial et al., 1989); (2) M o environment contains at least (Eidsness et al., 1986; Conradson et al., 1987) 2 O(N) at 2.10 8, (first coordination sphere), 3 S at 2.36 8, (first coordination sphere), and 3 Feat 2.68 8, (second coordination sphere); (3) Fe environment contains at least (Antonio et al., 1982; Arber et al., 1988) 1-2 O(N) at 1.87 8, (first coordination sphere), 3 S at 2.20 8, (first coordination sphere), 2 Fe at 2.64 %, (second coordination sphere), 1 M o at 2.70 8, (second coordination sphere), and 1 Fe at 3.68 A; (4) Irons are in five different magnetic environments (True et al., 1988). Although it is tempting to consider the cubane 4Fe : 4 s clusters as a starting point for visualizing the FeMo-co, there are several important differences. First, there appears to be only a limited number of proteinbased ligands, perhaps as few as two, one nitrogen and one thiol, per cofactor. That is, there are few terminal ligands for a cluster of six to nine metals. The thiol appears to be Cys-275 of the a subunit, based on mutagenesis (Kent et al., 1989) and thiol labeling of the UW45 protein (Magneson et al., 1991). Exogenous thiolates reversibly bind to the cofac-

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tor and are displaced by substrates (Conradson et al., 1989). A nitrogen ligand is predicted by electron spin echo (Thomann et al., 1987) and MoFe protein mutagenesis (Scott et al., 1990). Second, some of the bond distances are outside the ranges for cubane models. Third, the iron environments are considerably more asymmetrical, as judged by the Mossbauer and electron nuclear double resonance (ENDOR) spectra (True et al., 1988; Huynh et al., 1979). Perhaps the most significant and unexpected recent development in the FeMo-co structure function inquiry is the discovery that homocitric acid [ (R)-2-hydroxy-1,2,4-butanetricarboxylic acid] is a component (Hoover et al., 1987). It has long been known that several gene products besides the structural proteins for MoFe protein are needed for the biosynthesis of active nitrogenase. For mutants in one of these, n i N , activity in in vitro cofactor synthesis could be restored if homocitric acid was added. Thus, it appears that nifV encodes a homocitrate synthetase analogous to citrate synthase. Previously, it had been shown that nitrogenase in nip- mutants could not reduce dinitrogen, but retained the ability to reduce protons and the nonphysiological substrate acetylene (McLean et al., 1983). Recently, Liang et al. (1990) showed that in this mutant citric acid is substituted for homocitric acid, with the resultant change in substrate specificity. In a series of elegant papers, Ludden and co-workers (Imperial et al., 1989; Madden et al., 1990) worked out much of the structural requirements for the organic component of the cofactor. These are summarized with reference to Fig. 15: (1) OH and COOH are required on C-2; (2) C-2 has the R configuration; (3) a COOH is required on C-1; (4)Substituents on C-1 are trans to OH on C-2; (5) a variety of functional groups can be substituted for Y leading to altered substrate specificity; (6)X can be H, OH, or F; and (7) C-1 has the S configuration.

HOOC-C,-OH

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The array of altered substrate specificity is impressive. For example, only (R)-homocitrate and (S,R)-F-homocitrate allow dinitrogen reduction, while several di- and tricarboxylic acids permit the reduction of alternate substrates such as CN- and acetylene. For a third, less restrictive, group of acids, protons are reduced, but not the triple-bonded substrates. Not only is the substrate specificity partially defined by the organic component of the cofactor, but so is the pattern of inhibition by reagents such as CO. It appears that both steric and inductive effects are important in determining the specificity conveyed by a particular carboxylic acid. It is tempting to speculate that there may be a direct chemical role of the acid, as well as the more obvious role as a bi- or tridentate ligand that confers a required conformation to the cluster. Clearly, the homocitrate component must be included in any bioinorganic models, and failure to do so may explain the difficulty in mimicking the cofactor to date. Substrate specificity is also controlled in part by amino acid side chains, as demonstrated by mutagenesis experiments (Scott et al., 1990). Whether these substitutions are disrupting cofactor-substrate interactions directly or by altering the binding of homocitrate is not known. It should be noted that some of the analogs of homocitric acid may have alternate binding modes. Likewise, steric effects of the protein may alter the binding of analogs.

C. Aconitase: A Nonredox Fe :S-Containing Enzyme As shown in Table V, a number of Fe : S-containing proteins perform reactions other than redox o r electron transfer. That is, the function of the cluster does not include a change in oxidation state, even as a transient step in catalysis. This role is best illustrated by aconitase, one of the most extensively studied Fe : S proteins, regardless of function. The elegant recent work on this enzyme is largely under the guiding hand of H. Beinert and is summarized in the Krebs Memorial Lecture (Beinert and Kennedy, 1989). Aconitase was the third protein and the first enzyme for which a 3Fe cluster was postulated by Mossbauer spectroscopy (Kent et al., 1982); the meticulous inorganic sulfide analysis by Beinert et al. (1983) established the composition of this class of cluster as 3Fe : 4s. Although the 3Fe form of the protein is inactive, it can be reactivated by the addition of Fe2+ under anaerobic reducing conditions (Kennedy et al., 1983). The resulting cluster was shown by Mossbauer spectroscopy to be similar to ferredoxin 4Fe:4S clusters in the +2 oxidation state; the more reduced cluster was only -30% active. Mossbauer spectroscopy also indicated that

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one iron site (referred to as site “a”) had unique properties, including being the exclusive site of the added iron in the activation process. On mild oxidation (e.g., aerobic isolation of the enzyme) the iron of site a was lost. Likewise, this site rapidly exchanged iron with solution, while the other three required more vigorous conditions (Kennedy et al., 1983). Finally, the 4Fe :4 s cluster could be generated by reducing the 3Fe form without the addition of exogenous iron. In the latter reaction only -75% 4Fe form was recovered, suggesting that some of the 3Fe clusters had been sacrificed to the formation of 4Fe clusters. The combination of these results strongly argues that the 3Fe :4 s cluster is a cubane cluster missing one of the corner iron atoms (see Fig. 16a). These unique properties of the cluster, expecially regarding the site “a” iron, were further emphasized when substrate was added. T h e Mossbauer isomer shift of the site “a” iron increased from 0.45 mm/sec, typical of iron in [4Fe :4SI2+ clusters, to 0.89 mm/sec, which approaches that of octahedral high-spin ferrous iron (Kent et al., 1985). Significantly, only the parameters for the “a” site iron changed. These properties of Fe, and the fact that no thiolate ligand could be detected when the 4Fe-to-3Fe conversion occurred (Kennedy and Beinert, 1988; Plank and Howard, 1988) strongly argue for a ligand other than cysteine. Thus, it was satisfying when 1 7 0 ENDOR spectroscopy identified a possible ligand as H 2 0 from solvent (Kennedy et al., 1987). This was confirmed by the crystal structure of the 3Fe and 4Fe forms (Robbins and Stout, 1989a,b), in which no fourth cysteine was close enough to be a potential ligand and a hydroxyl group was found on Fe,. The aconitase structure is composed of four domains (see Fig. 3), domains 1,3, and 4 being roughly 200 residues and domain 2 being 100 residues (Robbins and Stout, 1989a,b). The cluster ligands were identified by both X-ray diffraction and chemical modification studies as residues 358,421, and 424 (Robbins and Stout, 1989a; Plank et al., 1989). Although these residues are contained in domain 3, all four domains contribute to the putative active-site pocket buried at the interface of domains 1-3; domain 4 forms a flap above the cluster. One interesting feature of the domains is their resemblance to nucleotide-binding proteins in terms of the secondary-structure motifs of P sheets and a helices, although the overall structures are different (Robbins and Stout, 1989a). T h e fourth domain is attached by a loop which may allow flexibility and access to the active site. Notwithstanding these studies, the role of the iron cluster in the catalysis remains an enigma. Clearly, the substrate binds to the Fe, site by the Mossbauer spectra. Also, the l7O ENDOR spectra of aconitase with specifically 70-labeled substrates and inhibitors imply that the P-carboxyl

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FIG. 16. Environment of the iron-sulfur center in aconitase.

(the middle carboxyl), and likely the hydroxyl of substrate, bind to the iron (Kennedy et al., 1987). Werst et al. (1990) reported that the exogenous ligand is a hydroxyl which becomes protonated to H 2 0 when the substrate binds (see Fig. 16b). It is tempting to suggest that the iron acts as a Lewis acid to labilize the substrate hydroxyl. However, there is no obvious explanation for the early stereochemical studies on the aconitase reaction (Rose and OConnell, 1967). Namely, the hydroxyl group from the substrate is exchanged with solvent, while the proton removed from the a carbon is conserved in the transfer to the fi carbon. Because the elimination and addition are trans, the mechanism would seem to require

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the cis-aconitate intermediate to flip 180” for the conversion between isocitrate and citrate. Furthermore, the requirement for a 4Fe :4s cluster to serve as the hypothetical Lewis acid is not evident; for example, mononuclear trigonal bypyramidal iron serves as a Lewis acid in the dioxygenase reactions (see Section 11,A). Hence, even with the crystal structure our level of sophistication is inadequate at this point to rationalize a mechanism. Aconitase has not only provided a tantalizing puzzle in biochemical reaction mechanisms, but also a labyrinth of interconverting Fe :S clusters. Besides those discussed above, the aconitase Fe : S cluster can be substituted with other metals and selenium (see Beinert and Kennedy, 1989).One fascinating example is the interconversion of the 3Fe and 4Fe forms to the linear 3Fe cluster at pH >9 or in denaturing buffers (Kennedy et al., 1984)(see Fig. 16a).This reaction is not a simple random destruction of a cluster with the subsequent formation of a new structure; rather, as Plank et al. (1989)showed, the ligands (Cys-421, -424, -250, and -257) of the linear “purple” cluster are derived from the active cluster. This can be best explained by the rotation of a single peptide bond between the second and third domains which allows the two cysteines, 250 and 257, to bridge the new cluster. These two residues are on the same face of an (Y helix and at a distance which allows spanning the linear 3Fe structure. Three different arrays for the disposition of the iron atoms are possible (see Fig. 16a): b-c-d-, c-b-d, or c-d-b. We favor c-b-d because it requires the fewest bonds to be broken and allows the two terminal cysteines from the original 3Fe cluster to remain on their original iron atoms. Although the linear 3Fe cluster conversion is not relevant to the enzyme biological activity, it does demonstrate the wealth of information about Fe : S chemistry afforded by the study of this protein. D. Fe :S Proteins with Other Prosthetic Groups

Fe : S-containing enzymes frequently have other redox-active prosthetic groups, notably, flavins FAD or FMN. Likewise, the redox partner for many Fe : S proteins is a flavoprotein; this provides a convenient mechanism for turning a one-electron transfer reaction into a two-electron donor/acceptor. Hence, the structures elucidating the interactions between Fe : S clusters and other cofactors are of considerable interest. At present there are only two examples for which we have crystallographic structures, yet both provide a basis to propose possible mechanisms for electron transfer to Fe : S clusters.

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1. Trimethylamine Dehydrogenase (TMAD) TMAD is a [4Fe : 4S] flavoprotein found in several methylotropic bacteria and catalyzes the oxidative demethylation of trimethylamine (Steenkamp et al., 1978). Although the amino acid sequence has yet to be completed, the 2.4 A crystallographic structure is sufficient to assign most of the primary sequence and the putative side chains (Lim et al., 1986; Mathews and Lim, 1987). (As these authors note, one should be circumspect when extracting mechanistic details based on the “X-ray sequence.”) The two identical subunits ( M , -65,000) are arranged about a molecular two-fold axis. The subunits have three domains, each of which is composed substantially of (Y helix and p sheet secondary structures (see Fig. 2). The first domain, approximately the amino-terminal half of the molecule, has the motif of [@]a and can be considered a large parallel barrel surrounded by the parallel (Y helices. This barrel structure has been found for two other flavoproteins: flavocytochrome 6 2 and glycolate oxidase. The second domain has two parts, the two halves being interrupted by short loops, and the third domain which “buds” off one of the loops. The loops provide the main contacts between subunits. The fold for domains 2 and 3 resemble the glutathione reductase superfamily of NAD(P)H/flavin oxidoreductases. However, in TMAD there are no cofactors associated with these domains, and the reasons for the conservation of secondary structure organization is not obvious. For the purposes of this chapter, the most interesting region of the molecule is the first domain and the cofactor-binding sites, as shown in Fig. 17. The FMN is attached to Cys-30 via the benzenoid C-6. Its location near the end of the first /3 strand is similar to the FMN binding in the other two flavoproteins. T h e isoalloxazine ring of the flavin is bent -20” along the N-5, N-10 axis and is positioned at an angle to the barrel axis. T h e si face of the flavin projects into the barrel central channel, which leads to the open interface between subunits. Presumably, this is the path for substrate approach to the active site. T h e [4Fe : 4S] cluster is nearby (-12 A, between cofactor centers) and is bound between an a helix and an antiparallel loop which terminates the first domain. Interestingly, the cluster ligands are in a “classical” 4Fe ferrodoxin-like sequence of residues Cys-347, -350, -353, and -366. Both the Fe cluster and the FMN are buried -20 8, from the surface and in an aromatic-rich hydrophobic environment. Although the separation between cofactor centers is long for efficient electron transfer, the methyl C-8a and the sulfur of Cys-353 are in van der Waals contact (-4 A). Difference electron density maps at 6 A resolution for enzyme complexes with substrate or inhibitor are consistent with the ligand-binding site’s being near 0 - 4 and N-5 of the

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FIG. 17. Active-site environment in trimethylamine dehydrogenase. (Reproduced with permission from Lim et al., 1986.)

flavin ring (Bellamy et al., 1989). In addition there appears to be some movement in the region between the two cofactors when substrate is bound. Hence, the structure accounts for a mechanism where substrate binds to the flavin, which is rapidly reduced. In the rate-determining step the reduced flavin transfers one electron to the Fe : S cluster, resulting in a flavin ( S - t ) and a [4Fe :4S]+ (S = *), which are spin coupled to S = 1. It is this triplet state that reacts with 0 2 . The spin-coupling model based on EPR studies (Stevenson et al., 1986) predicted a close approach between centers, similar to the 4 8, found in the crystal structure, which

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provides one of the rare examples of a defined electron transfer pathway.

2. Suljite Reductase Sulfite reductase catalyzes the six-electron reduction by NADPH of SO;- to S2- and NO, to NHJ. In E. coli this enzyme is a complex structure with subunit composition asp4 (Siege1et al., 1982). The enzyme active site is on the p subunit, which contains both a 4Fe :4 s cluster and a siroheme prophyrin. Substrates and ligands have been found to bind to the siroheme. T h e a subunit binds NADPH and serves to shuttle electrons to the active site through bound FAD and FMN groups. Isolated p subunits can catalyze sulfite reduction in the presence of a suitable electron donor. Spectroscopic studies indicate that the siroheme and 4Fe :4 s cluster are exchange coupled in all oxidation states (Christner et al., 1984; Cline et al., 1985a,b). The structural basis of this coupling has been provided by a preliminary 3 resolution X-ray structure of sulfite reductase in the oxidized state (McRee et al., 1986). The siroheme and the 4Fe : 4s cluster are packed against each other and appear to share a common ligand (Fig. 18). The distance from the siroheme iron to the cluster center is

FIG. 18. Model of the active-site environment of sulfite reductase. (Reproduced with permission from Ostrowski et al., 1989.)

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5.5 A, while the distance from the siroheme iron to the nearest cluster iron atom is 4.4 A. One of the cluster sulfur atoms is in van der Waals contact with the siroheme ring. T h e X-ray structure and spectroscopic experiments (Madden et al., 1989) suggest that a cysteine thiol sulfur serves as the bridging ligand between the cluster and the siroheme. The sixth coordination site of the siroheme appears to be vacant and solvent exposed. This site presumably represents the location of substrate binding to sulfite reductase. Amino acid sequence homology has been identified (Ostrowski et al., 1989) between the sulfite reductase p subunit and nitrite reductase, which also contains siroheme and a 4Fe :4 s cluster. A model for the active center of these two proteins based on sequence comparisons has been proposed. Only four cysteins occur in regions of sequence similarity between the two proteins. The conserved cysteines are located in two sequence regions, each of which contains two cysteines. The sequences of these two groups, Cys-(X)s-Cys and Cys-(X)a-Cys, are reminiscent of the cluster ligand sequences in ferredoxins, which suggests that these four cysteines provide the 4Fe : 4 s cluster ligands. Crystallographic and modeling studies cannot presently distinguish which cysteine serves as the bridging ligand between the cluster and the siroheme. The mechanism of substrate reduction by sulfite reductase has not been established. T h e close contact between the 4Fe : 4 s cluster and the siroheme could provide an efficient pathway for multielectron transfer from the enzyme to the substrate (McRee et al., 1986). Of special significance is the possibility that the cluster-siroheme overlap could stabilize high-oxidation states of the siroheme that might be involved in the catalytic mechanism. With the availability of genetic, biochemical, spectroscopic, and crystallographic approaches, it is anticipated that rapid progress will be made in working out the details of substrate reduction by sulfite reductase.

E . Fe :S Proteins with Non-thiolate Ligands: Rieske-Type Fe :S Proteins

One of the earliest recognized Fe : S proteins was that associated with mitochondria1 electron transport (Rieske et al., 1964). Even in the first partial in vivo characterization it was apparent that the protein had spectral properties that set it apart from the bacterial and plant-type ferredoxins which had just been discovered. Namely, the EPR spectrum had a gave near 1.91 and the high-field g value was shifted upfield. Furthermore, the protein had an Eo of approximately +250 mV, 600 mV more positive than the ferredoxins. Due to the instability of the protein, a more detailed analysis was not possible until the 198Os, when an analogous protein was isolated from bacterial sources (Fee et al., 1984). T h e ensuing

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characterization of the cluster is a paradigm of the power that multiple spectroscopic techniques can have in elucidating an iron center in lieu of a protein crystal structure. These studies unambiguously established for the first time that Fe : S clusters could have nonsulfur ligands in proteins, namely, histidyl nitrogen ligands. Subsequently, clusters with similar spectroscopic signatures have been found in a variety of proteins with both redox and nonredox functions, for example, NADH- and pterdinelinked aromatic oxygenases (see Section I1,A) and dihydroxy-acid dehydratase (Flint and Emptage, 1988). The evidence for the histidyl ligands is summarized here. By elemental analysis the cluster contained two irons. T h e EPR and Mossbauer spectra of the oxidized protein indicated that the cluster was diamagnetic and both irons were in the high-spin ferric state with tetrahedral coordination (Fee et al., 1984). Unlike plant-type ferredoxins, the Mossbauer spectra indicated that the two ferric irons of oxidized Rieske protein were in different environments (AEQ = 0.91 and 0.52 mm/sec), suggesting terminal ligands other than sulfur for one of the irons. When the protein was reduced, the cluster became a paramagnetic S = 4 system with the “trapped-valence” electronically coupled irons, as had been found for other two iron ferredoxins. Because the ferrous site had an unusually large AEQ (i.e., 3.05 mm/sec), it was likely to be the iron with the nonsulfur ligand(s). A Cpv symmetry with two nonsulfur ligands to one of the irons was predicted by resonance Raman spectra (Kuila et al., 1987). ENDOR and electron spin-echo spectra of the related proteins from several sources demonstrated that the nonsulfur ligands were nitrogens and likely from histidines (Cline et al., 1985a,b; Telser et al., 1987; Gurbiel et al., 1989). Gurbiel et al. (1989) presented a model in which the two cysteinyl sulfurs, the two irons, and the two histidyl nitrogens were in a common plane. Furthermore, there was little rotation of the histidyl rings with respect to the ligand iron plane. The EXAFS analysis confirmed the ENDOR model and found that the Fe-S and Fe-Fe bonds and angles were within the limits of other Fe : S models and proteins (Tsang et al., 1989). This suggests that the nitrogen ligands do not significantly alter the core structure, although they apparently have a major influence on the redox potential. T h e protein sequences from six different sources have been determined and compared to attempt identification of possible ligands (Gatti et al., 1989). The conserved consensus sequence around the cysteines is shown in Fig. 19. Clearly, there are several possible combinations of cysteines and histidines that fit established “bite” distances in other Fe : S proteins. On the basis of preferred sequences, bite distances, and the required order of ligands, we suggest that Cys-129-Cys-134 for the ferric

27 1

NON-HEME IRON PROTEIN CHEMISTRY

130

135

150

145

155

'-/VVbG I C T H L G C V P I G 2AA/ G G W F C P C H G S H Y Dn/

u t2t

LLf

m

t

w

FIG. 19. Sequence features of the Rieske-type Fe : S proteins. C, Cysteine; D, aspartic acid; F, phenylalanine; G , glycine; H, histidine; I, isoleucine; L, leucine; P, proline; S, serine; T, threonine; V, valine, W, tryptophan; Y, tyrosine.

site and His- 151-His- 154 for the ferrous site seem highly probable as the ligands. It is worth noting that the sequences Cys-Val-Pro-Ile and Cys-Gly-Ser-Cys (for the Rieske protein it would be His-Gly-Ser-His) are common in ferredoxins and other Fe : S proteins.

V. CONCLUSIONS It is expected that structural studies of proteins will provide a molecular basis for understanding the details of their function. This goal is particularly relevant to non-heme iron proteins because of the diversity of chemical processes performed by these enzymes. As indicated in this chapter, our ability to develop mechanistic schemes and to predict the outcome of structural changes remains in its infancy. Nevertheless, expectations for the future remain high. As evident from unpublished results in meetings and informal discussions, the field of non-heme iron proteins is advancing more rapidly than can be adequately reviewed, leaving the reader with the best to come. Certainly, exciting areas for future developments include mixed-metal clusters, new types of iron centers in proteins from bacteria growing in extreme environments, and the application of rapid structural methods based on diffraction and NMR techniques. Although the goal of predicting function from structure has not been reached, it is reasonable to proceed with mutagenesis techniques to design and modify existing iron proteins. For example, the three oxygenutilizing enzymes with binuclear clusters have partial mechanisms and intermediates in common. It should be possible to convert one type of reaction to another by appropriate structural manipulation. Likewise, the hypothesis of what protein structural constraints impose a specific type of Fe : S cluster can be tested by a combination of mutagenesis, spectroscopy, and crystallography of smaller Fe : S proteins. The explosive

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growth of developments in protein systems, converging with increasingly realistic model compound studies, portend an exciting future for nonheme iron proteins. NOTEADDEDIN PROOF. The amino acid sequence of Peptococctu aerogenes ferredoxin has been revised based upon new, high resolution X-ray diffraction studies (Backes et al., 1991). The new numbering has an insertion after residue 25 such that the last four cysteines are one higher.

ACKNOWLEDGMENTS We thank J. Lipscomb and A. Orville for helpful discussions about the mechanisms of dioxygenases; L. Que for providing a copy of his review prior to publication; J. Bolin for communicating preliminary results; E. N. Baker, H. Eklund, M. Ludwig, and D. H. Ohlendorf for figures; J. P. Allen, A. Chirino, M. Day, and K. Mertes for discussions; and P. Ray and J. Hantsch for preparation of the manuscript. This work was supported in part by National Institutes of Health grants GM 34321 (to J.B.H.), GM 45162 (to D.C.R.), and National Science Foundation grant 88-86920 (to J. B. H.).

REFERENCES Adams, M. (1990). Biochim. Biophys. Acta 1020, 115-145. Adams, M., Eccleston, E., and Howard, J. (1989). Proc. Natl. Acad. Sci. U.S.A. 86, 49324936. Adman, E. T. (1982). In “Electron Transport and Oxygen Utilization” (C. Ho, ed.), pp. 36 1-364. ElsevierINorth-Holland, Amsterdam. Adman, E. T., Sieker, L. C., and Jensen, L. H. (1973).J.Biol. Chem. 248,3987-3996. Adman, E., Watenpaugh, K. D., and Jensen, L. H. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 4854-4858. Adman, E. T., Sieker, L. C., and Jensen, L. H. (1976).J.Biol. Chem. 251,3801-3806. Adman, E. T., Sieker, L. C., Jensen, L. H., Bruschi, M., and LeGall, J. (1977).J.Mol. Biol. 112,113-120. Aisen, P., and Listowsky, I. (1980). Annu. Rev. Biochem. 49,357-393. Allen, J. P., Feher, G., Yeates, T. O., Komiya, H., and Rees, D. C. (1987).Proc. Natl. Acad. Sci. U.S.A. 84,5730-5734. Allen, J. P., Feher, G., Yeates, T. O., Komiya, H., and Rees, D. C. (1988). Proc. Natl. Acad. Sci. U.S.A. 85,8487-8491. Anderson, G., and Howard, J. B. (1984). Biochemistry 23,2118-2122. Anderson, B. F., Baker, H. M., Dodson, E. J., Norris, G. E., Rumball, S. V., Waters, J. M., and Baker, E. N. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 1769-1773. Anderson, B. F., Baker, H. M., Norris, G. E., Rice, D. W., and Baker, E. N. (1989).J.Mol. Biol. 209,711-734. Anderson, B. F., Baker, H. M., Norris, G. E., Rumball, S. V., and Baker, E. N. (1990).Nature (London) 344,784-787. Antonio, M., Teo, B.-K., Orme-Johnson, W., Nelson, M., Groh, S. Lindahl, P., Kauzlarich, S., and Averill, B. (1982).J.Am. Chem. SOC. 104,4703-4705.

NON-HEME IRON PROTEIN CHEMISTRY

273

Arber, J., Flood, A., Garner, C., Gormal, A., Hasnain, S., and Smith, B. (1988). Bi0chem.J. 252,421-425. Arciero, D., and Lipscomb, J. (1986).J.Biol. Chem. 261,2170-2178. Arciero, D., Lipscomb, J., Huynh, B.-H., Kent, T. A., and Munck, E. (1983).j.Biol. Chem. 258,1498 1- 14991. Atkin, C., Thelander, L., Reichard, P., and Lang, G . (1973).j.Biol. Chem. 248,7464-7472. Axcell, B. C., and Geary, P. J. (1975). Bi0chem.J. 146, 173-183. Backes, G., Mino, Y., Loehr, T., Meyer, T., Cusanovich, M., Sweeney, W., Adman, E., and Sanders-Loehr, J. (1991).J. Am. Chem. Sot. 113,2055-2064. Bailey, S., Evans, R. W., Garratt, R. C., Gorinsky, B., Hasnain, S., Horsburgh, C., Jhoti, H., Lindley, P. F., Mydin, A., Sarra, R., and Watson, J. L. (1988). Biochemistly 27, 58045812. Baker, E. N., Rumball, S., and Anderson, B. F. (1987). Trends Biochem. Sci. 12,350-353. Beinert, H. (1990). FASEBJ. 4, 2483-2491. Beinert, H., and Kennedy, M. C. (1989). Eur.J. Biochem. 186,5-15. Beinert, H., Emptage, M., Dreyer, J. L., Scott, R., Hahn, J., Hodgson, K., and Thomson, A. (1983).Proc. Natl. Acad. Sci. U.S.A. 80,393-396. Bellamy, H. D., Lim, L., Mathews, F. S., and Dunham, W. (1989). J . Biol. Chem. 264, 1 1887- 1 1892. Berg, J., and Holm, R. (1982).In “Iron-Sulfur Proteins” (T. Spiro, ed.), pp. 3-66. Wiley (Interscience), New York. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977).J. Mol. Biol. 112, 535-542. Bill, E., Berhardt, F., and Trautwein, A. (1981). Eur.J. Biochem. 121,39-46. Blomstrom, D. C., Knight, E., Jr., Phillips, W. D., and Weiher, J. F. (1964).Proc. Natl. A d . 5’1%.U.S.A. 51, 1085-1092. Blundell, T. L., and Johnson, L. N. (1976). “Protein Crystallography.” Academic Press, New York. Bolin, J. T., Ronco, A. E., Mortenson, L. E., Morgan, T. V., Williamson, M., and Xuong, N.-h. (1990). “Nitrogen Fixation: Achievements and Objectives” (P. M. Gresshoff, F. Roth, G. Stacy, and W. E. Newton, eds.), pp. 117-124. BrandCn, C.-I.,and Jones, T. A. (1990).Nature (London) 343,687-689. Bruschi, M., and Guerlesquin, F. (1988). FEMS Microbiol. Rev. 54, 155-176. Bull, C., Ballou, D., and Ostsaka, S. (1981).j.Biol. Chem. 256, 12681-12686. Burgess, B. K. (1984). “Advances in Nitrogen Fixation Research” (C. Veeger and W. Newton, eds.), pp. 103-1 14. Nijhoff, Boston, Massachusetts. Burgess, B. K. (1990). Chem. Rev. 90, 1377-1406. Camrnack, R. (1973). Biochem. Biophys. Res. Commun. 54,548-554. Campbell, H., and Zerner, B. (1973). Biochem. Bzophys. Res. Commun. 54, 1498-1503. Carlioz, A,, Ludwig, M. L. Stallings, W. C., Fee, J. A,, Steinman, H. M., and Touati, D. (1988).J.B i d . Chem. 263, 1555-1562. Carney, M. J., Papaefthymiou, G .C., Spartalian, K., Frankel, R., and Holm, R. (1988).J.Am. Chem. SOC.110,6084-6095. Carrell, C. J., Carrell, H. L., Erlebacher, J., and Glusker, J. P. (1988).J. Am. Chon. Sac. 110, 865 1-8656. Carter, C. W., Jr., Kraut, J., Freer, S. T., Alden, R. A., Sieker, L. C., Adman, E., and Jensen, L. H. (1972). Proc. Natl. Acad. Sci. U.S.A. 69,3526-3529. Carter, C. W., Jr., Kraut, J., Freer, S. T., Xuong, N.-H., Alden, R. A., and Bartsch, R. G. (1974).J . Biol. Chem. 249,4212-4225.

274

JAMES B. HOWARD AND DOUGLAS C. REES

Chen, T., Bazer, F., Cetorelli, J., Pollard, W., and Roberts, R. (1973).J . Biol. Chem. 248, 8560-8566. Chen, V., Orville, A,, Harpel, M., Frolik, C., Surerus, K., Munck, E., and Lipscomb, J. (1989).J.Biol. Chem. 264,21677-21681. Christner, J. A,, Munck, E., Kent, T. A., Janick, P. A., Salerno, J. C., and Siegel, L. M. (1984).J.Am. Chem. SOC. 106,6786-6794. Clark, I. E., and Webb, J. (1981). Biochemistry 20,4628-4632. Cline, J. F., Janick, P. A., Siegel, L. M., and Hoffman, B. M. (1985a). Biochemistry 24, 7942-7947. Cline, J., Hoffman, B., Mims, W., Lattaire, E., Ballou, D., and Fee, J. (1985b).J.Biol. Chem. 260,3251-3254. Conradson, S., Burgess, B., Newton, W., Mortenson, L., and Hodgson, K. (1987).J. Am. Chem. SOC. 109,7507-7515. Conradson, S., Burgess, B., Vaughn, S., Roe, A., Hedman, B., Hodgson, K., and Holm, R. (1989).J. Biol. Chem. 264, 15967-15974. Coucouvanis, D. (1991). Acc. Chem. Res. 24, 1-8. Cox, D., and Que, L. (198S).j.Am. Chem. Soc. 110,8085-8092. Cunningham, R., Asahara, H., Bank, J., Scholes, C., Surerus, K., Miinck, E., McCracken, J., Peisach, J., and Emptage, M. (1989). Biochemistry 28,4450-4455. Cupp, J., and Vickery, L. (1988).J.Biol. Chem. 263, 17418-17421. Davis, K., and Hatefi, Y. (1971).Biochemistry 10,2509-2916. Dawson, J., Gray, H., Hoenig, H., Rossman, G., Schredder, J., and Wang, R. (1972). Biochemistry 11,461-465. Debus, R. J., Feher, G., and Okamura, M. Y. (1986). Biochemistry 25,2276-2287. deGroot, J., Garssen, G., Veldink, G., Vilegenhart, J., and Boldingh, J. (1975). FEBS Lett. 56950-54. Deisenhofer, J., Epp, O., Miki, K., Huber, R., and Michel, H. (1984).J . Mol. Biol. 180, 385-398. Deisenhofer, J., Epp, O., Miki, K., Huber, R., and Michel, H. (1985). Nature (London) 318, 618-624. Diets, T . L., and Howard, J. B. (1989).J. Biol. Chem. 264,6614-6628. Doi, K., Antanaitis, B., and Aisen, P. (1988). Struct. Bonding 70, 1-26. Dreyer, J.-L. (1985). Eur.J. Biochem. 150, 145-154. Dreyer, J.-L. (1987). Eur. J. Biochem. 166, 623-630. Dunham, W., Hagen, W., Braaksura, A., and Haaker H. (1985). In “Nitrogen Fixation Research Progress” (H. Evans, P. Botteanley, and W. Newton, eds.), pp. 591-596. Nijhoff, Boston, Massachusetts. Eccleston, E., Magneson, J., Park, J., Adams, M., and Howard, J. (1991). Submitted. Eidsness, M., Flank, A., Smith, B., Flood, A., Garner, C., andcramer, S. (1986).J.Am. Chem. SOC.108,2746-2747. Eklund, H., Nordstrom, B., Zeppezauer, E., Soderlund, G., Ohlsson, I., Boiwe, T., Soderberg, B.-O., Tapia, O., Branden, G I . , and Akeson, A. (1976).J.Mol. Biol. 102,27-60. Emptage, M. (1988). ACS Symp. Ser. 372,343-371. Fee, J., Findling, K., Yoshida, T., Hille, R., Tarr, G., Hearshen, D., Dunham, W., Day, E., Kent, T., and Munck, E. (1984).J.Biol. Chem. 259, 124-133. Feher, G., and Okamura, M. Y. (1978). “The Photosynthetic Bacteria” (R. K. Clayton and W. Sistrom, eds.), pp. 349-386. Plenum, New York. Feher, G., Allen, J. P., Okamura, M. Y., and Rees, D. C. (1989). Nature (London) 339, 111-116. Flint, D., and Emptage, M. (1988).J.Biol. Chem. 263,3558-3564.

NON-HEME IRON PROTEIN CHEMISTRY

275

Fontecave, M., Eliasson, R., and Reichard, P. (1989).J.Biol. Chem. 264, 9164-9170. Fox, B., Surerus, K., Munck, E., and Lipscomb,J. (1988).J. Biol. Chem. 263,10553-10556. Fox, B., Froland, W., Dege, J., and Lipscomb, J. (1989).J.Biol. Chem. 264, 10023-10033. Frey, M., Sieker, L., Payan, F., Haser, R., Bruschi, M., Pepe, G., and LeGall,J. (1987).J.Mol. Biol. 197, 525-541. Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara, T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., and Matsubara, H. (1981).Nature (London) 286,522-524. Fukuyama, K., Nagahara, Y., Tsukihara, T., Katsube, Y., Hase, T., and Matsubara, H. (1988).J.Mol. Biol. 199, 183-193. Fukuyama, K., Matsubara, H., Tsukihara, T., and Katsube, Y. (1989).J . Mol. Biol. 210, 383-398. Gatti, D., Meinhardt, S., Ohnishi, T., and Tzagoloff, A. (1989).J.Mol. Biol. 205,421-435. George, D. G., Hunt, L. T., Yeh, L.-S., and Barker, W. C. (1985).J.Mol. Evol. 22,20-31. Georgiadis, M. M., Chakrabarti, P., and Rees, D. C. (1990). “Nitrogen Fixation: Achievements and Objectives” (P. M. Gresshoff, E. Roth, G. Stacy, and W. E., Newton, eds.), pp. 1 1 1-1 16. Chapman & Hall, New York. Gibson, J. F., Hall, 0. A., Thornley, J. H., and Watley, F. R. (1966). Proc. Natl. Acad. Sci. U.S.A. 56,987-991. Grandoni, J., Switzer, R., Makaroff, C., and Zalkin, H. (1989).J. Biol. Chem. 264, 60586064. Green, J., and Dalton, H. (1985).J.Biol. Chem. 260, 15795-15801. Gurbiel, R., Batie, C., Sivaraja, M., True, A., Fee, J., Hoffman, B., and Ballou, D. (1989). Biochemistry 28,4861-4871. Hagen, W. R., Wassink, H., Eady, R. R.,Smith, B. E., and Haaker, H. (1987).Eur.J. Biochem. 169,457-465. Hatefi, Y. (1985). Annu. Rev. Biochem. 54, 1015-1069. Hausinger, R. P., and Howard, J. B. (1983).J. Biol. Chem. 258, 13486-13492. Hausinger, R., Moura, I., Moura, J., Xavier, A., Santos, H., LeGall, J., and Howard, J. B. (1982).J.Biol. Chem. 257, 14192-14197. Hausmann, E. (1967). Biochim.Biophys. Acta 133,591-598. Hawkes, T., McLean, P., and Smith, B. (1984).Biochem.J. 217,317-321. Hendrich, M., Munck, E., Fox, B., and Lipscomb, J. (1991).J. Am. Chem. SOC.112, 58615865. Herskovitz, T., Averill, B. A., Holm, R. H., Ibers, J . A., Phillips, W. D., and Weiher, J. F. (1972).Proc. Natl. Acad. Sci. U.S.A. 69, 2437-2441. Honzatko, R. B., Crawford, J. L., Monaco, H. L., Ladner, J. E., Edwards, B. F. P., Evans, D. R., Warren, S. G., Wiley, D. C., Ladner, R. C., and Lipscomb, W. N. (1982).J. Mol. Biol. 160,219-263. Hoover, T., Robertson, A,, Cerny, R., Hayes, R., Imperial, J., Shah, V. K., and Ludden, P. (1987). Nature (London) 329,855-857. Howard, J. B., Davis, R., Moldenhauer, B., Cash, V. L., and Dean, D. (1989).J. Biol. Chem. 264, 11270-11274. Huynh, B.-H., Munck, E., and Orme-Johnson, W. (1979).Biochim. Biophys. Acta 576, 192203. Huynh, B.-H., Henzl, M. T., Christner,J. A., Zimmermann, R., Orme-Johnson, W. H., and Munck, E. (1980).Biochim. Biofhys. Acta 623, 124-138. Imperial, J., Hoover, T., Madden, M., Ludden, P., and Shah, V. K. (1989).Biochemistry 28, 7796-7799. Johnson, M. K., Kowal, A., Morningstar, J., Oliver, M., Wittaker, K., Gusalus, R., Ackress, B., and Cecchini, G. (1988).J. Biol. Chem. 263, 14732-14738.

276

JAMES B. HOWARD AND DOUGLAS C. REES

Kaufman, S., and Kaufman, E.(1985).I n “Folates and Pterius” (R. Blakley and S. Benkovic, eds.), Vol. 2, pp. 251-352. Wiley, New York. Kennedy, M. C., and Beinert, H. (1988).J.Biol. Chem. 263,8194-8198. Kennedy, M. C., Emptage, M., Dreyer, J., and Beinert, H. (1983). J . B i d . Chem. 258, 11098-1 1105. Kennedy, M. C., Kent, T., Emptage, M., Merkle, H., Beinert, H., and Munck, E. (1984). J . Biol. Chem. 259, 14463- 1447 1 . Kennedy, M. C., Werst, M., Telser, J., Emptage, M., Beinert, H., and Hoffman, B. (1987). Proc. Natl. Acad. Sci. U.S.A. 84,8854-8858. Kent, H., Ioanvidis, I., Gormal, C., Smith, B., and Buck, M. (1989). Biochem. J . 264, 257-264. Kent, T., Dreyer, J., Kennedy, M. C., Huynh, B.-H., Emptage, M., Beinert, H., and Munck, E. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 1096-1100. Kent, T., Emptage, M., Merkle, H., Kennedy, M. C., Beinert, H., and Miinck, E. (1985). J . Biol. Chem. 260,6871-6881. Kent, T., Munck, E., Pynz,J., Windom, J., and Que, L. (1987).Inorg. Chem. 26,1402-1408. Keyser, P. K., Pujar, B. G., Eaton, R., and Ribbons, P. W. (1976). E H P , Environ. Health Perspert. 18, 159-166. Kissinger, C. R., Adman, E. T., Sieker, L. C., and Jensen, J. H. (1988).J.Am. Chem. Soc. 110, 8721-8723. Kissinger, C. R., Adman, E. T., Sieker, L. C., Jensen, L. H., and LeGall,J . (1989).FEBS Lett. 244,447-450. Klotz, I., and Kurtz, D. (1984). Acc. Chem. Res. 17, 16-22. Kuila, D., Fee, J., Schoonover, J., Woodruff, W., Batie, C., and Ballan, D. (1987).J . Am. Chem. Soc. 109, 1559-1561. Kurtz, D. (1990). Chem. Rev. 90, 585-606. Laskowski, E. J., Reynolds, J. G., Frankel, R. B., Foner, S., Papefthymiou, G. C., and Holm, R. H. (1979).J.Am. Chem. Soc. 101,6562-6570. Lehnert, R., and Seel, F. (1978). Z. Anorg. Allg. Chem. 444,91-96. LeGall,J., Prickril, B., Moura, I., Xavier, A., Moura, J., and Huynh, B.-H. (1988).Bzochemistty 27, 1636-1642. Liang, J., Madden, M., Shah, V., and Burris, R. (1990). Biochemzrtry 29,8577-8581. Lim, L. W., Shamala, N., Mathews, F. S., Steenkamp, D. J., Hamlin, R., and Xuoug, N. H. (1986).J. Biol. Chem. 261, 15140-15146. Lindahl, P. A., Day, E. P., Kent, T. A., Orme-Johnson, W. H., and Miinck, E. (1985).J.B i d . Chem. 260,11160-1 1173. Lindahl, P. A., Papaefthyrniou, V., Orme-Johnson, W. H., and Miinck, E. (1988).J. B i d . Chem. 263,19412-19418. Lippard, S. (1988). Angew Chem., Int. Ed. Engl. 27,344-361. Lipscomb, J., Whittaker, J., Arciero, D., Orville, A., and Wolgel, S. (1988).In “Microbial Metabolism and the Carbon Cycle” (S. Hagedorn, R. Hanson, and D. Kung, eds.), pp. 259-281. Harwood Academic, New York. Lovenberg, W., ed. (1973a). “Iron-Sulfur Proteins,” Vol. 1. Academic Press, New York. Lovenberg, W., ed. (1973b). “Iron-Sulfur Proteins,” Vol. 2. Academic Press, New York. Lovenberg, W., ed. (1977). “Iron-Sulfur Proteins,” Vol. 3. Academic Press, New York. Lowery, R. G., Chang, C. L., Davis, L. C., McKenna, M.-C., Stephens, P. J., and Ludden, P. W. (1989). Biochemistty 28, 1206-1212. Ludwig, M. L., Metzger, A. L., Pattridge, K. A., and Stallings,W. C. (1991).J.Mol. Bzol. 219, 335-338. Lusty, C., Machinist, J., and Singer, T. (1965).J.B i d . Chem. 240, 1804-1810.

NON-HEME IRON PROTEIN CHEMISTRY

277

Lynch, J., Jaurez-Garcia, C., Munck, E., and Que, L. (1989).J. Biol. Chem. 264,8091-8096. Madden, J. F., Han, S., Siegel, L. M., and Spiro, T. G. (1989). Biochemistry 28,5471-5477. Madden, M., Kindon, N., Ludden, P., and Shah, V. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 651 7-652 1 . Magneson, J., Paustian, T., Roberts, G., and Howard, J. B. (1991). Submitted. Martin, A. E., Burgess, B. K., Stout, C. D., Cash, V. L., Dean, D. R., Jensen, G. M., and Stephens, P. J . (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 598-602. Maroney, M. J., Kurtz, D., Nocek, J., Pearce, L., and Que, L. (1986).J.Am. Chem. Sac. 108, 6871-6879. Massey, V. (1973). In “Iron-Sulfur Proteins” (A. Ehrenberg, ed.), pp. 301-360. Academic Press, New York. Mathews, F. S., and Lim, L. (1987).I n “Flavins and Flavoproteins” (D. B. McCormick, and D. E. Edmondson, eds.), pp. 663-672. de Gruytcr, Berlin. McLean, P., Smith, B., and Dixon, R. (1983). Biochem. J. 211,589-597. McLean, P. A., Papaefthymiou, V., Orme-Johnson, W. H., and Munck, E. (1987).J. B i d . Chem. 262,12900- 12903. McRee, D. E., Richardson, D. C., Richardson, J. S., and Siegel, L. M. (1986).J.Biol. Chem. 261,10277-1028 1. Moews, P. C., and Kretsinger, R. H. (1975).J.Mol. Biol. 91, 201-223. Montfort, W. R., Perry, K. M., Fauman, E. B., Finer-Moore, J. S., Maley, G. F., Hardy, F., Maley, F., and Stroud, R. M. (1990).Biochemistry 29,6964-6977. Morgan, T. V., McCracken, J., Orme-Johnson, W. H., Mims, W. B., Mortenson, L. E., and Peisach, J. (1990). Biochemistry 29,3077-3082. Morningstar, J.. Johnson, M., Cecchini, G., Ackrell, B., and Kearney, E. (1985). J. Biol. Chem. 260,13631-13638. Moulis,J.-M., Auric, P., Gaillard, J., and Meyer,J. (1984).J.Biol. Chem. 259,11396-1 1402. Moura, I., Moura, J., Munck, E., Papaefthymiou, V., and LeGall,J. (1986).J.Am. Chem. Sac. 108,349-351. Moura, J., Xavier, A., Hatchinkian, E., and LeGall, J. (1978). FEBS Lett 89, 177-179. Moura, J. J., Moura, I., Kent, T., Lipscomb, J., Huynh, B., LeGall, J., Xavier, A., and Munck, E. (1982).J. Biol. Chem. 257,6259-6263. Murray, T., and Groves, J. (1986). I n “Cytochrome P-450, Structure, Mechanism and Biochemistry” (R. Ortiz de Montellano, ed.), pp. 1-28. Plenum, New York. Murrell, S. A., Lowery, R. G., and Ludden, P. W. (1988). Bi0chem.J. 251,609-612. Nelson, M., Levy, M., and Orme-Johnson, W. (1983). Proc. Natl. Acad. Sci. U.S.A. 80, 147-150. Nocek, J., Kurtz, P., Sage, J., Xia, Y.-M., Debrunner, P., Shiemke, A., Sanders-Loehr, J., and Loehr, T. (1988). Biochemistry 27, 1014-1024. Noodleman, L. (1988). Inorg. Chem. 27,3677-3779. Nordlund, P., Sjoberg, B.-M., and Eklund, H. (1990).Nature (London) 345,593-598. Nuttleman, P., and Roberts, R. (1990). 1. B i d . Chem. 265, 12129-12199. Ohlendorf, D. H., Lipscomb, J. D., and Weber, P. C. (1988). Nature (London) 336, 403405. Okamura, M. Y., and Feher, G. (1989). Bi0phys.J. 55,221a. Okamura, M., and Klotz, I. (1973). In “Inorganic Chemistry” (G. Eichhorn, ed.), pp. 320-343. Elsevier, New York. Okamura, M., Klotz, I., Johnson, C., Winter, M., and Williams, R. (1969). Biochemistry 8, 1951-1958. Orme-Johnson, W. H. (1985). Annu. Rev. Biophys. Biophys. Chem. 14,419-459. Orville, A., and Lipscomb, J. (1989).J.B i d . Chem. 264, 8791-8801.

278

JAMES B. HOWARD AND DOUGLAS C. REES

Ostrowski, J., Wu, J.-Y., Rueger, D. C., Miller, B. E., Siegel, L. M., and Kredich, N. M. (1989).J.Biol. Chem. 264,15736-15737. Palmer, G. (1973). I n “Iron-Sulfur Proteins” (W. Lovenberg, ed.), Vol. 2, pp. 285-325. Academic Press, New York. Pankalainen, M., Aro, H., Simons, K., and Kivirikko, K. (1970). Biochim. Biophys. Actu 221, 559-568. Papaefthymiou, V., Girerd,J. J., Moura, I., Moura, J. J., and Miinck, E. (1987).J.Am. Chem. SOC. 109,4703-47 1 1. Petering, D., Fee, J., and Palmer, G. (1971).J.Biol. Chem. 246,643-653. Petersson, L., Graslund, A., Ehrenberg, A., Sjoberg, B.-M., and Reichard, P. (1980).J. Bzol. Chem. 255,6706-6712. Plank, D., and Howard, J. B. (1988).J. Biol. Chem. 263,8184-8189. Plank, D. W., Kennedy, M. C., Beinert, H., and Howard, J. B. (1989).J. Biol. Chem. 264, 20385-20393. Pope, M. P., Murrell, S. A., and Ludden, P. W. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 3173-3177. Que, L., and True, A. (1991). Prog. Znorg. Chem. 38,97-200. Rabinowitz, J. (1971). In “Bioinorganic Chemistry” (R. F. Could, ed.), pp. 322-345. Am. Chem. SOC.,Washington, D.C. Rawlings, J., Shah, V., Chishnell, J., Brill, W., Zimmermann, R., Miinck, E., and OrmeJohnson, W. (1978).J.Biol. Chem. 253, 1001-1004. Reem, R., McCormisk, J., Richardson, D., Devlin, F., Stevens, P., Musselman, R., and Soloman, E. (1989).J.Am. Chem. SOC. 111,4688-4704. Rees, D. C., and Farrelly, D. (1990). Enzymes 19, in press. Richards, F. M. (1974).J. Mol. Biol. 82, 1-14. Richardson, J. S. (1981).Adu. Protein Chem. 34, 167-339. Rieske, J., Zangg, W., and Hansen, R. (1964).J.Biol. Chem. 239,3023-3030. Ringe, D. M., Petsko, G., Yamakura, F., Suzuki, K., and Ohmori, D. (1983).Proc. Nutl. Acud. Sci. U.S.A. 80,3879-3883. Robbins, A. H., and Stout, C. D. (1989a).Proteins: Struct. Funct. Genet. 5,289-312. Robbins, A. H., and Stout, C. D. (1989b).Proc. Nutl. Acud. Sci. U.S.A. 86,3639-3643. Robson, R, (1984). FEBS Lett. 173,394-398. Rose, I., and OConnell, E. (1967).J.Biol. Chem. 242, 1870-1879. Sahlin, M., Graslund, A., Petterson, L., Ehrenberg, A., and Sjoberg, B.-M. (1989).Biochemist? 28,2618-2625. Sahlin, M., Sjoberg, B.-M., Backes, G., Leohr, T., and Sanders-Loehr, J. (1990). Biochem. Biophys. Res. Commun. 167, 813-818. Sanders-Loehr, J. (1989). In “Iron Carriers and Iron Proteins” (T. Loehr, ed.), pp. 375466. VCH Publ., New York. Scarrow, R., Maroney, M., Palmer, S., Que, L., Roe, A., Salowe, S., and Stabie, J. (1987). J . Am. Chem. SOC.109,7857-7864. Schweiger, G., Dutscho, R., and Buckel, W. (1987). Eur. J . Biochem. 86,441-448. Scott, D., May, H., Newton, W., Bringle, K., and Dean, D. (1990). Nature (London) 343, 188-190. Shah, V. K., and Brill, W. (1977). Proc. Nutl. Acud. Sci. U.S.A. 74, 3249-3253. Sheriff, S., Hendrickson, W. A., and Smith, J. L. (1987).J.Mol. Biol. 197, 273-296. Shiemke, A., Loehr, T., and Sanders-Loehr, J. (1986).J.Am. Chem. SOC.108,2437-2443. Siegel, L. M., Rueger, D. C., Barber, M. J., Krueger, R. J., Orme-Johnson, M. R., and Orme-Johnson, W. H. (1982).J. Biol. Chem. 257,6343-6350. Sjoberg, B.-M., Reichard, P., Graslund, A., and Ehrenberg, A. (1978).J. Biol. Chem. 253, 6863-6865.

NON-HEME IRON PROTEIN CHEMISTRY

279

Sjoberg, B.-M., Sanders-Loehr, J., and Loehr, T. (1987). Biochemistry 26,4242-4247. Spiro, T., ed. (1982). “Iron-Sulfur Proteins.” Wiley (Interscience), New York. Stallings, W. C., Powers, T. B., Pattridge, K. A., Fee, J. A., and Ludwig, M. (1983).Proc. Natl. Acad. Sci. U.S.A. 80,3884-3888. Steenkamp, D. J., McIntire, W., and Kenney, W. C. (1978).J. Biol. Chem. 253,2818-2824. Stenkamp, R. E., Sieker, L. C., and Jensen, L. H. (1984).J. Am. Chem. SOC.106,618-622. Stenkamp, R., Sieker, L., Jensen, L., McCallum,J., and Sanders-Leohr, J. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 713-716. Stevens, P., McKenna, M X . , Ensign, S. A., Bonam, D., and Ludden, P. (1989).J.Biol. Chem. 264,16347-16350. Stevenson, R. C., Dunham, W. R., Sands, R. H., Singh, T . P., and Beinert, H. (1986). Biochim. Biophys. Acta 869, 81-88. Stoddard, B. L., Howell, P. L., Ringe, D. M., and Petsko, G. A. (1990). Biochemistry 29, 8885-8893. Stout, C. D. (1988).J. Biol. Chem. 263,9256-9260. stout, c. D. ( 1 9 8 9 ) . ~~. o inioi. . 205,545-555. Stout, G. H., Turley, S., Sieker, L. C., and Jensen, L. H. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 1020-1022. Stubbe, J. (1989). Annu. Rev. Biochem. 58,257-285. Subramanian, V., Liu, T. N., and Gibson, D. T. (1979). Biochem. Biophys. Res. Commun. 91, 1131-1139. Sugiura, Y., Kuwahara, J., Nagasawa, T., and Yamada, H. (1987).J. Am. Chem. SOC.109, 5848-585 0. Surerus, K. (1989). Ph.D. thesis. University of Minnesota, Minneapolis, Minnesota. Surerus, K., Munck, E., Moura, I., Moura, J., and LeGall, J. (1987).J. Am. Chem. SOC.109, 3805-3807, Surerus, K., Munck, E., Snyder, B., and Holm, R. H. (1989). J . Am. Chem. SOC. 111, 5501-5502. Sussman, J. L., Shoham, M., and Harel, M. (1989). In “Computer-Assisted Modeling of Receptor-Ligand Interactions: Theoretical Aspects and Applications to Drug Design” (R. Rein and A. Golombek, eds.), pp. 171-187. Liss, New York. Telser, J., Hoffman, B., LoBrutto, R., Ohnishi, T., Tsai, A.-L., Simptin, D., and Palmer, G. (1987). FEBS Lett. 214, 117-121. Thelander, L., and Reichard, P. (1979). Annu. Rev. Biochem. 48, 133-158. Thomann, H., Morgan, T., Jin, H., Burgmayer, S., Bare, R., and Stiefel, E. (1987).J. Am. Chem. SOC.109,7913-7914. True, A., Nelson, M., Venters, R., Orme-Johnson, W., and Hoffman, B. (1988). J. Am. Chem. SOC.110,1935-1943. True, A., Orville, A., Pearce, L., Lipscomb, J., and Que, L. (1991). Biochemistry 29, 1084710854. Tsang, H.-T., Batie, C., Ballou, D., and Penner-Hahn, J. (1989). Biochemistry 28, 72337240. Tsukihara, T., Fukuyama, K., Mizushima, M., Harioka, T., Kusunoki, M., Katsube, Y., Hase, T., and Matsubara, H. (199O).J. Mol. Biol. 216,399-410. Tsutsui, T., Tsukihara, T., Fukuyama, K., Katsube, Y., Hase, T., Matsubara, H., Nishikawa, Y., and Tanaka, N. (1983).J. Biochem. (Tokyo) 94,299-302. Watenpaugh, K. D., Sieker, L. C .,and Jensen, L. H. (1980).J. Mol. Biol. 138,615-633. Watt, G., and MacDonald, J. (1985). Biochemistry 24,7276-7281. Watt, G. D., Burns, A., and Tennent, D. L. (1981). Biochemistry 20,7272-7277. Werst, M., Kennedy, M. C., Houseman, A., Beinert, H., and Hoffman, B. (1990). Biochemis19 29,10533-10540.

280

JAMES B. HOWARD AND DOUGLAS C. REES

Whittaker, J., and Lipscomb, J. (1984).J.Biol. Chem. 259,4487-4495. Whittaker, J., Lipscomb, J., Kent, T., and Miinck, E. (1984).J . B i d . Chem. 259, 44664475. Williams,J. C., Paddock, M. L., Feher, G., and Allen, J. P. (1991). Biophys.J. 59, 142a. Willing, A., and Howard, J. B. (1990).J.Biol. Chem. 265,6596-6599. Wolle, D., Dean, D., and Howard, J. B. (1991). Submitted for publication. Wong, G. B., Kurtz, D., Holm, R., Mortenson, L., and Upchurch, R. (1979).J.Am. Chem. SOC.101,3078-3090. Woodland, M., and Dalton, H. (1984).J.Biol. Chem. 259,53-59. Woodland, M., Patil, D., Cammack, R., and Dalton, H. (1986). Biochim. Biophys. Actu 873, 237-242. Yamamoto, I., Saike, T., Liu, S.-M., and Ljungdahl, L. (1983).J. Biol. Chem. 258, 18261832. Yamane, T.,Weininger, M. S., Mortenson, L. E., and Rossmann, M. G. (1982).J . Biol. Chem. 257,1221-1223. Yamashita, M. M., Wesson, L., Eisenman, G., and Eisenberg, D. (1990).Proc. Nutl. Acud. Sci. U.S.A. 87,5648-5652. Yao, Y.,Wakaboyashi, S., Matsubata, H., Yu, L., and Yu, C. (1986).In “Iron-Sulfur Protein Research” (H. Matsubara, Y. Katsuba, and K. Wada, eds.), pp. 240-244. Jpn. Sci. SOC. Press, Tokyo. Yost, F., and Fridovich, I. (1973).J. Biol. Chern. 248,4905-4908. Zhang, K., Stern, E., Ellis, F., Sanders-Loehr, J,, and Shiemke, A. (1988). Biochemistry 27, 7470-7479. Zimmerman, R., Mtinck, E., Brill, W. J., Shah, V. K., Hanzl, M. T., Rawlings, J., and Orme-Johnson, W. H. (1978).Biochim. Biophys. Actu 537, 185-207.

STRUCTURAL BIOLOGY OF ZINC

.

By DAVID W CHRISTIANSON Department of Chemlstry. University of Pennsylvania. Phlladeiphia. Pennsyivanla 19104

I . Introduction . . . . . . . . . . . . . . . . . A . Protein-Zinc Recognition . . . . . . . . . . . B. Bioinorganic Chemistry of Zinc . . . . . . . . I1. Stereochemistry of Biological Zinc-Ligand Interactions A. Carboxylates: Aspartate and Glutamate . . . . . B . Phosphate . . . . . . . . . . . . . . . . . C. Carbonyl; Amino Acid Chelates . . . . . . . . D . Histidine . . . . . . . . . . . . . . . . . E. Cysteine . . . . . . . . . . . . . . . . . . F. Solvent . . . . . . . . . . . . . . . . . . G . Methionine and Selenocysteine . . . . . . . . . I11. Long-Range Protein-Metal Interactions . . . . . . A . Long-Range Electrostatic Effects . . . . . . . . B. Hydrogen Bond Networks Involving Zinc Ligands . IV . Examples of Zinc in Biological Catalysis and Regulation A . Carbonic Anhydrase I1 . . . . . . . . . . . . B. Carboxypeptidase A and Other Zinc Proteases . . . C. Metallothionein . . . . . . . . . . . . . . . D . Transcription Factors IIIA and GAL4 . . . . . . V . Protein Engineering of Zinc-Binding Sites . . . . . . VI . Summary . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

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281 282 285 287 287 290 295 297 300 301 302 304 304 307 310 311 322 333 337 344 348 349

I . INTRODUCTION Among the first-row transition metals. zinc is second only to iron in terms of abundance and importance in biological systems. As a d'' metal ion. Zn2+ is not susceptible to ligand field stabilization effects. nor does it exhibit biologically relevant redox reactivity . Nevertheless. the coordination chemistry of zinc is as versatile as the metal ion functions in biology. and this function is governed to a great degree by the metal ligands . Zinc coordination polyhedra exhibit variation in number. charge. structure. and amino acid composition. depending on the structural. catalytic. or regulatory role of the metal ion . Molecular structure data bases are particularly useful in the analysis and engineering of zinc coordination polyhedra. and statistical results from the Brookhaven Protein Data Bank (Bernstein et al., 1977) and the Cambridge Structural Database (Allen et al., 1983) are presented ADVANCES IN PROTEIN CHEMISTRY. Val. 42

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Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

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throughout this chapter. Recurring stereochemical motifs identified among the independent three-dimensional structures in these data bases are effectively applied toward the understanding of protein structure, conformation, and folding. Consequently, these molecular data bases are increasingly used as tools in protein engineering and protein structure prediction. This chapter centers on the structural biochemistry of zinc, particularly on the role of the metal ligands in modulating this chemistry. Hence, general aspects of zinc chemistry and recognition are first reviewed from the bioinorganic perspective. Second, the stereochemistry of biological zinc-ligand interactions is discussed, and then long-range metal-protein interactions are considered. Examples of catalytic or regulatory zinc metalloproteins are subsequently reviewed, and their relevance to the engineering of de nova zinc-binding sites in proteins is discussed in the final section. A . Protezn-Zinc Recognition The recognition of a transition metal by a protein-binding site requires the discrimination of ionic size, charge, and chemical nature [i.e., “hardness’’ or “softness” (see Pearson, 1963; Glusker, this volume)]. Among biologically important divalent metal ions, zinc possesses a distinguishable ionic radius (Table I) and it is typically found in tetrahedral or distorted tetrahedral protein sites. Zinc is a metal of borderline hardness, and it easily accommodates nitrogen, oxygen, and sulfur atoms in its biological coordination polyhedra. In contrast the divalent ions of calcium or magnesium are hard cations and prefer all, or nearly all, oxygen atoms in their typically octahedral coordination polyhedra (Pearson, 1963). A hard cation has low polarizability, a small ionic radius, and a large charge. For example, Mg‘+ is harder than Zn2+since the charge 2+ is distributed over a smaller volume in the Mg2+ cation. Hence, an optimal Mg2+-bindingsite is probably not an optimal Zn2+-binding site (and vice versa) due to the chemical composition of the ligand atoms (which are usually of complementary hardness). In addition to chemical hardness and/or softness, simple electrostatic effects are important for protein-metal ion recognition. In proteins zinc binds to negatively charged residues (e.g., carboxylates and thiolates) and/or neutral dipolar residues (e.g., carbonyls and imidazoles). From a purely electrostatic perspective the interaction of a glutamate carboxylate or a cysteine thiolate with zinc constitutes a charge-charge interaction which should contribute favorably to the enthalpy of zinc-ligand association. T h e potential energy V of two infinitesimal point charges q1 and q 2

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283

TABLE I Prope7ties of Certain Biological Metal Ions Ion

Radius (Ay

Hard-softb

Mg2+ Ca2+ Mn2+ Fez+ FeS+

0.65 0.99 0.80 0.76

co2+

0.74 0.69 0.96 0.72' 0.74 0.97

Hard Hard Hard Borderline Hard Borderline Borderline Soft Borderline Borderline Soft

Ni2+

cu+

cup+ Zn2+ Cd2+

0.53"

a Radii were calculated by the method of Pauling, abstracted from Cotton and Wilkinson (1980). From Pearson (1963). ' Goldschmidt radii; that of Cu2+ is somewhat uncertain due to Jahn-Teller effects.

separated by a distance r in a medium of dielectric constant D ( D = 80 for water) is described by Coulomb's law, V , qlq2/Dr. The interaction of a charge with a dipole (e.g., as zinc interacts with a neutral histidine imidazole, a carbonyl oxygen, or a water molecule) reflects a dependence on the orientation of the dipole with respect to the charge. The potential energy V of an infinitesimal point charge q interacting with an electronic dipole of magnitude p, where r is the distance between the charge and the midpoint of the dipole (Fig. I), in a medium of bulk dielectric constant D is approximated from Coulombs law, V , qp cos O/Dr2. The expressions above for charge-charge and charge-dipole interactions are not physically rigorous for the description of carboxylate-zinc or imidazole-zinc interactions; for example, neither the carboxylate anion nor the zinc is an infinitesimal point charge, and the carboxylate anion is not even spherically symmetric. However, the expressions illustrate two important points: (1) the charge-charge interaction is stronger and less sensitive to separation than the charge-dipole interaction, and (2)the charge-charge interaction is sensitive only to separation, whereas the charge-dipole interaction is sensitive to both separation and orientation. Long-range interactions between proteins and bound metal ions are appreciable when they involve charged protein residues; these effects are discussed further in Section III,A. For an excellent review of polar

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DAVID W. CHRISTIANSON

FIG. 1. The potential energy V for the interaction of a metal ion with charge +p with a dipole of magnitude p is given by the expression V 0: qp cos e/D?, where rand 0 are defined as shown.

interactions in protein structure and function, see that by Burley and Petsko (1988). In addition to simple electrostatic effects, molecular orbital effects make an important contribution to the energetics of protein-metal ion recognition and discrimination. Berg and Merkle (1989) outlined a scheme based on ligand field theory which accounts for the favorable binding of zinc to tetrahedral protein sites. In general, if a transition metal is subject to ligand field stabilization effects, the change from an octahedral to a tetrahedral ligand field is energetically unfavorable. Zinc is not subject to ligand field stabilization effects, so the change from an octahedral to a tetrahedral ligand field is not energetically unfavorable. Therefore, the equilibrium between the octahedral hexaaquo species M'+(OH& in solution and the tetrahedral protein-metal ion complex M2+L4 (M, metal; L, protein ligand) is disfavored for the first-row transition metals M = Fe-Cu relative to M = Zn (Berg and Merkle, 1989; Berg, 1989a). In addition to enthalpic contributions, the entropy effects accompanying protein-metal ion interactions are substantial. These effects manifest themselves in the desolvation of the metal ion and its binding site. However, as the metal ion binds to a protein, the entropy gain of solvent release may be offset to some degree by the reduction of the conformational entropy of the polypeptide chain as it becomes more firmly bound

285

STRUCTURAL BIOLOGY OF ZINC

to the metal ion. The entropy loss from metal-binding site organization is not expected to surpass the entropy gain of metal desolvation as zinc binds to a small peptide (Berg, 1989a). Moreover, in the metal-binding sites of larger proteins, metal ligands are engaged in hydrogen bond networks which minimize the conformational entropy loss conferred by metal binding (see e.g., Adman et al., 1975; Argos et al., 1978; Christianson and Alexander, 1989, 1990). Work from Sturtevant's laboratory detailed the kinetics and thermodynamics of zinc binding to apocarbonic anhydrase (carbonate dehydratase); selected data are recorded in Table I1 (Henkens and Sturtevant, 1968; Henkens et al., 1969). T h e thermodynamic entropy term AS at p H 7.0 is 88 e.u. (1 e.u. = 1 cal/mol-K), and this is essentially matched by the binding of zinc to the hexadentate ligand cyclohexylenediamine tetraacetate where AS = 82 e.u. At p H 7.0 the enthalpy of zinc-protein association is 9.8 kcal/mol, but this unfavorable term is overwhelmed by the favorable entropic contribution to the free energy (AG = AH - T AS), where -TAS = -26.2 kcal/mol at 298 K (25°C). Hence, the kinetics and thermodynamics of protein-zinc interaction in this example are dominated by very favorable entropy effects.

B . Bioinorganic Chemistry of Zinc Zinc displays coordination numbers 4-6 in small-molecule complexes, and the coordination number 5 is particularly common (Cotton and Wilkinson, 1980). In aqueous solution zinc exists as the hexahydrate, Zn2+(OH& although other zincate species may be observed under certain conditions [e.g., Zn2+(-OH)4 and Zn2+(0H2)2(-OH)2 (see, e.g.,

TABLE 11 Kinetic and Thermodynamic Data for Zinc-Ligand Association" ki

Zn2+ + L s zn2+ - L

AH

pH

(mol-'sec-')

AHS (kcal/mol)

ASS

Ligand

(e.u.")

(kcal/mol)

apo-CA apo-CA CHDTA

5.5

a04 104

21 21

28 30

3.9 9.8 4.7'

ki

7.0 7.0

-

-

-

AS (e.u.*) 61 88

81"

AG (kcal/mol) - 14.3 - 16.4

- 19.4

"Data are from Henkens and Sturtevant (1968) and Henkens et al. (1969). apo-CA, Bovine apocarbonic anhydrase 11; CHDTA, cyclohexylenediamine tetraacetate. One entropy unit (e.u.) = 1 cal/rnol-K. Values were calculated by Henkens et al. (1969) based on data reported by Anderegg (1963).

286

DAVID W.CHRISTIANSON

Sharma and Reed, 1976)l. In proteins the coordination number 4 is most common, where the zinc ion is typically coordinated in tetrahedral or distorted tetrahedral fashion. T h e coordination polyhedron of structural zinc is dominated by cysteine thiolates, and the metal ion is typically sequestered from solvent by its molecular environment; the coordination polyhedron of catalytic zinc is dominated by histidine ligands, and the metal ion is exposed to bulk solvent and typically binds a solvent molecule (Vallee and Auld, 1990). The inner-sphere coordination number of catalytic zinc may increase to 5 during the course of enzymatic turnover, and several five-coordinate zinc enzyme-substrate, enzyme product, and enzyme-inhibitor complexes have been studied by high-resolution X-ray crystallographic methods (reviewed by Matthews, 1988; Christianson and Lipscomb, 1989). The coordination polyhedron of zinc in five coordinate examples may tend toward either trigonal bipyramid or octahedral-minus-one geometry. Zinc may function to promote the nucleophilicity of a bound solvent molecule in both small-molecule and protein systems. T h e pK, of metalfree H20 is 15.7, and the pK, of hexaaquo-zinc, Zn2+(0H&, is about 10 (Woolley, 1975) (Table 111). In a novel small-molecule complex the coordination of H20 to a four-coordinate zinc ion reduces the pK, to about 7 (Groves and Olson, 1985) (Fig. 2). This example is particularly noteworthy since it has a zinc-bound solvent molecule sterically constrained to attack a nearby amide carbonyl group: as such, it provides a model for the carboxypeptidase A mechanism (see Section IV,B). T o be sure, the zinc ligands play an important role in modulating the chemical function of the metal ion in biological systems and their mimics. Biomimics of noncatalytic, or structural, zinc generally focus on zincthiolate clusters designed after the metal-thiolate clusters of metallothionein (see Section IV,C). Adamantoid anions of formula [ (p-SPh)s(ZnSPh)#--are targets of synthetic and structural study, where each metal ion is coordinated in tetrahedral fashion by bridging and terminal thiolate ligands (see Hencher et al., 1985; Dean and Vittal, 1987, and references cited therein). TABLE 111 Solvent PKa as a Function of Metal Coordination

Reaction

PKa

H 2 0 S -OH + H + Zn2+(OH& 2 (H20)5Zn2+(-OH)+ H+

15.7 -10

LsZn2+(0H2) L3Zn2+(-OH) + H+

-7

Reference

Woolley (1975) Groves and Olson (1985)

STRUCTURAL BIOLOGY OF ZINC

287

FIG. 2. The zinc-bound water molecule of this rigid metalloamide complex exhibits a pK, of about 7 (Groves and Olson, 1985). The complex is a biomimic of the tetracoordinate metal ion in the carboxypeptidase A active site.

11. STEREOCHEMISTRY OF BIOLOGICAL ZINC-LIGAND INTERACTIONS The geometric preferences of zinc coordination by biologically important Lewis bases are discussed in this section. These Lewis bases include the carboxylate groups of aspartate and glutamate, phosphates, peptide carbonyl groups, histidine imidazoles, cysteine thiolates, and solvent (both water and hydroxide ion). Here, the focus is on the stereochemistry of zinc coordination by individual functional groups, rather than the stereochemistry of individual metal coordination polyhedra. Interactions not directly observed in protein structures, but potentially observable or engineerable within a biological context (e.g., methionine-zinc and selenocysteine-zinc complexes) are considered in the final section.

A . Carboxylates: Aspartate and Glutamate As shown in Fig. 3, Lewis acids (i.e., metal ions and hydrogen bond donors) display syn or anti stereochemistry as they interact with the carboxylate anion. However, in a study of enzyme active sites, Gandour (1981) first noticed that hydrogen bond donors to the carboxylates of aspartate and glutamate residues preferentially occur with syn stereochemistry. As a carboxylate-hydrogen bond donor interaction C02-H

288

DAVID W. CHRISTIANSON

I

,, ,, #

anti

syn (preferred)

anti

.., ,. $

, ,

FIG. 3. Lewis acids (i.e., metal ions or hydrogen bond donors) generally prefer syn in-plane stereochemistryas they interact with the carboxylate anion.

compares with a protonated carboxylic acid COBH,Gandour’s observation is in accord with the results of Peterson and Csizmadia (1979), who calculated that the syn-carboxylic acid (the proton being covalently bound to the carboxylate) is more stable than the anti isomer by 4.5 kcal/mol; Wiberg and Laidig (1987) attributed the stability of the syn isomer to the favorable opposition of C=O and 0 - H bond dipoles (Scheme 1). However, the energetics of the carboxylate-hydrogen bond donor interaction (COG-H) and the protonated carboxylic acid C02H may not be entirely comparable. Li and Houk (1989) proposed that the carboxylate anion shows only a modest preference (0.6 kcal/mol) for a synoriented hydrogen bond donor on the basis of ab initio calculations at the 6-31G* level (Scheme 1). A survey of carboxylate-hydrogen bond donor stereochemistry in proteins suggests a modest energetic preference (i.e., <1 kcal/mol) for syn-oriented hydrogen bonds (Ippolito et al., 1990). Nevertheless, the expectation of Gandour (1981) is realized in that synoriented hydrogen bond donor interactions with the carboxylate are preferred. As for carboxylate-hydrogen bond donor interactions, carboxylatemetal ion interactions are expected to be most favorable with syn stereochemistry. In a survey of the Cambridge Structural Database, Carrel1 et al. (1988) presented a stereochemical analysis of carboxylate-metal ion interactions which supports this expectation (Scheme 1).These investigators found that the syn-oriented lone electron pair of the carboxylate

STRUCTURAL BIOLOGY OF ZINC

Anti

SYn

A,I

H

-

289 Energetics

AG = -4.5 kcallmol Peterson & Csizmadia, 1979

AG = -0.6 kcallmol Li & Houk, 1989

I

Carrell et al., 1988 Christianson & Lipscomb, 1988a

I

I I

I

znz+ SCHEME 1

oxygen is preferred for cation binding, and bidentate carboxylate-metal ion interactions typically occur when the metal-oxygen distance is in the range 2.3-2.6 A. In particular, 67 carboxylate-zinc interactions are retrieved, of which 52 are syn (five are bidentate) and 15 are anti (i.e., 78% syn/22% anti). As for interactions with other first-row transition metals, the zinc ion displays a strong preference to be in the plane of the carboxylate; This optimizes its interaction with the oxygen sp2 lone electron pair. Some metal ions do not display a preference for in-plane interactions with the carboxylate; Na+, K’, and Hg2+ions are examples in which the maximum deviation from the plane of the carboxylate exceeds 1.5 A. A scatterplot of carboxylate-zinc interactions retrieved from the Cambridge Structural Database, with superimposed “probability” contours (Rosenfield et al., 1984), is reproduced in Fig. 4 (Carrell et al., 1988).

290

DAVID W. CHRISTIANSON

Currently, only a handful of examples of unique protein carboxylatezinc interactions are available in the Brookhaven Protein Data Bank. Each of these entries, however, displays syn coordination stereochemistry, and two are bidentate (Christianson and Alexander, 1989) (Fig. 5 ) . Other protein structures have been reported with syn-oriented carboxylate-zinc interactions, but full coordinate sets are not yet available [e.g., DNA polymerase (Ollis et al., 1985) and alkaline phosphatase (Kim and Wyckoff, 1989)l. A survey of all protein-metal ion interactions reveals that syn-carboxylate-metal ion stereochemistry is preferred (Chakrabarti, 1990a). It is been suggested that potent zinc enzyme inhibition arises from syn-oriented interactions between inhibitor carboxylates and active-site zinc ions (Christianson and Lipscomb, 1988a; see also Monzingo and Matthews, 1984), and the structures of such interactions may sample the reaction coordinate for enzymatic catalysis in certain systems (Christianson and Lipscomb, 1987). B . Phosphate The anionic phosphinyl portion (PO;) of the phosphate group, as it comprises the backbone of nucleic acids or zinc enzyme inhibitors (see Section IV,B), may interact with Lewis acids (i.e., metal ions and hydro-

FIG.4. Two perpendicular orientations of 67 carboxylate-zinc interactions retrieved from the Cambridge Structural Database. The Y-shaped stick figure on the right represents the carboxylate group as oriented in Fig. 3; contours indicate that interactions tend to cluster with syn coordination stereochemistry. [Reprinted with permission from Carrell, C. J., Carrell, H. L., Erlebacher,J., & Glusker,J. P. (1988)J. Am. Chem. SOC.110,8651-8656. Copyright 0 1988 American Chemical Society.]

STRUCTURAL BIOLOGY OF ZINC

29 1

a

0 0 0 .. . . . . . .. .. .. .. .. . . . .. .. .. .. .. .. . . . .. ... ... ..

b

. . . .. .. . . . . . . . .. . .. . .. .. .. .. .

(J . . . . . .. .. . . . . . . . .

0 . . . . . . . .. . . .. . .. . ... ......... . . . . . . .. .. .. . . . . .. .. .. .. .. .. .. .. . . . . .

FIG.5. (a and b) Two perpendicular orientations of direct carboxylate-zinc interactions retrieved from four metalloprotein structures contained in the Brookhaven Protein Data Bank. Orientation (a) represents the carboxylategroup as found in Fig. 3. The coordination stereochemistry is syn for each example.

292

DAVID W. CHRISTIANSON

0

anli

'.,

FIG. 6. Phosphinyl anion-Lewis acid stereochemistry is described as syn or anti. Metal ions generally prefer syn coordination stereochemistry; however, they do not exhibit a preference to lie within the plane of the phosphinyl group (PO2 -).

gen bond donors) with stereochemistry described by the synlanti nomenclature (Fig. 6). In a study of the Cambridge Structural Database, Alexander et al. (1990) reported that the phosphinyl group displays a 63% preference for syn-oriented metal interactions. A scatterplot of 108 unique and independent phosphinyl-metal ion interactions involving 17 different metals appears in Fig. 7 (Alexander et al., 1990). A tendency toward out-of-plane geometry is observed, and unidentate P=O+Mn+ coordination is preferred with an average angle of 141". Importantly, phosphate-metal ion interactions observed in the structure and stabilization of the tRNA molecule are in accord with these stereochemical results (Fresco et al., 1966; Jack et al., 1977; Holbrook et al., 1977). For the subset of 12 independent phosphinyl-zinc interactions retrieved from the Cambridge Structural Database, unidentate interactions are similarly preferred, and the synlanti ratio is 8 :4, or 67% : 33%. T h e average P=O+Zn'+ angle is 136",and the average zinc-oxygen distance is 1.94 A. A scatterplot of phosphinyl-zinc interactions is found in Fig. 8. An intriguing contrast can be made between carboxylates and phosphates as each interacts with metal ions. In carboxylate-metal ion interactions, zinc and other metal ions typically prefer to be in the plane of the carboxylate group (Carrel1 et al., 1988). However, the metal ion engaged by phosphate prefers a location that is nearly 1 8, out of the plane of the phosphinyl group. Additionally, even though there are several examples of bidentate carboxylate-metal ion interactions, a symmetrically bidentate phosphinyl-metal ion interaction is not preferred. These differ-

STRUCTURAL BIOLOGY OF ZINC

293

a

b

FIG. 7. (a and b) Two perpendicular orientations of a scatterplot of 108 unique and independent phosphinyl-metal ion interactions. Orientation (a) represents the phosphinyl group as in Fig. 6. Contours indicate where interactions tend to cluster, and syn coordination stereochemistry is preferred by 63%.

DAVID W. CHRISTIANSON

294

. Q

a

b

\\ ' \

/

FIG.8. (a and b) Two perpendicular orientations of a scatterplot of 12 unique and independent phosphinyl-zinc interactions. Orientation (a) represents the phosphinyl group as in Fig. 6. Contours indicate where interactions tend to cluster, and syn coordination stereochemistry is preferred by 67%.

STRUCTURAL BIOLOGY OF ZINC

295

ences are apparent in the comparison of carboxylate-zinc interactions (Fig. 4) and phosphinyl-zinc interactions (Fig. 8). Molecular orbital theory may provide an explanation for stereochemical differences between carboxylate-metal ion and phosphate-metal ion interactions. Detailed ab initio calculations demonstrate that the semipolar P=O double bond of R3P=0 is electronically different from the C=O double bond, for example, as found in H&=O (Kutzelnigg, 1977; Wallmeier and Kutzelnigg, 1979). The P=O double bond is best described as a partial triple bond, that is, as one full (T bond and two mutually perpendicular half-n bonds (formed by backbonding between the electrons of oxygen and the empty d orbitals of phosphorus). Given this situation, a lone electron pair should be oriented on oxygen nearly opposite the P=O bond, and these molecular orbital considerations for P=O may extend to the phosphinyl monoanion -O-P=O. If this extension is valid, then the electronic structure of -O-P=O should not favor bidentate metal complexation by phosphate; this is in accord with the results by Alexander et al. (1990).

C. Carbonyl; Amino Acid Chelates The peptide carbonyl is a significant metal ligand in biological systems, although it is more commonly observed in protein structures as a calcium ligand than as a zinc ligand (Chakrabarti, 1990b). The stereochemistry of carbonyl-calcium ion interactions (Einspahr and Bugg, 1984) and carbonyl-Lit, -Na+, -K+, -Mg2+, and -Ca2+ interactions (Chakrabarti et al., 1981) have been studied in small-molecule systems. More recently, Chakrabarti (1990b) outlined the stereochemistry of peptide carbonylmetal ion interactions as observed in refined protein structures. Among a total of 72 observations (62 calciums, six zincs, two coppers, one magnesium, and one sodium), 11 are defined as bidentate; that is, another atom from one of the amino acids forming the peptide linkage also interacts with the metal ion. A scatterplot of peptide carbonyl-metal ion interactions as retrieved from the Brookhaven Protein Data Bank is reproduced in Fig. 9. For unidentate interactions the C=O+Mn+ angles tend to lie within the range 140"-170", but for bidentate examples this angle tends to lie within the range 110"-130"; not unexpectedly, most metal ions tend to lie within a 35" deviation from the peptide plane. Unidentate examples do not tend toward interaction with carbonyl oxygen lone electron pairs, but instead are situated in a region colinear with the C=O bond. Chakrabarti (1990b) noted that this geometry contrasts with the geometry of peptide-water interactions, in which solvent molecules tend to align with carbonyl oxygen lone electron pairs (see e.g., Thanki et al., 1988). Of

296

DAVID W. CHRISTIANSON

0

0

0

0

FIG. 9. Scatterplot of peptide carbonyl-metal ion interactions from the Brookhaven Protein Data Bank, with unidentate and bidentate examples indicated by circles and x's, respectively. [Reprinted with permission from Chakrabarti, P. (1990).Biochemistry 29,651658. Copyright 0 1990 American Chemical Society.]

course, carbonyl-metal ion stereochemistry is strikingly different in bidentate examples, as revealed in Fig. 9 (Chakrabarti, 1990b). A special case of bidentate carbonyl-metal ion coordination is found in glycine chelates, in which both the carbonyl oxygen and the amino nitrogen of glycine coordinate to a metal ion to form a five-membered chelate (Fig. 10). This binding mode has been observed in important examples of biological zinc complexation. For example, avian pancreatic polypeptide hormone requires zinc for satisfactory crystallization (Blundell et al., 1981; Pitts, 1990). Electron density maps of the protein reveal that the metal ion cross-links three individual molecules and thereby stabilizes the crystal lattice; the amino-terminal glycine of one of these protein molecules engages zinc via a five-membered chelate. A five-membered glycine chelate is also observed in the binding of the pseudosubstrate glycyl-L-

'I FIG. 10. The peptide carbonyl and the free a-amino group of an amino acid may complex a metal ion to form a five-rnembered chelate.

STRUCTURAL BIOLOGY OF ZINC

Y248

297 Y248

P R145

FIG. 1 1. The slowly hydrolyzed substrate glycyl-L-tyrosinebinds to carboxypeptidase A in a nonproductive complex where the amino-terminal glycine complexes the active-siteion (large sphere) to form a five-membered chelate, as in Fig. 10. Protein-bound zinc ligands Glu-72, His-69, and His- 196 complete the coordination polyhedron of pentacoordinate zinc. Active-site residues are indicated by one-letter abbreviations and sequence numbers: E, glutamate; H, histidine; R, arginine; Y, tyrosine. [Reprinted with permission from Christianson, D. W., & Lipscomb, W. N. (1986) Proc. Natl. Acad. Sci. U.S.A. 83,7568-7572.1

tyrosine to the zinc protease carboxypeptidase A (Christianson and Lipscomb, 1986) (Fig. 1l), and is reminiscent of the binding of a hydroxamate inhibitor to the zinc protease thermolysin through an analogous five-membered chelate structure (Holmes and Matthews, 1981). D . Histidine

The geometry of Zn2+ and Co2+ interactions with sp2 nitrogencontaining heterocycles (e.g., imidazole and its derivatives) has been examined by Vedani and Huhta (1990) in a study by the Cambridge Structural Database. The metal ion expectedly prefers a head-on and in-plane approach to the sp2 lone electron pair of the nitrogen atom (Vedani and Huhta, 1990). (Fig. 12). This stereochemical result is in accord with the analysis of imidazole hydrogen bond geometry in small molecules (Vedani and Dunitz, 1985),histidine hydrogen bond geometry in protein structures (Ippolito et al., 1990), and the histidine-metal ion geometry in protein structures (Chakrabarti, 1990~). Hydrogen bond interactions are important for the function of histidine as a zinc ligand. For example, in a survey of zinc-binding motifs, Christianson and Alexander (1989, 1990) reported that head-on and

298

DAVID W. CHRISTIANSON

b

a /

/

/

/

/

/

\

\

FIG. 12. The geometry of Zn" interactions with sp2 nitrogen-containing heterocycles retreived from the Cambridge Structural Database shows that the metal prefers a head-on and in-plane approach to the sp' lone electron pair of nitrogen. (a and b) Two perpendicular orientations are shown. Dashed lines are at 230" from the C-N-X (X = C, N ) bisector. [Reprinted with permission from Vedani, A., & Huhta, D. W. J . Am. Chem. SOC. 112, 4759-4767. Copyright 0 1990 American Chemical Society.]

in-plane geometry is observed as histidine residues coordinate to zinc in certain metalloproteins, and head-on and in-plane geometry is likewise favored for carboxylate and carbonyl groups hydrogen bonded to these histidine metal ligands. In general, there are several chemical ambiguities regarding the imidazole side chain of histidine as it is observed in protein structures. For example, the protonation state of histidine cannot be ascertained from X-ray crystallographic experiments, and this is of particular concern because the near-physiological first pK, of the imidazole side chain is about 6.0-6.5. T h e second pK, of histidine is about 14.0-14.5, and this value may be lowered -2 units by imidazole coordination to a metal ion (Tainer et al., 1982). Hence, histidine is found in protein structures as the protonated imidazolium or as the neutral imidazole, and even sometimes as the deprotonated imidazolate. There is also a conformational ambiguity characteristic of the histidine side chain as it is built into electron density maps: T h e two possible orientations about the Cp-Cy bond are indistinguishable, so the crystallographer must select one of these orientations based on chemical intuition [e.g., the availability of hydrogen bond interaction(s) in one particular orientation and not the other]. Additionally, the proper histidine tautomer is likewise selected by chemical intuition. Reynolds et al. (1973) demonstrated that the NE-H tautomer is the predominant form of free histidine in solution, and these investigators showed that the ratio NE-H tautomerIN6-H tautomer is approximately 80% :20% (Fig. 13).

STRUCTURAL BIOLOGY OF ZINC

299

FIG.13. The NE-H tautomer of histidine is favored for the free amino acid, but within a protein structure either tautomer may be preferentially stabilized due to hydrogen bond arrangements and other environmental factors. For example, the N6-H tautomer is a frequently observed zinc ligand (Chakrabarti, 1990~).

However, the environment of individual histidine residues within a protein structure may affect this tautomeric equilibrium. When the imidazole side chain of histidine coordinates to a metal ion, some of these chemical ambiguities are resolved. The histidine side chain complexing a metal ion is certainly not in the protonated imidizolium form. However, histidine may be present as the negatively charged imidazolate, as found in copper, zinc-superoxide dismutase, in which His-6 1 bridges the copper and zinc ions. Alternatively, the imidazolate form of a histidine-zinc ligand may be stabilized by hydrogen bonding. Molecular orbital calculations (Nakagawa et al., 1981) suggest that a system in which a carboxylate hydrogen bonds to a histidine which, in turn, coordinates to a zinc ion is isoenergetic with a system in which the carboxylate is protonated and the histidine is negatively charged. That is, C0;-HHis-*Zn2+ is isoenergetic with C02H-His-+Zn2+, and both forms could be in equilibrium at the zinc-binding site (see Section 111,B).Hence, a hydrogen bond ambiguity may remain for the imidazole side chain of histidine as it coordinates to zinc. The conformational ambiguity of the histidine side chain is resolved when histidine coordinates to a metal ion. Logically, only N:, and not C-H, may coordinate to a metal. T h e tautomeric ambiguity of the neutral histidine side chain is similarly resolved when histidine coordinates to a metal ion. Interestingly, the complexation of histidine with zinc may affect the tautomeric equilibrium, since the majority of histidine ligands found in zinc protein structures are in the N6-H form (Chakrabarti, 1990c).

300

DAVID W. CHRISTIANSON

E. Cysteine The sulfur atom is a favorable zinc ligand because of its size and polarizability. The thiol side chain of cysteine (pK, -8.5) is negatively charged as it complexes a metal ion in a protein; in addition to metal coordination, the cysteine thiol may simultaneously accept hydrogen bonds from other protein residues (Adman et al., 1975; Ippolito et al., 1990). Hydrogen bond networks with cysteine metal ligands are discussed further in Section II1,B. A detailed stereochemical analysis of cysteine-metal ion interactions was presented by Chakrabarti (1989) in a survey of the Brookhaven Protein Data Bank. The average S+Zn2+ distance for 14 independent observations is 2.1 A, and the average C@-S+Zn2+ angle is 112". Although 28 independent CP-S+Fe2+ interactions dominate the total number of 47 Cp-S+Mn+ observations, some important torsional relationships are observed in the ensemble which are relevant to the understanding of individual cysteine-zinc interactions. Chakrabarti (1989) found that the Ca-CP-S+Mn+ torsion angle distribution is trimodal with peaks at ?90" and 180" (Fig. 14); torsion angles for the 12 independent Ca-C@-S+Zn'+ interactions are consistent with this distribution. Chakrabarti (1989) observed the following: (1) cysteine residues binding metal ions are found predominantly in turn or coil regions of the poly-

M-SG-CP-Ca

FIG. 14. The distribution of cysteine-metal ion torsion angles, Ca-CP-S-M"+, in protein structures is trimodal, with peaks at +90" and 180"; this distribution holds for M"+ = Zn2+. [Reprinted with permission from Chakrabarti, P. (1989). Biochemistry 28, 60816085. Copyright 0 1989 American Chemical Society.]

STRUCTURAL BIOLOGY OF ZINC

30 1

peptide backbone; (2)although the conformation of the cysteine side chain, as measured by the N-Ca-CP-S torsion angle X I , tends toward gauchef ( X I = 300"; preferred) or gauche- ( X I = 60") in the absence of metal coordination (McGregor et al., 1987), this angle preferentially tends toward a trans (XI = 180"; preferred) or gauche- orientation in which cysteine coordinates to a metal ion; and (3) when X I = 60" the Ca-CP-S+Mn+ torsion angles tend toward go", and when X I = 180" the Ca-CP-S-+M"+ torsion angles tend toward 18O"-the latter combination results in an extended conformation which sets the metal ion at the greatest possible distance from the protein backbone (Chakrabarti, 1989).

F. Solvent Water is often observed as a zinc ligand in metalloprotein structures, particularly where zinc-water or zinc-hydroxide species are important for the catalytic function of enzymes. It is known that the coordination of a water molecule to a four-coordinate zinc ion reduces the solvent pKa to about 7 (Groves and Olson, 1985; Groves and Baron, 1989). Hence, in zinc proteins in which metal-bound water is a known participant in catalysis, it may not be clear whether the water molecule is ionized to hydroxide ion in the resting enzyme. Vedani and Huhta (1990) studied the structure of zinc-water interactions retrieved from the Cambridge Structural Database, and a scatterplot of these interactions is reproduced in Fig. 15. It is obvious that zinc-bound water orients an sp3 lone electron pair toward the metal ion b

a

H

H'

FIG. 15. Zn2+-OH2 interactions retrieved from the Cambridge Structural Database indicate that an sp3 lone electron pair of water engages the metal ion. In view (a), dashed lines indicate the H-0-H bisector. In view (b), which is perpendicular to (a), dashed lines indicate sp2 (0') and sp3 (60")directions. [Reprinted with permission from Vedani, A., & Huhta, D. W. (1990) J . Am. Chem. SOC. 112, 4759-4767. Copyright 0 1990 American Chemical Society.]

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DAVID W. CHRISTIANSON

for effective coordination. Zinc-bound solvent typically donates a hydrogen bond to an amino acid side chain in the metalloenzyme, and this restricts the rotational freedom of the solvent molecule about the Zn'+-O axis (Merz, 1990); see Section 111,B). G . Methionine and Selenocysteine

Although methionine is not a common metal ligand in proteins, it is possible that its function as a zinc ligand may be observed in a naturally occurring or engineered protein. For example, it is particularly intriguing to see the possible participation of methionine in the binding of a third metal ion to the glucocorticoid receptor (Pan et al., 1990). Alternatively, a methionine residue may be engineered as a protein zinc ligand by site-directed mutagenesis methods. Rosenfield et al. ( 1977) reported the geometric preference for the interaction of metal ions with divalent sulfur (i.e., as found in the sulfide of methionine, -CH2-S-CH3). Specifically, Lewis acids such as metal ions approach the sulfide sulfur with an average polar angle 8 = 21", and the longitudinal angle 4 tends to lie within the range 150"-180" (spherical polar coordinates are defined in Fig. 16). This geometry indicates the participation of a sulfur lone electron pair in metal complexation, and the average elevation of metal ions from the sulfide plane is 69" (Kosenfield et al., 1977).

normal

. .

FIG. 16. Spherical polar coordinate system used to define metal ion (M) interactions with the side chain of methionine. In proteins the average polar angle 0 is 38O, and the average longitudinal angle 4 is 169". [Reprinted with permission from Chakrabarti, P. (1989) Biochemistry 28,608 1-6085. Copyright 0 1989 American Chemical Society.]

STRUCTURAL BIOLOGY OF ZINC

303

More recently, Chakrabarti (1989) analyzed the geometry of 14 independent methionine-metal interactions (metals are predominantly iron, then copper and mercury) and found that the average polar angle 8 = 38" and the average longitudinal angle 4 = 169" (Chakrabarti, 1989) (Fig. 16). This indicates that metal ions tend to approach the methionine sulfur at an elevation of 52" from the C-S-C plane, indicating an interaction with a lone electron pair orbital of sulfur. Although these average values contrast with those calculated in the analysis of small-molecule crystal structures summarized above (Rosenfield et al., 1977), Chakrabarti ( 1989) logically attributed this discrepancy to the greater inaccuracy accompanying the determination of the position of the terminal carbon of methionine in protein structures. Finally, Chakrabarti ( 1989) found that methionine residues involved in metal complexation are generally found in coiled regions of the polypeptide backbone. Selenocysteine has been implicated in binding the divalent nickel ion in the active site of the [NiSeFeIhydrogenase from Desulfovibrio baculatus (Eidsness et al., 1989). Given the highly polarizable nature of the selenol, -SeH, and its relative acidity with a pKa of 5.2 (Huber and Criddle, 1967), selenocysteine is negatively charged and available for metal ion complexation at physiological pH. Although no examples of selenocysteine-zinc interactions are known in biological systems, the complexation of a firstrow transition metal ion by this novel amino acid in the hydrogenase active site suggests a use for selenocysteine in the complexation of other biological transition metals. In a comparative study of C-S--zZn2+ and C-Se-+Zn2+ interactions in small-molecule complexes, Ueyama et al. (1988) reported some important structural distinctions between sulfur and selenium as contained in thiophenolato and selenophenolato metal li ands. These investigators found that both the Zn(SPh)z- and Zn(SePhi- complexes reveal a distorted tetrahedral coordination polyhedron about the metal ion, and that steric congestion resulting from the bulky phenyl rings likely gives rise to the distortion. Additionally, Se+Zn2+ bond lengths are consistently about 0.1 8, longer than S+Zn2+ bond lengths, and C-Se bond lengths are likewise about 0.1 8, longer than C-S bond lengths. Finally, a smaller C-X-+Zn'+ angle and a longer X-+Zn2+ distance for X = Se than for X = S is said to indicate an increase in the ionic character of the X+Zn2+ bond; this may correlate with the increased strength of metal complexation. The unique size, polarizability, and geomertric properties of the C-Se- anion may ultimately be exploited toward zinc complexation in a selenocysteine-engineered protein; alternatively, this group might be incorporated into a potent zinc-enzyme inhibitor.

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DAVID W. CHRISTIANSON

111.

LONG-RANGE

PROTEIN-METALINTERACTIONS

Protein residues far removed from a metal-binding site may affect metal recognition and specificity; this is particularly so for charged residues. Thus, the entire protein-notjust the direct metal ligands-plays a role in metal recognition and binding. Here, long-range effects are considered within two general categories: effects which do not involve apparent hydrogen bonds (i.e., they involve bulk electrostatic or through-space effects within the protein scaffolding) and effects across hydrogen bond networks involving metal ligands. A. Long-Range Electrostatic Effects N o systematic studies of long-range electrostatic effects on proteinZn2+ interactions are available. However, studies of long-range electrostatic effects on protein-H+ and protein-Ca2+ interactions illustrate concepts which apply to long-range protein-Zn2+ interactions. Studies of long-range protein-H+ interactions have been reported from Fersht's laboratory, where the relationship between surface charge of the serine protease subtilisin and the pK, of active-site residue His-64 has been detailed (Thomas et al., 1985; Russell and Fersht, 1987; Russell et al., 1987). In a series of site-specific mutants of subtilisin in which two negatively charged carboxylate residues on the protein surface, Asp-99 and Glu- 156, are replaced by uncharged serines and positively charged lysines, Russell and Fersht (1987) observed shifted pH activity profiles, altered catalytic activities,and altered substrate specificities for each mutant enzyme. In particular, the pK, variation of His-64 with changes in protein surface charge demonstrates the effects of long-range electrostatics on histidine basicity: If the overall negative charge of the protein is decreased, protonated His-64 is destabilized and thus its pKa decreases. A diagram illustrating the spatial relationships among His-64 and residues 99 and 156 of subtilisin is shown in Fig. 17. Protein structure intervenes between His-64 and position 99 (- 13 A), and solvent intervenes between His-64 and position 156 (-15 A). Wild-type subtilisin has Asp-99 and Glu-156, and when either of these side chains is mutated to serine, the pK, of His-64 decreases by 0.4 (assayed at ionic strength 0.001 M). If both side chains are mutated to serines, the pKa of His-64 in the double-mutant decreases by 0.65. Hence, decreasing the amount of negative surface charge results in the expected pKa decrease for His-64. Further perturbation of the surface charge results in even greater effects. The mutation of either Asp-99 or Glu-156 to a positively charged lysine results in a pKa decrease of 0.6 for His-64, and the pKa of the

STRUCTURAL BIOLOGY OF ZINC

305

su bt i I i s i ti

FIG. 17. Schematic diagram of subtilisin showing the spatial relationships among His-64, Asp-99, and Glu-156.

double-lysine mutant decreases by about 1 unit. The same electrostatic effect is exerted on His-64 from either position 99 or position 156, and this long-range effect apparently does not depend on the composition of the intervening milieu [i.e., protein or solvent (Russell and Fersht, 1987)l. Additionally, coulombic effects exhibit near-additivity ; that is, the ApK, for the double-mutant is almost equal to the sum of the ApK, values for each of the two corresponding single-site mutants. Minor reorganization of the protein structure and/or the solvent structure in the mutant enzymes may result in less than perfect additivity (Russell and Fersht, 1987). The salt dependence of the His-64 pK, differs between the Asp99-Lys mutant and the Glu-l56+Lys mutant. On increasing the ionic strength from 0.00 1 M to 0.1 M, the ApK, of His-64 between wild-type and mutant subtilisins is increasingly smaller for the Glu-l56+Lys mutant (Russell and Fersht, 1987). Apparently, counterions tend to cluster about Lys- 156, and the negatively charged counterion cluster stabilizes the protonated form of His-64. As a result, His-64 does not experience as much of a pK, decrease with increasing ionic strength in the Glu1 5 h L y s mutant as compared with the Asp-99+Lys mutant. Negatively charged counterions clustering about Lys-99 would not be in the vicin-

306

DAVID W. CHRISTIANSON

ity of His-64, so the protonated form of His-64 would not be stabilized in this mutant. Th e stabilizing effect of counterion clustering in the active site is more pronounced for counterdianions, for which coulombic interactions are twice as strong (Russell and Fersht, 1987). Russell and Fersht (1987) established rules for altering p H activity profiles by long-range electrostatic effects; some of these rules are pertinent to the understanding of long-range effects on protein-metal interactions. Making a protein surface more negatively charged raises the pKa values of protein residues, and making a protein surface more positively charged lowers the pKa values of protein residues; these effects will be most pronounced in media of low ionic strength. Hence, making a protein surface more negatively charged results in stronger metalbinding sites. Experimental results on the long-range modulation of subtilisin-Ca2+ interactions (Pantoliano et al., 1988) are in accord with the expectations raised by the results of subtilisin-H+ interactions (Russell and Fersht, 1987). The three-dimensional structure of subtilisin BPN’ has been refined to R = 0.14 at 1.3 A resolution (Pantoliano et al., 1988), and two divalent metal-binding sites are observed (Finzel et al., 1986; Bryan, 1987). T h e first metal-binding site is a high-affinity site, with log K,,,,, = -8, termed CaA.The second metal-binding site is a low-affinity site, with log K,,,,, = 1.49, termed CaB. Site-specific mutagenesis work centers on improving the affinity of site CaB;two single-site mutants at residues 172 and 131, plus the corresponding double-mutant, have been studied. T h e structure of the Pro-172-A~~ mutant shows that the carboxylate side chain of aspartate is 6.8 8, from CaB, and measurements indicate that log K,,,, = 2.02. T h e structure of the Gly-l3l+Asp mutant shows that the carboxylate side chain of Asp-131 is 13.2 A from CaB, and log K,,,,, = 1.82. The double-mutant exhibits a log K,,,,, of 2.27, which results in a ApK of 0.78 compared with the wild-type enzyme. Although this is only a modest increase in metal affinity at CaB, it is important to note that protein-M”+ affinity can be regulated by electrostatic interactions occurring over long distances: A more negatively charged protein surface results in higher-affinity metal-binding sites. Other long-range effects which may be exploited for metal binding are those involving the so-called “macrodipoles” of a helices, that is, the sizable electrostatic potentials that characterize the amino and carboxy termini of a helices. T h e amino-terminal portion of an a helix is characterized by a positive electrostatic potential and is implicated in the binding of phosphate anions to proteins (Hol et al., 1978); the carboxyterminal end of the helix is likewise characterized by a negative electrostatic potential and may be similarly implicated in cation binding. Work

STRUCTURAL BIOLOGY OF ZINC

307

from Fersht’s laboratory on barnase demonstrates that helix termini effects are responsible for perturbing the pKa of His-18 (at the carboxyterminal end of an a helix) to 7.9, 1.6 units above the pKa value in the urea-denatured protein (Sali et al., 1988). If electrostatic effects enhance the basicity of carboxy-terminal histidine toward the proton, they might likewise enhance the basicity of carboxy-terminal histidine toward a metal ion. Zinc-binding a! helices have been designed with helix termini effects in mind, where His- Alas-His or Cys- Alas-His units have been placed at the carboxy termini of short a helices with the intention of enhancing Zn2+ complexation (Ghadiri and Choi, 1990). B. Hydrogen Bond Networks Involving Zinc Ligands The amino acid side chains which serve as zinc ligands in a metalloprotein are densely packed within the protein structure and often make hydrogen bond contacts with other residues. Argos et al. (1978) pointed out that such interactions orient the metal ligands, and these interactions may also enhance the electrostatic interaction between the metal ion and its ligands. For example, consider the carboxylate-histidine-zinc triad of the zinc enzyme active site (Argos et al., 1978; Christianson and Alexander, 1989, 1990). A carboxylate (usually aspartate, but sometimes glutamate) hydrogen bonded to histidine enhances the basicity of histidine toward the proton by as much as 2 pK, units (Carver and Bradbury, 1984; Bradbury and Carver, 1984); if histidine makes a hydrogen bond with a carbonyl, the ApKa is about half as much (Perutz et al., 1985). Likewise, the basicity of histidine toward a metal ion such as zinc is enhanced by a hydrogen bond with a negatively charged carboxylate. This metal-binding motif is described as indirect carboxylate-zinc interaction across bridging histidine, and it is probably optimal when the carboxylate engages the histidine through a syn-oriented hydrogen bond (Christianson and Alexander, 1989) (Fig. 18). Interestingly, Christianson and Alexander (1989) speculated that the analogous phosphate-histidine-zinc triad may be a feature of certain zinc-requiring DNA-binding proteins in their complexes with nucleic acids (see Section IV,D). The carboxylate-histidine hydrogen bond also reduces the entropic barrier to the organization of the metal-binding site as histidine coordinates to zinc, since this hydrogen bond orients the histidine with the proper geometry for metal coordination (Argos et al., 1978). In an X-ray crystallographic study of apocarboxypeptidase A, it was found that His69, a zinc ligand which is anchored by a hydrogen bond with Asp-142, does not undergo an appreciable conformational change as zinc disso-

308

DAVID W. CHRISTIANSON

0

0

\=I=/

Asp, G i A c - - - - -HN'L----Z"~+

His

==

9

Asp, GluAOH-----.NAN-Z"Z*

His

FIG. 18. The carboxylate-histidine-zinc triad represents indirect carboxylate-zinc interaction across bridging histidine. Both tautomers of histidine are observed, and the hydrogen bond stereochemistry with carboxylate (either aspartate or glutamate) is generally syn. Experimental results and theoretical calculations suggest that the carboxylatehistidin-zinc form may be in equilibrium with the carboxylic acid-histidinat-zinc form, as shown.

ciates from the enzyme; in contrast His-196, which is not anchored by a similarly strong hydrogen bond, undergoes a noticeable conformational change on zinc removal (Rees and Lipscomb, 1983). T h e carboxylatehistidine-zinc triad of carboxypeptidase A is found in Fig. 19. Theoretical studies of carboxypeptidase A suggest that the coordination of the aspartate-histidine couple to zinc, Asp-142-His-69+Zn2+, facilitates proton transfer from histidine to the carboxylate of aspartate (Nakagawa et al., 1981). Furthermore, Nakagawa et al. (1981) found that

FIG. 19. The carboxylate-histidine-zinc triad appears in the active site of carboxypeptidase A as Asp-l42-His-69+Zn2+, and this interaction may contribute to zinc affinity in the metalloenzyme active site. Atomic coordinates were retrieved from the structure of the native enzyme (Rees et al., 1983) deposited in the Brookhaven Protein Data Bank.

STRUCTURAL BIOLOGY OF ZINC

309

the forms carboxylate-histidine (C0;-H-His) and carboxyl-histidinate (COZH-His-) are essentially isoenergetic in the presence of zinc, but in the absence of zinc they find that the former pair is 24 kcal/mol more stable. T h e suggestion that the zinc-complexing motif may actually be an equilibrium population of CO&H-His+Zn'+ and COZH-His-+Zn'+ forms is novel and may find verification in the active site of the iron metalloprotein cytochrome c peroxidase, where the results of resonance Raman spectroscopic experiments suggest the presence of a carboxylhistidinate-iron triad (Smulevich et al., 1988). A double-well potential for the imidazole proton is proposed where the two minima are not very different in energy, since the two resonance Raman components corresponding to protonated histidine and protonated carboxylic acid are of comparable intensity in the spectrum. If the carboxylate-histidine-iron triad of cytochrome c peroxidase is in equilibrium with the carboxylhistidinate-iron form, as demonstrated by experiments (Smulevich et al., 1988), then it is a reasonable expectation that the carboxylate-histidinezinc triad of the zinc enzymes is in equilibrium with the carboxylhistidinate-zinc form, as predicted from theory (Nakagawa et al., 1981) (Fig. 18). A different type of histidinate-zinc interaction occurs in the active site of superoxide dismutase (Tainer et al., 1982), where the histidinate anion is stabilized by bridging Zn2+ and Cu2+. In addition to facilitating zinc complexation, the indirect carboxylatezinc interaction via bridging histidine is likely to "fine tune" the pK, and reactivity of zinc-bound solvent in an enzyme active site. As compared with other amino acid side chains, the imidazole of histidine is the optimal candidate for such modulation. Although the carboxylate itself may engage in hydrogen bonds while coordinated to a metal ion, such contact is not as effective in modulating basicity due to electronic reasons: A carboxylate engaging a metal ion with syn stereochemistry may receive hydrogen bonds with only the less favorable anti geometry. Additionally, the negatively charged carboxylate and thiolate groups may only accept hydrogen bonds; in doing so, the basicity of these groups toward metal complexation must diminish slightly. Although sulfur is not necessarily the most favorable partner in hydrogen bond arrangements, the cysteine thiol(ate) has been observed to engage in stereochemically well-defined hydrogen bond interactions (Adman et al., 1975; Ippolito et al., 1990). In particular, NH-S hydrogen bonds, involving backbone NH groups of the polypeptide chain and the thiolate sulfurs of cysteine metal ligands, are observed in the structures of iron-binding proteins (Adman et al., 1975).These interactions are important for metal-binding site organization, as well as the folding and stabilization of zinc protein structure. For example, Berg (1988) proposed a

310

DAVID W. CHRISTIANSON

model for a zinc finger domain of transcription factor IIIA from Xenopus oocytes, in which two cysteine sulfur atoms (which are also zinc ligands) receive hydrogen bonds from backbone NH groups of the peptide. Berg (1988) formulated his prediction by analogy with hydrogen bonds to cysteine first noticed in rubredoxin and ferredoxin (Adman et al., 1975). Among nonprotein zinc ligands metal-bound water typically engages in significant and functionally important hydrogen bond interactions with charged protein residues. Such interactions not only enhance the basicity of zinc-bound solvent in its coordination to the metal ion, but they also minimize rotational entropy about the O+Zn2+ axis (Merz, 1990). For example, Glu-270 of carboxypeptidase A and Glu-143 of thermolysin accept syn-oriented hydrogen bonds from zinc-bound water molecules (Rees et al., 1983; Holmes and Matthews, 1982); Thr-199 of carbonic anhydrase (which also donates a syn-oriented hydrogen bond to Glu- 106) accepts a hydrogen bond from zinc-bound hydroxide (Fig. 20). These hydrogen bond networks comprise indirect carboxylate-zinc interactions via bridging hydroxyl(s), and these networks function to keep the nucleophilic solvent molecule oriented properly for attack at the substrate C=O; too much rotational freedom about the O+Zn'+ axis would confer an entropic barrier to solvent reactivity which would compromise efficient catalysis (Merz, 1990) (Fig. 21).

IV. EXAMPLES OF ZINC I N BIOLOGICAL CATALYSIS AND REGULATION Four examples of catalytic or regulatory zinc proteins are reviewed here, and the discussion of metalloprotein function is set within the context of the metal ion and its coordination polyhedron. In the zinc enzymes carbonic anhydrase (carbonate dehydratase) I1 and carboxypeptidase A, the coordination polyhedron of the metal ion changes as the

a

b

FIG.20. Schematic diagram of indirect carboxylate-zinc interactions through bridging hydroxyl groups, as observed in the (a) zinc proteases and (b) carbonic anhydrases.

31 1

STRUCTURAL BIOLOGY OF ZINC

FIG. 2 1 . Indirect carboxylate-zinc interactions through bridging hydroxyl groups (Fig. 20) orient the nucleophilic lone electron pair (stippled dumbbell) of zinc-bound solvent. This reduces the conformational disorder about the Zn'+-O axis, and thereby reduces the entropic barrier to catalysis (Merz, 1990).

chemistry of catalysis proceeds. However, dynamic metal coordination behavior is not an important aspect for the function of metallothionein or the zinc-requiring transcription factors; in these examples, zinc apparently serves a purely structural role.

A . Carbonic Anhydrase 11 Human carbonic anhydrase 11, found primarily in the erythrocyte, is the prototypical member of the family of carbonic anhydrases and has been extensively reviewed (Pocker and Sarkanen, 1978; Lindskog, 1983, 1986; Silverman and Lindskog, 1988). Within the erythrocyte carbonic anhydrase I1 hydrates CO:! to form bicarbonate ion plus a proton via tandem chemical processes (Silverman and Lindskog, 1988) (Scheme 2). Most of the carbon dioxide generated during the process of respiration requires this carbonic anhydrase 11-catalyzed event for transport out of the cell. The resultant protons of COa hydration are taken u p by His146p, His- 122a, and the amino terminus of the a subunits of the hemoglobin tetramer. As a reference, Scheme 3 outlines the interconversions

H20= H20

+ C02-

HOHCO;

SCHEME 2

+

H*

+

H*

312

DAVID W. CHRISTIANSON

C02+ H2O 0 H* + HCO,

H2C03

SCHEME 3. KH = 0.0026; K , = 10-6.35;K H ~ C Q= 10”~”M.

among relevant species of the carbonic acid system (Lindskog and Coleman, 1973, and references cited therein). Importantly, carbonic anhydrase I1 is one of the most efficient biological catalysts known and it catalyzes the hydration of COn with a turnover rate of lo6 sec-’ at 25°C (Khalifah, 1971; Steiner et al., 1975). With k,,,lK, = 1.5 X lo8 M-’ sec-’, carbonic anhydrase I1 is one of a handful of enzymes for which catalysis apparently approaches the limit of diffusion control. Since transfer of the product proton away from the enzyme to bulk solvent comprises a kinetic obstacle [an enzyme-bound group with a pKa of about 7 cannot transfer a proton to bulk solvent at a rate faster than lo3 sec-’ (for a review see Eigen and Hammes, 1963)],the observed turnover rate of lo6 sec-’ requires the participation of buffer in the proton transfer. X-Ray crystallographic studies of native human carbonic anhydrase I1 have been reported at 2.0 8, resolution (Eriksson et al., 1986, 1988a). Important active-site residues include Thr- 199, Thr-200, Glu- 106, His64, Trp-209, Val-121, Val-143, the zinc ion (liganded by His-94, His-96, and His-119),and the zinc-bound hydroxide ion. A schematic view of the active site is found in Fig. 22. The globular protein has a molecular His-64

, I

I

Glu-106 405 -

His-94

’I

Zn2+ ‘His-1 His-96

19

FIG.22. Important residues in the active site of carbonic anhydrase 11.

313

STRUCTURAL BIOLOGY OF ZINC

weight of about 29K and is comprised of a single polypeptide chain of 260 amino acids. The active site of the enzyme can be envisaged as a conical cavity extending into the core of the protein. The zinc ion resides at the bottom of the conical cavity, and this cavity is roughly divisible into hydrophobic and hydrophilic halves. The catalysis of COz hydration by carbonic anhydrase I1 occurs via the two chemically independent steps outlined in Scheme 2; a general mechanistic profile is found in Fig. 23. The first step involves the association of substrate with enzyme and the chemical conversion of substrate into product. The second step is product dissociation and the regeneration of the catalytically active nucleophile zinc hydroxide (Coleman, 1967). Below, we address the structural aspects of zinc coordination in each of these steps. The participation of zinc in the association of substrate C O z with the 0

0

H

H

\ 0-

\\ \(0 -

c\2

II

H

\ 0/c\o-

I -

I I I

I I

I

/'& \

-H* to

H

buffer via His 64

H

\0

/

\0 /H I

I I

/'& FIG. 23. A general mechanism of COBhydration as catalyzed by carbonic anhydrase 11. Certain structural details (e.g., the function of pentacoordinate zinc or the degree of C02-Zn2+ interaction in enzyme-substrate association)remain to be elucidated.

314

DAVID W. CHRISTIANSON

-202

4 s @ C 6 -106

C D 1 -209

I

-64

-67

FIG.24. “Snapshots” of a possible COB diffusion pathway into the active site of carbonic anhydrase 11, as calculated in a molecular dynamics simulation. The COB molecule is represented as a “stick,” and its diffusion pathway is shown by dashed arrows. [Reprinted with permission from Liang, J.-Y., & Lipscomb, W. N. (1990)Proc. Natl. Acad. Sci. U.S.A.87, 3675-3679.1

enzyme has been the subject of much discussion (Lindskog, 1983, 1986; Silverman and Lindskog, 1988, and references cited therein), but a precatalytic inner-sphere C=O+Zn2+ interaction is probably not operative in the carbonic anhydrase mechanism. The results of spectroscopic studies suggest a discrete binding site for substrate COP,and this site is probably not the zinc ion (e.g., Bertini et al., 1987). However, the outer-sphere interaction of CO:! with zinc is not precluded, and it may interact, over a large distance (i.e., 3 or greater), with an incipient fifth coordination site on the metal (Eriksson et al., 1988a,b). Riepe and Wang (1968) presented spectroscopic evidence which apparently does not support a zinc-COP interaction, but subsequent molecular orbital calculations demonstrate that the spectroscopic results are not inconsistent with inner-sphere zinc-CO:! coordination (Liang and Lipscomb, 1989).

STRUCTURAL BIOLOGY OF ZINC

3 15

Inspection of the carbonic anhydrase I1 active site reveals hydrophobic clefts or patches where the recognition of the hydrophobic gas molecule may occur with some affinity. The question of protein-gas affinity has been recently approached from the theoretical molecular dynamics level. Liang and Lipscomb (1990) have reported the possibility of three C02binding sites in the carbonic anhydrase I1 active-site cleft, and Merz (1990, 1991) has identified two CO2-binding sites. Both research groups identified a hydrophobic gas-binding site near Trp-209, about 3-4 hi from zinc, and this site is probably the precatalytic location of C02 just prior to nucleophilic attack by zinc-bound hydroxide. In this site substrate C02 is about 3.5 8, from zinc (Liang and Lipscomb, 1990; Merz, 1990). Figure 24 shows a possible CO2 diffusion pathway into the carbonic anhydrase I1 active site, and Fig. 25 shows the binding of C 0 2in the hydrophobic pocket (Liang and Lipscomb, 1990). Also in Fig. 25 is the binding of CO2 in this pocket as independently calculated by Merz (1990, 1991). The hydrophobic pocket corresponds to the location of the socalled “deep” water molecule in the native enzyme, and Lindskog (1986) proposed that this water is displaced by COPassociation. Clearly, the results of molecular dynamics simulations are consistent with this proposal. The results of kinetic and X-ray crystallographic experiments on mutant carbonic anhydrases 11, in which side-chain alterations have been made at the residue comprising the base of the hydrophobic pocket (Val-143),illuminate the role of this pocket in enzyme-substrate association. Site-specificmutants in which smaller hydrophobic amino acids such as glycine, or slightly larger hydrophobic residues such as leucine or isoleucine, are substituted for Val-143 do not exhibit an appreciable change in CO2 hydrase activity relative to the wild-type enzyme; however, a substitution to the bulky aromatic side chain of phenylalanine diminishes activity by a factor of about lo4, and a substitution to tyrosine results in a protein which displays activity diminished by a factor of about lo5 (Fierke et al., 1991). The X-ray crystal structure of the Val-143-Gly mutant reveals a hydrophobic pocket that is essentially unchanged except for its depth, and the X-ray structures of the Val-l43+Phe and Val-143-Tyr mutants show that the larger side chains at position 143 nearly obliterate the pocket; moreover, the phenolic hydroxyl of Val-143-Tyr engages the zinc-bound hydroxide ion in a long hydrogen bond-polar van der Waals contact (Alexander et al., 1991) (Fig. 26). Hence, a working hypothesis is that the Val-1434Phe mutation and near-obliteration of the hydrophobic pocket severely hinders C02 association, and the combination of pocket obliteration and hydrogen bond engagement of the zinc-bound nucleophile in the Val-143-Tyr mutant results in an additional 10-fold loss of activity.

316

DAVID W. CHRISTIANSON

a

b

FIG. 25. (a) Proposed binding of substrate COP(the dark “stick in the center) in the hydrophobic pocket of carbonicanhydrase 11, as calculated in a moleculardynamics simulation. [Reprinted with permission from Liang, J.-Y., & Lipscomb, W. N. (1990) Proc. Nutl. Acud. Sci. U.S.A. 87, 3675-3679.1 (b) Proposed carbonic anhydrase II-COP complex, as calculated in an independent investigation (Merz, 1990, 1991, personal communication).

STRUCTURAL BIOLOGY OF ZINC

317

FIG.26. Electron density map, calculated with Fouriercoefficients21Fol - IF,) and phases calculated from the final model, of the hydrophobic pocket of the Val-149Tyr mutant of human carbonic anhydrase I1 (Alexander et al., 1991). This mutation nearly obliterates the pocket and results in a 105-f01dloss of activity (Fierke et al., 1991).

It is interesting that although the Val-l43+His mutation leads to a bulky side chain at the base of the hydrophobic pocket, the mutant enzyme exhibits only a 1OZ-foldloss of COz hydrase activity relative to the wild-type enzyme (Fierke et al., 1991). In this mutant the Val-143-His side chain packs differently in the pocket relative to the side chains of the Val-l43+Phe and Val-143-Tyr mutants (Alexander et al., 1991). It is likely that differences in side-chain packing, as well as differences involving active-site solvent structure, are responsible for differences in enzyme-substrate association behavior among the residue- 143 mutants of carbonic anhydrase 11. Subsequent to COz association in the hydrophobic pocket, the chemistry of turnover requires the intimate participation of zinc. The role of zinc is to promote a water molecule as a potent nucleophile, and this is a role which the zinc of carbonic anhydrase I1 shares with the metal ion of the zinc proteases (discussed in the next section). In fact, the zinc of carbonic anhydrase I1 promotes the ionization of its bound water so that the active enzyme is in the zinc-hydroxide form (Coleman, 1967; Lindskog and Coleman, 1973; Silverman and Lindskog, 1988).Studies of small-molecule complexes yield effective models of the carbonic anhydrase active site which are catalytically active in zinc-hydroxide forms (Woolley, 1975).In addition to its role in promoting a nucleophilic water molecule, the zinc of carbonic anhydrase I1 is a classical electrophilic catalyst; that is, it stabilizes the developing negative charge of the transition state and product bicarbonate anion. This role does not require the inner-sphere interaction of zinc with the substrate C==O in a precatalytic complex.

318

DAVID W. CHRISTIANSON

The carbonic anhydrase I1 mechanism may proceed through one or more pentacoordinate zinc intermediates involving two nonprotein ligands (Ericksson et d., 1988a,b). For example, zinc may be involved in the syn-bidentate stabilization of product bicarbonate anion and its preceding transition state, and the metal ion may pass through a five-coordinate intermediate involving one oxygen of bicarbonate and one oxygen of a water molecule as the catalyst is regenerated. Pentacoordinate zinc complexes, involving one inner-sphere nonprotein ligand and another nonprotein ligand tending toward outer-sphere coordination, have been observed in carbonic anyhydrase II-inhibitor complexes (Eriksson et al., 1988b), as well as in enzyme-inhibitor complexes with the related zinc enzyme carboxypeptidase A (Christianson and Lipscomb, 1989). Hence, the prospect of a five-coordinate zinc intermediate(s) in the carbonic anhydrase I1 mechanism is not unreasonable. A hydrogen bond network in the carbonic anhydrase I1 active site functions to optimize the geometry of zinc-hydroxide attack at COP. I n a detailed molecular dynamics study of the Glu-106-Thr- 199HO-+Zn2+ hydrogen bond network, Merz (1990) proposed that this network functions to keep the nucleophilic hydroxide ion oriented properly for attack at the substrate C=O. The stereochemical analysis of this hydrogen bond network, in which Thr-199 serves as a bridge, shows that it is composed of optimally oriented, nearly linear hydrogen bonds in the native enzyme (Ippolito et al., 1990): syn-oriented Glu-106 accepts a hydrogen bond from Thr-199 with a trans-oriented OH group, and this OH group, in turn, accepts a gauche--oriented hydrogen bond from zinc-bound hydroxide (Fig. 27). The Thr- 199 hydroxyl oxygenhydroxide oxygen-zinc angle of 103" indicates that a hydroxide lone electron pair is directed almost precisely toward the metal ion. There may be two proton transfers in the carbonic anhydrase IIcatalyzed mechanism of COP hydration that are important in catalysis, and both of these transfers are affected by the active-site zinc ion. T h e first (intramolecular) proton transfer may actually be a tautomerization between the intermediate and product forms of the bicarbonate anion (Fig. 28). This is believed to be a necessary step in the carbonic anhydrase I1 mechanism, due to a consideration of the reverse reaction. The coulombic attraction between bicarbonate and zinc is optimal when both oxygens of the delocalized anion face zinc, that is, when the bicarbonate anion is oriented with syn stereochemistry toward zinc (this is analogous to a syn-oriented carboxylate-zinc interaction; see Fig. 28a). This energetically favorable interaction probably dominates the initial recognition of bicarbonate, but the tautomerization of zinc-bound bicarbonate is subsequently required for turnover in the reverse reaction (Fig. 28b).

319

STRUCTURAL BIOLOGY OF ZINC

Thr-199

FIG.27. Optimal hydrogen bond stereochemistry in the active site of carbonic anhydrase 11. Lone electron pairs are represented by stippled dumbbells.

Experimental evidence in support of bicarbonate tautomerization has been presented by Pocker and Deits (1983), who reported that alkyl carbonates ROCO; bind to carbonic anhydrase 11, but are not active in the reverse reaction. If only the reorientation of the zinc-bound carbonate were required for catalytic activity, it would not be unreasonable to expect carbonic anhydrase I1 to catalyze the degradation of alkyl carbonates. However, alkyl carbonates cannot undergo the proton transfer

0

ONH

I

/:\

0-0

a

H\o/C\o

ii;, b

FIG. 28. (a) Product bicarbonate anion and (b) intermediate bicarbonate anion are tautomers.

DAVID W. CHRISTIANSON

320

His-64

I

-

Thr 199-0 H

--

I I

GIU -106-CO;

His-64

I

Thr

Glu

11

I I I I

-

buffer His-64

J.

2n2+

Glu 106-Co;

FIG. 29. The regeneration of zinc-bound hydroxide is achieved by a proton transfer from zinc-bound water through a solvent network to His-64, which then releases a proton to buffer. His-64 may undergo changes in tautomerization and/or conformation during this process.

STRUCTURAL BIOLOGY OF ZINC

32 1

required for tautomerization of the zinc-bound species, and the inactivity of alkyl carbonates toward carbonic anhydrase II-catalyzed degradation is consistent with the suggestion that a proton transfer rather than a reorientation of zinc-bound bicarbonate is responsible for the interconversion of forms a and b in Fig. 28. Molecular orbital calculations support this conclusion, and additionally suggest that the proton transfer of bicarbonate-zinc tautomerization is facilitated by one or two additional solvent molecules, or perhaps the hydroxyl group of Thr-199 (Liang and Lipscomb, 1987). Other calculations suggest that a simple rotation of zinc-bound bicarbonate is energetically more favorable than a pathway involving intramolecular proton transfer (Jacob et al., 1990). A second proton transfer of catalytic importance involves zinc-bound solvent, that is, the ionization of zinc-bound water to regenerate the active catalyst species zinc-hydroxide. This step is rate limiting for COPhydration (for a review see Silverman and Lindskog, 1988)and is mediated by a hydrogen-bonded solvent network between zinc-bound water and His64, and then by buffer (Fig. 29). This conclusion is verified by recent studies of site-specific mutants of carbonic anhydrase I1 at position 64, which show that, although other proton transfer pathways exist, the pathway involving His-64 is optimal and less dependent on the chemical nature of the buffer (Forsman et al., 1988; Tu et al., 1989).The results of molecular orbital calculations are also in accord with these data (Liang and Lipscomb, 1988),and other calculations suggest that the bicarbonate ion itself may serve as a bridge in the proton relay mechanism (Merz et al., 1989). X-ray crystallographic work suggests that the role of His-64 as a proton shuttle may involve rapid conformational changes about the Cp-C, and C,-Cp bonds of the side chain (Krebs et al., 1991; Nair and Christianson, 1991). A final consideration of proton transfer concerns the hydrogen bond network between Glu- 106, Thr-199, and zinc-bound solvent. There remains an unfavored (Silverman and Vincent, 1983) hypothesis that Glu106 participates in a proton transfer event required for the regeneration of the catalyst (Kannan et al., 1977).A possible proton relay is implicated between Thr- 199 and syn-oriented Glu- 106of carbonic anhydrase 11, and the optimal hydrogen bond stereochemistry among these species could facilitate such a proton transfer (see Fig. 27). However, a proton transfer involving Glu-106 is not a feature of the generally accepted mechanism (see, e.g., Silverman and Lindskog, 1988). Interestingly, though, Glu- 106 is in contact with His-64 (and, hence, bulk solvent) through a hydrogen bond network (Eriksson et al., 1988a; Vedani and Huhta, 1990).

322

DAVID W. CHRISTIANSON

B . Carboxypeptidase A and Other Zinc Proteases Zinc proteases carboxypeptidase A and thermolysin have been extensively studied in solution and in the crystal (for reviews, see Matthews, 1988; Christianson and Lipscomb, 1989). Both carboxypeptidase A and thermolysin hydrolyze the amide bond of polypeptide substrates, and each enzyme displays specificity toward substrates with large hydrophobic Pi side chains such as phenylalanine o r leucine. T h e exopeptidase carboxypeptidase A has a molecular weight of about 35K and the structure of the native enzyme has been determined at 1.54 8,resolution (Rees et al., 1983). Residues in the active site which are important for catalysis are Glu-270, Arg-127,Zn2+ (liganded by His-69, His-196, and Glu-72 in bidentate fashion), and the zinc-bound water molecule (Fig. 30). T h e endopeptidase thermolysin has a molecular weight of about 35K, and the structure of the native enzyme has been reported at 1.6 resolution (Holmes and Matthews, 1982). Residues in the active site which are important for binding and catalysis are Glu-143, His-23 1, Zn2+ (liganded by His-142, His-146, and Glu-166 in unidentate fashion), and the zinc-bound water molecule (Fig. 30). Zinc proteases carboxypeptidase A and thermolysin are related by convergent evolution, and the common catalytic features of each zinc protease active site are as follows (Matthews, 1988; Christianson and Lipscomb, 1989): (1) the zinc ion, liganded by two histidine residues, a negatively charged glutamate, and a bound water molecule; (2) a second glutamate residue (Glu-270 in carboxypeptidase A, Glu- 143 in thermolysin) hydrogen bonded to zinc-bound water and functioning as a general base/acid; and (3)a positively charged electrophilic enzyme residue (Arg127 in carboxypeptidase A, His-231 in thermolysin) flanking the substrate-binding groove on the side opposite that of the general base/acid (Fig. 30). Carboxypeptidase A was the first zinc enzyme to yield a threedimensional structure to the X-ray crystallographic method, and the structure of an enzyme-pseudosubstrate complex provided a model for a precatalytic zinc-carbonyl interaction (Lipscomb et al., 1968). Comparative studies have been performed between carboxypeptidase A and thermolysin based on the results of X-ray crystallographic experiments (Argos et al., 1978; Kester and Matthews, 1977; Monzingo and Matthews, 1984; Matthews, 1988; Christianson and Lipscomb, 1988b). Models of peptide-metal interaction have recently been utilized in studies of metal ion participation in hydrolysis (see e.g., Schepartz and Breslow, 1987). In these examples a dipole-ion interaction is achieved by virtue of a chelate interaction involving the labile carbonyl and some other Lewis base (e.g.,

323

STRUCTURAL BIOLOGY OF ZINC

Arg*-127

His-69

’I

in‘* ‘Glu-72 His1196

His+-231 TLN:

His-146

*Electrophile

t

Zn2+ ! ‘GluHis’ HIS FIG. 30. Important active-site residues of carboxypeptidase A (CPA) and thermolysin (TLN) and a general scheme for the active sites of related zinc proteases.

a primary amino group) on the model compound. However, the biological relevance of such a chelate interaction in peptide hydrolysis is questionable. X-Ray crystallographic studies of carboxypeptidaseA suggested that a non-zinc, or outer-sphere zinc, carbonyl-bindingsite in the Michaelis complex between the protease and the substrate C=O is conceivable

324

DAVID W. CHRISTIANSON

(Christianson et al., 1985). A wealth of chemical and kinetic information on carboxypeptidase A suggest that this is an attractive possibility (Christianson and Lipscomb, 1989): (1) K , values for peptides with various metallosubstituted enzymes are not significantly variable (Auld and Holmquist, 1974); (2) peptides have been shown to bind to the apoenzyme through kinetic methods (Auld and Holmquist, 1974); (3) X-ray crystallographic studies with the apoenzyme show that peptides bind to Arg-127 (Rees and Lipscomb, 1983); (4) peptides bind to the Co3+substituted enzyme, and, since cobalt in this oxidation state is substitution inert, a non-metal-binding site for the peptide carbonyl is implicated (Van Wart and Vallee, 1978); and (5) one arginine residue is required for proteolysis (Riordan, 1973), and site-directed mutagenesis work suggests that this residue is Arg-127 (Phillips et al., 1990). Hence, a wealth of experimental data suggests that a precatalytic zinc-carbonyl interaction is not a mechanistic requirement for the zinc protease. Coleman (1986) commented on the possibility of a non-zinc-binding site for the substrate carbonyl prior to nucleophilic attack by zinc-promoted water (incipient hydroxide).

a

I CHZ

I HC-CO0

I

ti

,, 2

H,N-Asn-144

--- -- Are.145

\ \

C Glu-270’

‘0-

Ser-197

‘co

I

Ty-198 /NH

FIG. 3 1. Mechanistic proposal for peptide hydrolysis catalyzed by carboxypeptidase A (Christianson and Lipscomb, 1989). (a) The precatalytic Michaelis complex with substrate carbonyl hydrogen bonded to Arg-127 allows for nucleophilic attack by a water molecule promoted by zinc and assisted by Glu-270 (an outer-sphere C=O+Zn2+ interaction is not precluded). (b) The stabilized tetrahedral intermediate collapses, with required proton donation by Glu-270 (Monzingo and Matthews, 1984); Glu-270 may play a bifunctional catalytic role (Schepartz and Breslow, 1987), which results in the product complex (c). [Reprinted with permission from Christianson, D. W., & Lipscomb, W. N. (1989)Acc.Chem. Res. 22,62-69. Copyright 0 1989 American Chemical Society.]

325

STRUCTURAL BIOLOGY OF ZINC

b

IQ7 I

CH 2

Hy-CO;

HzN-As~144

'

.= = = = Arg2145 I

C

I

H,N-Asn-144

CHZ

I HC-COI

0

II

H2N

C Glu-270'

/

.-.-- -- --

/

-

Ars'145

'"0 -Tyr-248

'OH

Ser-197

\ co

I

Tyr-198 /NH

FIG.31. (continued)

Subsequent to substrate binding, a promoted-water mechanism is favored for the hydrolysis of the scissile peptide linkage based on the results of chemical, kinetic, and structural investigations of carboxypeptidase A. A general mechanism is shown in Fig. 3 1, in which the zinc-bound water of the native enzyme is a nucleophile promoted both by zinc and by the general base Glu-270 (Christianson and Lipscomb, 1989). Breslow and Wernick (1976, 1977) demonstrated that the results of a landmark ''0 labeling experiment are consistent with a promoted-water

326

DAVID W. CHRISTIANSON

pathway in the hydrolysis (and synthesis) of typical peptide substrates. Additionally, low-temperature studies of the enzyme are consistent with a promoted-water hydrolytic pathway for peptide and ester hydrolysis. Auld et al. (1984) achieved short-term isolation of metastable enzymebound intermediates that do not react with trapping reagents. In radiationless energy transfer experiments on Zn'+-carboxypeptidase A and Co2+-carboxypeptidase A with N-dansylated substrates, Auld et al. found that peptides and esters are hydrolyzed via the pathway E + S ES1 ESz @ E + P. Results from subsequent cryokinetic studies suggest that peptides and esters coordinate to the metal ion prior to the ratedetermining step, and that structurally distinct intermediates are observable (Geoghegan et al., 1983, 1986; Galdes et al., 1986). Hydrolysis of the ester occurs prior to the rate-determining step so that the esterolytic intermediate ESz is actually a product complex EPlPz (Galdes et al., 1986). In contrast, peptide hydrolysis occurs during (or after) the ratedetermining step. Instead of remarkably differing binding modes for peptides and esters (Vallee et al., 1983; Vallee and Galdes, 1984), differences between peptide and ester binding and catalysis may be interpreted by a common promoted-water mechanism with differing participation of zinc and Arg-127 (Christianson and Lipscomb, 1989). Results from X-ray crystallographic study of carboxypeptidase Asubstrate and carboxypeptidase A-inhibitor complexes are particularly suggestive of a promoted-water pathway for peptide hydrolysis, and these structural studies have been reviewed recently (Christianson and Lipscomb, 1989). For example, certain substrate analogs display apparent chemical reactivity when bound to the active site of the carboxypeptidase A. The ketonic analog of the substrate N-tert-boc-Lphenylalanyl-L-phenylalanine,N- (tert-butoxycarbonyl)-5-amino-2-benzyl-4-oxo-6-phenylhexanoic acid (BBP) (Fig. 32) exhibits a Ki of 6.7 X M (diastereomeric mixture) against carboxypeptidase A (Shoham et al., 1988). Although this inhibitor is hydrated at the carbonyl carbon to no greater than 0.2% in aqueous solution [using the hydration of acetone as a benchmark value (Lewis and Wolfenden, 1977)],it is significant that the inhibitor binds to carboxypeptidase A as 100% hydrate (Shoham et al., 1988). Given that (1) an enzyme binds the transition state more tightly than substrate o r product (Pauling, 1948), (2) enzymes bind stable analogs of the transition state more tightly than corresponding substrates (Wolfenden, 1972; Lienhard, 1972), and (3) carboxypeptidase A selects the hydrated gem-diol(ate) form to bind, instead of a form resembling unreacted substrate, or a form in which an enzyme-bound nucleophile adds to the ketone carbonyl of BBP; it is a reasonable inference that the enzyme favors a promoted-water pathway in hydrolysis, and that the

327

STRUCTURAL BIOLOGY OF ZINC

PH3

CH3-C-O-C-NH-C-C-NH-C-CO;

I

CH3

II

0

ph7H21 II

CH2Ph

I

H

0

a

CH3-C-O-C-NH-C-C-CH,-C-CO~

I CH3

II

0

Ph7Hz II

CH,Ph

I

H

0

b

FIG.32. (a) Carboxypeptidase A substrate N-t-boc-phenylalanylphenylalanine (an arrow denotes the scissle peptide carbonyl) and (b) its ketomethylene analog, N (tert-butoxycarbonyl)-5-amino-2-benzyl-4-oxo-6-phenylhexanoic acid (BBP) (Shoham et al., 1988).

bound gem-diol(ate) of the ketone mimics the gem-diolate of the proteolytic tetrahedral intermediate (Fig. 33). The collapse of the proteolytic tetrahedral intermediate of the promoted-water pathway requires a proton donor in order to facilitate the departure of the leaving amino group. Rees and Lipscomb (1982) considered Glu-270, but favored Tyr-248 for this role, but Monzingo and Matthews (1984) fully elaborated on a role for Glu-270 of carboxypeptidase A and Glu-143 of thermolysin as intermediate proton donors. This proposal for carboxypeptidase A is corroborated by the near-normal activity observed for the Tyr-248+Phe mutant of rat carboxypeptidase A (Gardell et al., 1985; Hilvert et al., 1986) and is reflected in the mechanistic scheme of Fig. 31 (Christianson and Lipscomb, 1989). Mock (1975) considered Glu-270 a proton donor in the carboxypeptidase A mechanism, but his mechanism does not favor a Glu-270/zinc-promoted water molecule as the hydrolytic nucleophile. Schepartz and Breslow (1987) observed that Glu-270 may mediate an additional proton transfer in the generation of the P1 product carboxylate. The binding mode of coproducts PI carboxylate and Pi amine in the carboxypeptidase A active site is suggested by the X-ray crystal structure of the enzyme complex with N-benzoyl-L-phenylalanineand phenylala-

328

DAVID W. CHRISTIANSON

FIG. 33. The promoted-water tetrahedral intermediate (a) in the carboxypeptidase A mechanism is simulated by the binding of the ketomethylene substrate analog BBP (Fig. 32b) as the gem-diol(ate) (b). [Reprinted with permission from Christianson, D. W., & Lipscomb, W. N. (1989) Acc. Chem. Res. 22,62-69. Copyright 0 1989 American Chemical Society.]

nine (Christianson and Lipscomb, 1987). In this complex Arg-127 engages the PI carboxylate in an anti-oriented hydrogen bond as this carboxylate coordinates to zinc in syn-bidentate fashion. Hence, Arg- 127 may participate throughout the catalytic cycle, and this role is depicted in Fig. 3 1 (Christianson and Lipscomb, 1989). Recent site-directed mutagenesis work from Rutter’s laboratory confirms the catalytic requirement for Arg-127 (Phillips et al., 1990).The mechanistic proposal for thermolysin generally parallels that for carboxypeptidase A (Matthews, 1988), with analogous roles for Glu-270 (carboxypeptidase A) and Glu-143 (thermolysin), and Arg- 127 (carboxypeptidase A) and His-23 1 (thermolysin). Parenthetically, it should be noted that there is evidence for the accumulation of an acylenzyme (i.e., a mixed anhydride,with Glu-270) in the carboxypeptidase A-catalyzed hydrolysis of esters at low temperature, but this evidence does not include confirmation by chemical trapping experiments (Makinen et al., 1979; Kuo and Makinen, 1982; Suh et al., 1985). This would imply a nucleophilic, rather than promoted-water, pathway for ester hydrolysis. Sander and Witzel(l985) provided the only

STRUCTURAL BIOLOGY OF ZINC

329

direct chemical evidence for an acyl enzyme intermediate in the hydrolysis of both peptides and esters, but these investigators did not achieve quantitative recovery of the trapping adduct. Finally, the accumulation of an intermediate at low temperatures was not indicated in the results of a resonance Raman spectroscopic study (Hoffman et al., 1983), and work by Auld et al. (1984) revealed the isolation of enzyme-bound intermediates which do not react with trapping reagents. The coordination polyhedron of zinc plays an important role in the mechanism and inhibition of carboxypeptidase A. Although tetrahedrally liganded in the native enzyme, zinc probably binds a pentacoordinate transition state and intermediate in proteolysis as discussed (see, e.g., Fig. 33). Additionally, the binding of substrate or inhibitor molecules to the enzyme is known to alter the zinc coordination geometry of Glu-72. The y-carboxylate of Glu-72 coordinates to zinc with bidentate geometry in native carboxypeptidase A (Rees et al., 1983).In contrast, the y-carboxylate of Glu- 166 coordinates to the zinc ion of thermolysin with unidentate geometry (Holmes and Matthews, 1982). On the binding of inhibitors to thermolysin which contribute one or two ligand atoms to the zinc coordination polyhedron, the coordination geometry of Glu-166 with zinc does not change significantly (for a review see Matthews, 1988). However, on the binding of inhibitors or substrates to carboxypeptidase A which complex the metal ion, the interaction of Glu-72 with zinc tends from bidentate to unidentate coordination (see, e.g., Christianson and Lipscomb, 1985);this is achieved both by the movement of the zinc ion [in the Arg-127 direction (Rees and Lipscomb, 1982)] and, to a lesser degree, by the movement of Glu-72. Flexibility of carboxylate-zinc coordination may be important for enzyme function, and a comparison of Glu-72+Zn2+ interactions for native and inhibitor-complexed carboxypeptidase A (inhibitors have been reviewed by Christianson and Lipscomb, 1989) is found in Fig. 34 (D. W. Christianson and W. N. Lipscomb, unpublished observations). The coordination polyhedron of zinc in carboxypeptidase A does not exhibit any peculiar pH dependence, as demonstrated in high-resolution X-ray crystallographic studies of the enzyme at pH values within the range 7.5-9.5 (Shoham et al., 1984). In particular, Glu-72 remains coordinated in bidentate fashion to the zinc, and no significant differences of histidine-zinc geometries are observed. However, the zinc-solvent distance decreases with increasing pH, possibly indicating an increased population of the zinc-hydroxide form at higher pH values (Shoham et al., 1984). The stereochemistry of anion-zinc interactions is important as inhibitors interact with the active site of carboxypeptidase A. For instance,

330

DAVID W.CHRISTIANSON

OE2

FIG. 34. Glu-72+Zn2+ interactions in native carboxypeptidase A and in carboxypeptidase A-inhibitor complexes (inhibitors have been reviewed by Christianson and Lipscomb, 1989).When substrates or inhibitors bind to the enzyme active site and interact with the zinc ion, the interaction of the metal with Glu-72 tends from bidentate toward unidentate coordination. The flexibility of protein-zinc coordination may be an important aspect of catalysis in this system, and the Glu-72+Zn2+ coordination stereochemistry observed here is consistent with the stereochemical analysis of carboxylate-zinc interactions from the Cambridge Structural Database (Carrel1 et al., 1988; see Fig. 4).

tetrahedral phosphonamidates and phosphonates have been utilized as analogues of the proteolytic o r esterolytic transition statehntermediate in the zinc protease mechanism (Jacobsen and Bartlett, 1981; Bartlett and Marlowe, 1983, 1987a,b; Hanson et al., 1989). From the correlation between inhibitor Ki values and the related substrate K , k,,, values, the transition-state analogy has been elegantly demonstrated (Bartlett and Marlowe, 1983; Hanson et al., 1989). The statistical analysis of structures in the Cambridge Structural Database suggests (Alexander et al., 1990; see Section II,B), and the molecular orbital studies by Kutzelnigg (1977; Wallmeier and Kutzelnigg, 1979) implied that symmetrically bidentate zinc coordination is unfavorable for the tetrahedral phosphonamidate and phosphonate groups of the zinc protease inhibitors (Jacobsen and Bartlett, 1981; Bartlett and Marlowe, 1983, 1987a,b; Hanson etal., 1989). This is in accord with the structures of binary enzyme-inhibitor complexes involving thermolysin (Tronrud et al., 1987; Holden et al., 1987) and carboxypeptidase A (Christianson and Lipscomb, 1988a; Kim and Lipscomb, 1990,199 l),in which phosphinyl-containing inhibitors generally tend toward asymmetric unidentate zinc coordination (Fig. 35). However, the bidentate interaction of the tetrahedral intermediate (and its flanking transition states) with zinc is proposed in the proteolytic mechanisms of the zinc proteases (Matthews, 1988; Christianson and Lipscomb, 1989). The electronic peculiarities of P=O may pose an upper limit to the

STRUCTURAL BIOLOGY OF ZINC

33 1

ARG

FIG. 35. The phosphonate analog of the carboxypeptidase A substrate Cbz-Ala-AlaPhe inhibits the enzyme with a Ki of 3 pM (Hanson et al., 1989) and binds with unidentate phosphinyl-zinc coordination, as revealed in an X-ray crystallographic study (Kim and Lipscomb, 1990). Stippled density shows the refined positions of zinc and phosphorus. [Reprinted with permission from Kim, H., & Lipscomb, W. N. (1990) Biochemistry 29, 5546-5555. Copyright 0 1990 American Chemical Society.]

transition-state analogy for phosphinyl-containing enzyme inhibitors. Alternatively, given the demonstration of the transition-state analogy for these inhibitors (Bartlett and Marlowe, 1983; Hanson et al., 1989), it is possible that the hydrolytic transition state does not tend toward symmetrically bidentate zinc coordination in the promoted-water mechanism. Questions regarding zinc coordination in the mechanism(s) and inhibition of peptide bond hydrolysis are even more perplexing in the case of leucine aminopeptidase. This exopeptidase possesses two divalent metalbinding sites per monomer which are involved in catalysis or the regulation of catalysis; the zinc-magnesium form is more active than the zinczinc form (Thompson and Carpenter, 1976). The three-dimensional structure of bovine Zng+-leucine aminopeptidase has been reported at 2.7 8, resolution and reveals a novel bimetallic cluster in which the two zinc ions are separated by 2.88 8, (Burley et al., 1990) (Fig. 36). This arrangement of metal ions has not been observed in any previously determined zinc protease structure, and the implications for substrate recognition and the hydrolytic mechanism are intriguing. Since aldehydes are potent inhibitors (Anderson et al., 1985) and phosphonates are very modest inhibitors (Giannousis and Bartlett, 1987) of leucine aminopeptidase, it is possible that the enzyme uses a nucleophilic mechanism, rather than a promoted-water mechanism, in the hydrolysis of peptide substrates (Giannousis and Bartlett, 1987).

332

DAVID W. CHRISTIANSON

FIG. 36. The three-dimensional structure of the binuclear zinc cluster of leucine aminopeptidase, as determined by X-ray crystallographicmethods (Burley et al., 1990;S. K. Burley and W. N. Lipscomb, personal communication).

Having discussed the carbonic anhydrases and the zinc proteases, it is useful at this point to indicate important relationships and contrasts between the two families of zinc enzymes, particularly with regard to the coordination polyhedron of zinc and the function of the metal ion in catalysis. As the general active sites of these two families are compared, it is evident that each protein structure has evolved in response to chemical requirements for catalysis. Since the biological substrate of the protease is a peptide, the participation of a proton donor (general acid) is mandatory for the departure of the leaving amino group from the tetrahedral intermediate in a promoted-water mechanism (Matthews, 1988; Christianson and Lipscomb, 1989). This is not a requirement of catalysis by carbonic anhydrase, since the biological substrate is the COZ molecule. Indeed, there is no leaving group from the “intermediate” in the carbonic anhydrase mechanism, since the intermediate bicarbonate anion is, at most, a tautomer of the product bicarbonate anion (Fig. 23 and 28). Additionally, there is no requirement for an additional electrophile in the carbonic anhydrase active site as there is in the zinc protease active site (e.g., Arg- 127 in carboxypeptidase A, His-23 1 in thermolysin). Therefore, the carbonic anhydrases and the zinc proteases share a primitive catalytic function: the attack of (incipient) hydroxide at substrate C=O. It is the chemical fate of the subsequently formed species that differentiates the function of each class of enzymes-in the carbonic anhydrases

STRUCTURAL BIOLOGY OF ZINC

333

the nucleophilic adduct is a product; in the zinc proteases the nucleophilic adduct is an intermediate (Scheme 4). Another contrast between the zinc proteases and the carbonic anhydrases concerns the zinc coordination polyhedron. The carbonic anhydrases ligate zinc via three histidine residues, whereas the zinc proteases ligate the metal ion through two histidine residues and a glutamate (bidentate in carboxypeptidase A, unidentate in thermolysin). Hence, the fourth ligand on each catalytic zinc ion, a solvent molecule, experiences enhanced electrostatic polarization in carbonic anhydrase I1 relative to carboxypeptidase A. Indeed, the zinc-bound solvent of carbonic anhydrase I1 is actually the hydroxide anion [via a proton transfer step mediated by His-64 (for a review see Silverman and Lindskog, 1988)l. A final chemical contrast between the zinc proteases and the carbonic anhydrases is found in the environment and the mechanistic role of active-site glutamate residues. Glu- 106 of carbonic anhydrase I1 is hydrogen bonded to a zinc-bound solvent molecule through the bridging hydroxyl of Thr-199, whereas Glu-270 of carboxypeptidase A and Glu-143 or thermolysin are hydrogen bonded directly to zinc-bound solvent (see Fig. 20). In the zinc proteases this structure is consistent with the participation of the active-site glutamate as a general baselacid (Matthews, 1988; Christianson and Lipscomb, 1989). However, in the carbonic anhydrases the role of Glu-106 is subject to at least some ambiguity (Kannan et al., 1977; Silverman and Vincent, 1983). C . Metallothionein

Metallothionein is remarkable among the zinc-binding proteins in that it contains 20 cysteines, in a polypeptide chain of 61 or 62 amino acids, that are highly conserved among the greater family of metallothioneins (Lerch et al., 1982; Winge et al., 1984; Kagi and Kojima, 1987; Kagi and Schaffer, 1988). T h e metallothioneins are so named because of their

SCHEME 4. Zinc proteases: nucleophilic adduct is a reaction intermediate; carbonic anhydrases: nucleophilic adduct is the product.

334

DAVID W. CHRISTIANSON

impressively high metal and sulfur content (Margoshes and Vallee, 1957; Kagi and Vallee, 1960). In spite of their high cysteine content, metallothioneins lack disulfide linkages and instead bind seven metal ions (zinc and/or cadmium) via the cysteines in two distinct metal clusters of three and four metals each (Otvos and Armitage, 1980; Frey et al., 1985). Although the specific biological function of the metallothioneins remains a topic of discussion, these metal-rich proteins may serve a role in the activation of zinc apoproteins in vivo, and/or they may serve a role as biological scavengers of free radicals, given that their biosynthesis is induced by ultraviolet radiation (Karin, 1985). T h e three-dimensional structure of metallothionein-2 from rat liver has been determined by X-ray crystallographic methods at moderate resolution (Furey et al., 1986) and revised at high resolution (Robbins et al., 1990). This structure reveals two domains, with each containing a novel multinuclear metal-thiolate cluster (Robbins et al., 1990) (see Fig. 37). Additionally, the three-dimensional structures of Cd7metallothionein-2 from rabbit liver (Arseniev et al., 1988) and rat liver (Schultze et al., 1988) have been solved by nuclear magnetic resonance (NMR) methods. Early NMR work on rabbit metallothionein-2 directly established the topologies and li and compositions of a three-Cd*+ cluster and a four-Cd2+cluster by '"Cd-'H heteronuclear correlation spectroscopy (Frey et al., 1985). However, the three-dimensional arrangement of the polypeptide chain and the cysteine-metal connectivity, as observed in the moderate-resolution X-ray structure (Furey et al., 1986), differed from the required by (Frey et al., 1985) and observed in (Arseniev et al., 1988; Schultze et al., 1988) solution NMR studies. Subsequent high-resolution X-ray work on rat liver metallothionein-2 has resulted in a refined structure that is in accord with the NMR structure (Robbins et al., 1990).Thus, metallothionein-2 represents the first multinuclear zinc-thiolate clusters protein which has been successfully studied by biophysical structural methods. Prior biophysical studies of zincthiolate coordination have centered on mononuclear sites, for example, as found in aspartate carbomoyltransferase (Honzatko et al., 1982) (Fig. 38). The three-dimensional structure of rat metallothionein-2 reveals that the bound metal ions of both clusters are tetrahedrally coordinated by combinations of terminal and bridging cysteine sulfur ligands (Robbins et al., 1990). The first cluster consists of three metal ions liganded by six terminal and three bridging cysteines, in which the three metals and three bridging sulfurs alternate to form a six-membered ring with a left-handed twist boat conformation (Fig. 39). The second cluster consists of four metal ions liganded by six terminal and five bridging cysteines, in

STRUCTURAL BIOLOGY OF ZINC

335

a

b

FIG. 37. (a and b) Two perpendicular views of rat liver metallothionein 11, where the metal clusters are highlighted (C. D. Stout, personal communication).

336

DAVID W. CHRISTIANSON

FIG. 38. The zinc-thiolate cluster of the regulatory subunit of aspartate carbamoyltransferase (Honzatko et al., 1982). Coordinates are from the Brookhaven Protein Data Bank.

which the four metals and five bridging sulfurs alternate to form two fused six-membered rings: one with a left-handed twist boat conformation and the other with an envelope conformation (Fig. 40). It is interesting that a unique secondary structural element, designated the "half-turn," was indentified in preliminary NMR studies of rabbit metallothionein-:! (Wagner et al., 1986). T h e half-turn element is defined as a type I1 turn with 4 3 rotated from 90" to -90"; its occurrence in the metallothionein-2 structure arises from the constraints placed on the relatively short polypeptide chain by the metal clusters. Although these constraints are not well understood and are certainly difficult to predict, the continued biophysical study of metallothionein-:! will certainly improve our understanding of protein-metal cluster interactions.

Bet. Domaln Cluster

Beta Domain Cluster

S2

s2 13

13

FIG.39. The three-metal-thiolate cluster in the P-domain of rat liver metallothionein I1 (C. D. Stout, personal communication).

STRUCTURAL BIOLOGY OF ZINC

Alpha Domain Cluster

337

Alpha Domain Cluater

“P s3

s3

SJB

S58

FIG.40. The four-metal-thiolate cluster in the (I domain of rat liver metallothionein I1 (C. D. Stout, personal communication).

D . Transcription Factors IIIA and GAL4

The requirement for zinc in the regulation of gene expression is exemplified by transcription factor IIIA, which has been shown to contain from two (Hanas et al., 1983) to seven to eleven (Miller et al., 1985) zinc ions bound to a 40K protein molecule. Transcription factor IIIA is obtained from immature Xenopzu oocytes in a complex with 5 S RNA, and the protein is required for transcription initiation; the apoprotein does not bind to the 5 S RNA gene (Hanas et al., 1983). Uncertainties regarding the stoichiometric requirement for zinc persist, given the report by Shang et al. (1989) that transcription factor IIIA, as either the isolated protein or the 5 S RNA complex, contains two firmly bound zinc ions which are required for transcription activation. The sequence analysis of transcription factor IIIA reveals nine (Miller et al., 1985) to 12 (Brown et al., 1985; Brown and Argos, 1986) tandem repeats, each with the general sequence Cys-Xaa2-5-Cys-Xaal2-HisXaaz-s-His (Xaa, any amino acid), which are proposed to bind zinc through the two cysteine and two histidine side chains. These potential metal-binding domains are referred to as zinc fingers (Miller et al., 1985), and the extended X-ray absorption fine-structure analysis of transcription factor IIIA confirms the composition of the zinc coordination polyhedron (Diakun et al., 1986). The systematic search of the National Biomedical Research Foundation protein library for amino acid sequences of the general form Cys-Xaa2-4-Cys-Xaa2- 15-Aaa-Xaa2-4-

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DAVID W. CHRISTIANSON

FIG. 41. Berg’s proposed structure of a zinc finger peptide: (left) hydrogen bond pattern; (center)ribbon diagram illustratingsecondary-structurecomponents; (right) molecular model illustratingconserved metal-bindingand hydrophobic residues. [Reprinted with permission from Berg, J. M. (1988) Proc. Nutl. Acud. Sci. U.S.A. 85,99-102.1

Aaa or Aaa-Xaa2-4-Aaa-Xaa2- 15-Cys-Xaa2-4-Cys (Xaa, any amino acid, Aaa, Cys o r His) reveals potential zinc finger domains in several classes of proteins involved in nucleic acid recognition and binding (Berg, 1986a). Further analysis of sequence retrievals yields the consensus form (Phe, Tyr)-Xaa-Cy~-Xaa2,~-Cys-Xaa3-Phe-Xaa5-Leu-Xaa2-HisXaa3-His-Xaas (Berg, 1986b; Vincent, 1986), and Berg (1988) proposed a structure for this zinc-binding domain by analogy with known metalloprotein structures (Fig. 4 1). Independently, Gibson et al. (1988) proposed a zinc finger structure based on the results of modeling and molecular dynamics refinement, which is somewhat similar to the model by Berg (1988). Berg’s (1988) structure prediction is essentially confirmed by the NMR structure determination of a synthetic 25-residue peptide corresponding to a single zinc finger from the Xenopus protein Xfin (Lee et al., 1989). However, Berg’s (1988) model does not account for the presence of a segment of 310 helix at the carboxy terminus of the peptide which was revealed in the NMR structure determination (Lee et al., 1989). T h e structure of the Xfin zinc finger is shown in Fig. 42, and it may be compared with Berg’s proposed structure shown in Fig. 41. The “lowresolution” NMR structure of a zinc finger domain from yeast ADRl (Parraga et al., 1988, 1990) differs more substantially from Berg’s (1988) prediction in that no evidence is found for the antiparallel p turn in the carboxy-terminal region of Berg’s model. The role of zinc in transcription factor IIIA is presumably structural, not catalytic, and Zn2+ is required for DNA-binding activity (Hanas et al.,

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FIG. 42. The structure of the zinc finger domain of the Xenopus Xfin protein, as determined by solution NMR methods. [Courtesy of Michael Pique and Peter E. Wright, Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California. From Lee et al., Science 245, 635-637, 1 1 August 1989. Copyright 0 1989 by the American Association for the Advancement of Science.]

1983). The metal ion probably stabilizes the nucleic acid-binding domain of the protein in order to maintain the proper topology for nucleic acid association. Although the direct interaction of the nucleic acid with the metal ion of a zinc finger is not expected, the hydrogen bond interaction of a histidine zinc ligand with the phosphate backbone or a base remains a possibility (Christianson and Alexander, 1989; Pavletich and Pabo, 1991). Hence, in addition to its structural role, the zinc ion may play a chemical role in protein-DNA recognition.

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It is noteworthy that the high-resolution NMR solution structure of a zinc finger domain from human enhancer-binding protein (Omichinski et al., 1990) reveals a significant structural similarity [backbone atomic root mean square (rms) difference of 0.95 A] to a region of protease inhibitor domain I11 of Japanese quail ovomucoid (Papamokos et al., 1982). T h e core of the &3a folding motif in both examples is formed by the close packing of hydrophobic side chains; the polypeptide chain appears to arrange itself about this core (Omichinski et al., 1990). This suggests that the @3a folding of the zinc finger domain from transcription factor IIIA and related proteins is not unique to DNA-binding proteins, but instead is a more general backbone folding motif adapted by some metal-binding domains (Omichinski et al., 1990). There exist zinc-binding domains of nucleic acid-binding proteins which are characterized not by Cys2His2ligand composition, but instead by CyssHis composition (Berg, 1986a). T h e NMR structure determination of one of these domains, an 18-mer derived from the gag protein p55 of human immunodeficiency virus (Summers et al., 1990), reveals a CyssHis zinc finger which exhibits a novel backbone fold which differs from that of a His2Cys2 zinc finger (Fig. 43). On inspection of the structure, Summers et al. (1990) proposed that the functionally important nucleic acid-binding region of the retroviral CyssHis finger motif is a four-residue tight turn which is more reminiscent of a knuckle than a finger. Unlike transcription factor IIIA or the gag protein p55 of human immunodeficiency virus, the zinc ligand composition of transcription activator GAL4 is Cys6Znft+,and the structure of the binuclear GAL4 zinc-binding domain provides an interesting contrast to the finger and knuckle domains discussed above. The GAL4 protein is required for galactose metabolism in Saccharomyces cerevisiae (St. John and Davis, 1981), and its biological function requires binding to a 17-base pair palindromic site in the galactose upstream activating sequence UASc (Giniger et al., 1985). Deletion mutagenesis studies show that the structural requirements for GAL4-UASG recognition are contained within the amino-terminal 74 amino acids of the 881-residue metalloprotein (Keegan et al., 198S), and this amino headpiece contains six cysteine residues in the arrangement Cys- 1l-Xaap-Cys- 14-XaaG-Cys-2 1Xaa6-Cys-28-Xaa2-Cys-3 1-Xaae-Cys-38. This cysteine-rich sequence is suggestive of a metal binding site(s). Although the sequence of cysteine residues does not correspond to the general sequences discussed above for transcription factor IIIA-like zinc finger domains (Berg, 1986a,b), it has been proposed that the amino headpiece of GAL4 binds a single Zn2+ ion in a tetrahedral arrangement similar to that proposed for transcrip-

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FIG.43. The structure of the CyssHis zinc-binding domain derived from the gag protein p55 from human immunodeficiency virus, as determined by solution NMR methods. [Reprinted with permission from Summers, M. F., South, T. L., Kim, B., & Hare, D. R. (1990) Biochemistry 29,329-340. Copyright 0 1990 American Chemical Society.]

tion factor lIIA (Johnston, 1987), although the recent work by Pan and Coleman (1989, 1990a,b), argued against this possibility. T h e conservation of the six-cysteine motif among several different proteins (summarized by Berg, 1989a) implies that all six cysteine residues are functionally important and potentially involved in metal binding. Pan and Coleman (1989) demonstrated the existence of two spectroscopically different metal-binding sites in a 149-amino truncation of the GAL4 protein by titration of the apoprotein with '13Cd2+ and subsequent analysis by '13Cd NMR. This amino-terminal truncation of the 881-residue GAL4 protein, consisting of GAL4 residues 1-147 plus two additional residues from the cloning vector, contains the 74-residue cysteine-rich DNA-binding domain discussed above. Pan and Coleman (1989) showed that the readdition of Zn2+or Cd2+to the apoprotein in a 1 : 1 molar ratio restores DNA-binding activity, and it is possible that activity is slightly improved on incubation with additional metal. In subsequent '13Cd and 'H NMR studies of a 63-residue GAL4 amino headpiece (which exhibits DNA-binding activity), Pan and Coleman

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(1990a,b) found that the zinc- and cadmium-substituted proteins are monomers in solution and have similar conformations. Moreover, these investigators observed two protein-bound metal ions coordinated by six cysteine thiolates. Although the two metal-binding sites each display similar affinity toward Cd2+, a difference in Zn2+ affinity is observed. This phenomenon may account for the nonintegral stoichiometry of zinc-protein analyses of GAL4 and its amino-terminal truncations (even though there is a 2 : 1 molar ratio of metal to protein in the presence of micromolar Zn2+ concentrations). Pan and Coleman ( 1990b) speculated that this property may allow for the regulation of transcription by metal concentration in certain environments, and these investigators proposed a model for the GAL4 63-mer in which four terminal and two bridging cysteines tetrahedrally ligate two metal ions in a novel binuclear cluster (Pan and Coleman, 1990a,b, 1991) (Fig. 44). Other multinuclear protein-zinc interactions may be implicated for the GAL4 protein in viva For example, since the GAL4 dimer binds to palindromic DNA sequences (Giniger et al., 1985), one possibility is that the dimer interface of the intact protein could comprise three zinc ions tetrahedrally coordinated by the 12 cysteines of two GAL4 monomers

Cyrl4 CLUSTER SEOUENCE

SPECIFICITY DOMAIN SEOUENCE GAL4 P K T K R S P L T R A H L T E V E S R LAC8 P O V V R T P L T R A H L T E M E O R PPRl D P A T G K 0 V P R C V V F F L E 0 R 42

FIG.44. Structure of the binuclear Zn2 or Cd:, complex in the DNA binding domain of the yeast transcription factor GAL4. The amino acid sequence surrounding the cluster and containing the six C ligands, as well as a short sequence immediately C-terminal required for specific DNA recognition is shown below. Sequences are given for three fungal transcription factors, GAL4, LACS, and PPRI. GAL4 and LAC9 recognize the same DNA sequence. [Figure from Gardner, Pan, and Coleman, personal communication, based on data in Pan and Coleman (1990a,b; 1991).]

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FIG. 45. The structure of' the glucocorticoid receptor DNA-binding domain, as determined by NMR methods (Hard et al., 1990a,b; R. Kaptein, personal communication).

(Berg, 1989a). However, the results of Pan and Coleman (1989, 1990a,b) argue against this alternative. Cysteine-rich sequences have been observed in other classes of proteins which may bind metal ions in multinuclear complexes. The steroid receptors contain a 61-residue cysteine-rich region in which nine cysteine residues are conserved among the family of receptor proteins (Hollenberg et al., 1985, 1987), and possible structures of multinuclear proteinmetal complexes have been considered (Berg, 1989a,b). At least eight of these cysteine residues are apparently required for receptor function (Severne et al., 1988), and it is possible that all nine are required for transcriptional activation (Schena et al., 1989).T h e NMR structure determination of the glucocorticoid receptor DNA-binding domain shows two metal ions bound in two separate domains, each tetrahedrally coordinated by cysteine residues (Hard et al., 1990a,b) (Fig. 45). Intriguingly, a third, unexpected, metal binding site has been identified in the glucocorticoid receptor in recent '13Cd NMR experiments; the zinc ligands at this site are proposed to be Cys-450, Cys-500, and Met-505 (Pan et al., 1990). Finally, the recent NMR structure determination of the DNA-binding domain of a related steroid receptor, the estrogen receptor, reveals a structure very similar to that of the glucocorticoid receptor (Schwabe et al., 1990) (Fig. 46). This work, as well as that discussed in the preceding examples, indicates that the study of zinc in transcription and biological recognition is certain to remain an exciting field of inquiry for years to come (see, e.g., Berg, 1989a, 1990a,b).

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V. PROTEIN ENGINEERING OF ZINC-BINDING SITES

Given the ubiquitous occurrence of zinc in biological function, it follows that the engineering of metal-binding sites is an important goal for the design of proteins “tailor-made” for specific structural, catalytic, or regulatory tasks. Factors to be considered in the design of zinc-binding sites are the number and composition of direct metal ligands, the stereochemistry of ligand-metal coordination, the geometry of ligand field (preferably tetrahedral), and long-range protein-metal ion interactions which occur in through-space and through-hydrogen bond fashion. The engineering of a de nova metal-binding site in a monomeric ahelical peptide has been reported by Ghadiri and Choi (1990), and the bound metal ion (Zn2+,as well as other transition metals) stabilizes the a helix in aqueous solution. The placement of histidine and cysteine residues at positions n and n+4 of an a helix mimics naturally occurring metal-binding sites. Moreover, the placement of metal-chelating residues near the carboxy terminus of the helix is thought to optimize the longrange interaction between the negative electrostatic potential of the helix carboxy terminus and the positively charged metal ion. Two 17-mers, one containing Cys-12 and His-16 and the other containing His-12 and His-16, are found to be stabilized by metals. Transition metal ions induce up to 90% helicity in these peptides at O”C, as observed in circular dichroism spectra. Independently, Ruan et al. (1990)demonstrated that unnatural metalligating residues may likewise be utilized toward the stabilization of short a helices by transition metal ions (including Zn2+)-these investigators reported that an 11-mer is converted from the random coil conformation to about 80% a helix by the addition of Cd2+at 4°C. These results suggest that the engineering of zinc-binding sites in small peptides or large proteins may be a powerful approach toward the stabilization of protein secondary structure. The engineering of zinc-binding sites in a-helical peptides, where metal binding stabilizes protein tertiary structure, has been reported by Handel and DeGrado (1990). In these experiments zinc-binding sites are incorporated into a dimeric helix-loop-helix peptide (H3a2) and a protein composed of four helices connected by three short loop sequences (H3a4). A model of one subunit of the H3a2 dimer is found in Fig. 47. In addition to metal complexation by two histidine residues at positions n and n+4 of one a helix, the metal is coordinated by a third histidine residue of an adjacent a helix. The composition of the zinc coordination polyhedron is like that of carbonic anhydrase (i.e., HisQ), and spectroscopic results suggest that all three histidine residues are involved in zinc complexation. This work sets an important foundation

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FIG.46. The structure of the estrogen receptor DNA-bindingdomain, as determined by NMR methods (Schwabe et al., 1990;J. W. R. Schwabe, personal communication).

for the understanding and design of de novo zinc-binding sites within the scaffolding of protein tertiary structure, and catalytic versatility is one feature that may be built on this foundation (Handel and DeGrado, 1990). Moreover, a rich variety of biological coordination chemistry remains to be explored in these systems. For example, recent work by Regan and Clarke (1990) demonstrated the incorporation of a tetrahedral Cys2His2 zinc-binding site in a four-helix bundle protein, The zinc-binding site of carbonic anhydrase serves as a structural paradigm for the incorporation of a de novo zinc-binding site in an antibody (Iverson et al., 1990; Roberts et al., 1990; Tainer and Roberts, 1990). Because individual antibody light- and heavy-chain libraries can be com-

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FIG. 47. Model of the a-helical peptide H3a2, showing backbone atoms and three histidine zinc ligands. The bound zinc ion is represented by a sphere. [Reprinted with permission from Handel, T., & DeCrado, W. F. (199O)J. Am. Chem. SOC. 112,6710-671 1. Copyright 0 1990 American Chemical Society.]

bined into random chimeric proteins, it is reasoned that one chain may be engineered by site-directed mutagenesis to contain a cofactor-binding site (i.e., a catalytic site), and the other complementary chain may be raised via the immune response against a specific substrate analogue. The engineering of hydrolytic chemistry is one goal of metalloantibody experiments. Given that the catalytic zinc ions found in the paradigm zinc enzymes carbonic anhydrase and carboxypeptidase A are bound by three protein ligands (His3 and HisnGlu-, respectively) and a nucleophilic solvent molecule, the metalloantibody is designed after a functioning biological precedent. In order to mimic the zinc coordination polyhedron of carbonic anhydrase, three histidine residues are introduced by sitedirected mutagenesis in the metalloantibody which ligate Zn2+. Moreover, the solvent-binding site of the metal is oriented toward a potential substrate-binding site in VH of the antibody Fv, although the catalytic versatility of engineered metalloantibodies remains to be explored. The Hiss-zinc metalloantibody exhibits a K,-J of M as measured by fluorescence quenching experiments; a model is found in Fig. 48. In addition to structure stabilization and catalytic applications, transition metal-binding sites may be designed and exploited for regulatory purposes. For example, a regulatory metal-binding site has been engineered into the active site of trypsin, where metal binding inhibits proteo-

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FIG.48. Transplantation of a cofactor-binding site into the antibody light-chain complementarity-determining region (CDR). The framework FVregions (dark gray) for the antibodies McPC603, D1.3, HyHel5, HEdlO, Newm, Kol, J539,4-4-20, and R19.9 are superimposed to show the Ca backbones of the light-chain variable region [CDRl (medium gray), CDR2 (white), and CDR3 (light gray)] with the zinc site from carbonic anhydrase (three histidines and the zinc ion) superimposed onto residues 34, 89, and 91. In this view the binding site (top) faces forward. While the CDRs show much more structural variation than the framework regions, all of the antibodies share the /3-strand structure that extends from the framework into the CDRs (Tainer and Roberts, 1990; Roberts et al., 1990). [Reprinted from Tainer and Roberts (1990) by permission from Nature, Vol. 348, p. 589. Copyright 0 1990 Macmillan Magazines Limited.]

lytic activity (Higaki et al., 1990). Modeling experiments suggest that for the site-specific mutant A r g - g h H i s , a 90" rotation of His-5'7 about the Ca-CP bond gives rise to a His2-M2+ coordination complex. The proposed structure of this complex is similar to that of part of the zinc coordination polyhedron of thermolysin (Holmes and Matthews, 1982). Importantly, His-57 is part of the catalytic triad of the serine protease, and its metal ion-induced movement would render the catalytic triad, hence the enzyme, inactive. It has been found that the trypsin mutant Arg-96+His is inhibited by Cu2+, Ni'+ , and Zn2+ with inhibition constants 21, 49, and 128 p M , respectively; inhibitory activity is attenuated by the addition of the metal chelator EDTA (Higaki et al., 1990). These

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results are suggestive of regulatory metal binding. Although the reported zinc binding is modest, this seminal experiment represents the first step in the protein engineering of regulatory zinc-binding sites. Given that carbonic anhydrase serves as a paradigm for the de novo construction of zinc-binding sites in peptides (Handel and deGrado, 1990) and metalloantibodies (Iverson et al., 1990; Roberts et al., 1990; Tainer and Roberts, 1990), it is logical that the zinc-binding site of carbonic anhydrase I1 itself is an optimal subject for protein engineering experiments. Th e His-1 19+Cys mutant of carbonic anhydrase I1 has been prepared and expressed in the construction of a novel biological zinc coordination polyhedron with composition His2Cys (C. A. Fierke and L. L. Kiefer, personal communication). Based on modeling experiments, the His- 119+Cys substitution results in nearly perfect cysteinemetal ion coordination stereochemistry (R. S. Alexander and D. W. Christianson, unpublished observations); the Ca-CP-Sy-Zn2+ torsion angle tends toward favorable trans stereochemistry (Chakrabarti, 1989; see Section 11,E).The X-ray crystallographic structure determination of the His-1 19+Cys mutant may verify the prediction of modeling experiments and also provide a structural basis for the exploration of catalytic function. These experiments will sustain the prominence of carbonic anhydrase I1 as a paradigm in the protein engineering of zinc-binding sites for structural, regulatory, and catalytic applications. VI. SUMMARY The biological function of zinc is governed by the composition of its tetrahedral coordination polyhedron in the metalloprotein, and each ligand group that coordinates to the metal ion does so with a well-defined stereochemical preference. Consequently, protein-zinc recognition and discrimination requires proper chemical composition and proper stereochemistry of the metal-ligand environment. However, it should be noted that the entire protein behaves as the “zinc ligand,” since residues that are quite distant from the metal affect recognition and function by throughspace (either solvent or the protein milieu) or through-hydrogen bond coulombic interactions. Additionally, long-range interactions across hydrogen bonds serve to orient ligands and therefore minimize the entropy loss incurred on metal binding. Since zinc is not subject to ligand field stabilization effects, it is easy for the tetrahedral protein-binding site to discriminate zinc from other first-row transition metal ions: It is only for Zn2+ that the change from an octahedral to a tetrahedral ligand field is not energetically disfavored. Structural considerations such as these must illuminate the engineering of de nouo zinc-binding sites in proteins.

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Zinc serves chemical, structural, and regulatory roles in biological systems. In biological chemistry zinc serves as an electrophilic catalyst; that is, it stabilizes negative charges encountered during an enzyme-catalyzed reaction. T h e coordination polyhedron of catalytic zinc is usually dominated by histidine side chains. In biological structure zinc is typically sequestered from solvent, and its coordination polyhedron is almost exclusively dominated by cysteine thiolates. Structural or regulatory zinc is found as either a single metal ion or as part of a cluster of two o r more metals. In multinuclear clusters cysteine thiolates either bridge two metal ions or serve as terminal ligands to a single metal ion. Even in complex multinuclear clusters, Zn2+ displays tetrahedral coordination. The structural biology of zinc continues to receive attention in catalytic and regulatory systems such as leucine aminopeptidase, alkaline phosphatase, transcription factors, and steroid receptors. For example, zincmediated hormone-receptor association has recently been demonstrated in the binding of human growth hormone to the extracellular binding domain of the human prolactin receptor (Cunningham et al., 1990). T o be sure, structural studies of zinc in biology will continue to be a fruitful source of bioinorganic advances, as well as surprises, in the future.

ACKNOWLEDGMENTS I thank the Chicago Community Trust for a Searle Scholar Award and the Office of Naval Research for a Young Investigator Award. The assistance of R. S. Alexander, A. M. Cappalonga, J. A. lppolito, Z. F. Kanyo, and S. K. Nair in the preparation of the figures is gratefully acknowledged. I thank C. A. Fierke, R. Kaptein, J. W. R. Schwabe, C. D. Stout, and J. A. Tainer for transmittal of their results prior to publication. I thank these investigators, as well as S. K. Burley, J. E. Coleman, W. F. DeGrado, W. N. Lipscomb, K. M. Merz, and P. E. Wright, for supplying figures. Additionally, I am grateful to many of my colleagues above, as well as J. M. Berg, J. T. Edsall, D. Eisenberg, A. M. Khoury, and B. Tidor, for critical reading of the manuscript.

REFERENCES Adman, E., Watenpaugh, K. D., and Jensen, L. H. (1975).Proc. Natl. Acad. Sci. U.S.A. 72, 4854-4858. Alexander, R. S., Kanyo, Z. F., Chirlian, L. E., and Christianson, D. W. (199O).J.Am. Chem. SOL. 112,933-937. Alexander, R. S., Nair, S. K., and Christianson, D. W. (1991).Biochemktry (submitted). Allen, F. H., Kennard, O., and Taylor, R. (1983). Acc. Chem. Res. 16, 146-153. Anderegg, G . (1963). Helu. Chim. Acta 46, 1833-1842. Anderson, L., MacNeela, J., and Wolfenden, R. (1985).Biochemistry 24,330-333.

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Argos, P., Garavito, R. M., Eventoff, W., Rossmann, M. G., and Branden, C. I. (1978). J. Mol. Biol. 126, 141-158. Arseniev, A., Schultze, P., Wogotter, E., Braun, W., Wagner, G., VaSak, M., Kagi, J. H. R., and Wuthrich, K. (1988).J. Mol. Biol. 201,637-657. Auld, D. S., and Holmquist, B. (1974). Biochemistry 13,4355-4361. Auld, D. S., Galdes, A., Geoghegan, K. F., Holmquist, B., Martinelli, R. A., and Vallee, B. L. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 5041-5045. Bartlett, P. A., and Marlowe, C. K. (1983). Biochemistry 22,4618-4624. Bartlett, P. A., and Marlowe, C. K. (1987a). Biochemistry 26, 8553-8561. Bartlett, P. A., and Marlowe, C. K. (1987b). Science 235,569-571. Berg, J. M. (1986a). Nature (London) 319,264-265. Berg, J. M. (1986b). Science 232,485-487. Berg, J. M. (1988). Proc. Natl. Acad. Sci. U.S.A. 85,99-102. Berg, J. M. (1989a). Prog. Inorg. Chem. 37, 143-185. Berg, J. M. (1989b). Cell 57, 1065-1068. Berg, J. M. (1990a). Annu. Rev. Biophys. Biophys. Chem. 19,405-421. Berg, J. M. (1990b).J. Biol. Chem. 265,6513-6516. Berg, J. M., and Merkle, D. L. (1989).J. Am. Chem. SOC. 111,3759-3761. Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, E. F., Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977). J . Mol. Biol. 112, 535-542. Bertini, I., Luchinat, C., Monnanni, R., Roelens, S., and Moratal, J. M. (1987).J. Am. Chem. SOL. 109,7855-7856. Blundell, T. L., Pitts, J. E., Tickle, 1. J., Wood, S. P., and Wu, C.-W. (1981). Proc. Null. Acad. Sci. U.S.A. 78,4175-4179. Bradbury, J. H., and Carver, J. A. (1984). Biochemistry 23,4905-4913. Breslow, R., and Wernick, D. L. (1976).J. Am. Chem. SOC.98,259-261. Breslow, R., and Wernick, D. L. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 1303-1307. Brown, R. S., and Argos, P. (1986). Nature (London) 324,215. Brown, R. S., Sander, C., and Argos, P. (1985). FEBS Lett. 186,271-274. Bryan, P. N . (1987). Biotechnol. Adu. 5,221-234. Burley, S. K., and Petsko, G. A. (1988). Adu. Protein Chem. 39, 125-189. Burley, S. K., David, P. R., Taylor, A., and Lipscomb, W. N. (1990). Proc. Natl. Acad. Sci. U.S.A. 87,6878-6882. Carrell, C. J., Carrell, H. L., Erlebacher, J., and Glusker, J. P. (1988).J. Am. Chem. SOC. 110, 8651-8656. Carver, J. A., and Bradbury, J. H. (1984). Biochemistry 23,4890-4905. Chakrabarti, P. (1989). Biochemistry 28,608 1-6085. Chakrabarti, P. (1990a). Protein Eng. 4,49-56. Chakrabarti, P. (1990b). Biochemistry 29,651-658. Chakrabarti, P. (1990~).Protein Eng. 4, 57-63. Chakrabarti, P., Venkatesan, K., and Rao, C. N. R. (1981). Proc. R . SOC.London, Ser. A 375, 127-153. Christianson, D. W., and Alexander, R. S. (1989).J. Am. Chem. SOC. 111,6412-6419. Christianson, D. W., and Alexander, R. S. (1990). Nature (London) 346, 225. Christianson, D. W., and Lipscomb, W. N. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 68406844. Christianson, D. W., and Lipscomb, W. N. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 75687572. Christianson, D. W., and Lipscomb, W. N. (1987).J. Am. Chem. SOC. 109,5536-5538.

STRUCTURAL BIOLOGY OF ZINC

35 1

Christianson, D. W., and Lipscomb, W. N. (1988a).J. Am. Chem. SOC.110,5560-5565. Christianson, D. W., and Lipscomb, W. N . (1988b). In “Mechanistic Principles of Enzyme Activity” (J. F. Liebman and A. Greenberg, eds.), pp. 1-25. VCH Publ., New York. Christianson, D. W., and Lipscomb, W. N. (1989). Acc. Chem. Res. 22,62-69. Christianson, D. W., Kuo, L. C., and Lipscomb, W. N. (1985). J . Am. Chem. SOC.107, 8281-8283. Coleman, J. E. (1967).J.Bid. Chem. 242,5212-5219. Coleman, J. E. (1986). In “Zinc Enzymes” (1. Bertini, C. Luchinat, W. Maret, and M. Zeppezauer, eds.), pp. 49-58. Birkhauser, Cambridge, Massachusetts. Cotton, F. A., and Wilkinson, G. (1980). “Advanced Inorganic Chemistry,” 4th Ed., p. 14. Wiley, New York. Cunningham, B. C., Bass, S., Fuh, G., and Wells, J. A. (1990).Science 250, 1709-1712. Dean, P. A. W., and Vittal, J. J. (1987). Inorg. Chem. 26, 278-283. Diakun, G. P., Fairall, L., and Klug, A. (1986). Nature (London) 324, 698-699. Eidsness, M. K., Scott, R. A., Prickril, B. C., DerVartanian, D. V., Legall, J., Moura, I., Moura, J. J. G., and Peck, H. D. (1989). Proc. Natl. Acud. Sci. U.S.A. 86, 147-151. Eigen, M., and Hammes, G. G. (1963). A d a Enzymol. 25, 1-38. Einspahr, H., and Bugg, C. E. (1984). Met. lorn Eiol. Syst. 17,51-97. Eriksson, A. E., Jones, T. A., and Liljas, A. (1986). In “Zinc Enzymes” (I. Bertini, C. Luchinat, W. Maret, and M. Zeppezauer, eds.), pp. 317-328. Birkhauser, Cambridge, Massachusetts. Eriksson, A. E., Jones, T. A., and Liljas, A. (1988a). Proteins: Struct., Funct. Genet. 4, 274-282. Eriksson, A. E., Kylsten, P. M., Jones, T. A., and Liljas, .4. (1988b). Proteins: Struct. Funct. Genet. 4,283-293. Fierke, C. A., Calderone, T. L., and Krebs, J. F. (1991).Biochemistry (submitted). Finzel, B. C., Howard, A. J., and Pantoliano, M. W. (1986).J.Cell. Biochem., Suppl. 10A, 272. Forsman, C., Behravan, G., Jonsson, B.-H., Liang, Z.-W., Lindskog, S., Ren, X., Sandstrom, J., and Wallgren, K. (1988). FEBS Lett. 229,360-362. Fresco, J. R., Adams, A., Ascione, R., Henley, D., and Lindahl, T. (1966).Cold Spring Harbor Symp. Quant. Biol. 31, 527-537. Frey, M. H., Wagner, G., VaSak, M., Sorensen, 0. W., Neuhaus, D., Worgotter, E., Kagi, J. H. R., Ernst, R. R., and Wiithrich, K. (1985). J . Am. Chem. SOC. 107, 6847685 1. Furey, W. F., Robbins, A. H., Clancy, L. L., Winge, D. R., Wang, B. C., and Stout, C . D. (1986). Science 231,704-710. Galdes, A., Auld, D. S., and Vallee, B. L. (1986). Biochemistry 25,646-651. Gandour, R. D. (1981).Eioorg.Chem. 10, 169-176. Gardell, S. J., Craik, C. S., Hilvert, D., Urdea, M. S., and Rutter, W. J. (1985). Nature (London) 317,551-555. Geoghegan, K. F., Galdes, A., Martinelli, R. A., Holmquist, B., Auld, D. S., and Vallee, B. L. (1983). Biochemistry 22,2255-2262. Geoghegan, K. F., Galdes, A., Hanson, G., Holmquist, B., Auld, D. S., and Vallee, B. L. (1986).Biochemistry 25,4669-4674. Ghadiri, M. R., and Choi, C . (199O).J.Am. Chem. SOC.112, 1630-1632. Giannousis, P. P., and Bartlett, P. A. (1987).J.Med. Chem. 30, 1603-1609. Gibson, T.J., Postma, J. P. M., Brown, R. S., and Argos, P. (1988).ProieinEng. 2,209-218. Giniger, E., Varnum, S. M., and Ptashne, M. (1985). Cell 40, 767-774. Groves, J. T., and Baron, L. A. (1989).J.Am. Chem. SOC. 111, 5442-5448. Groves, J. T., and Olson, J. R. (1985). Inorg. Chem. 24,2715-2717.

352

DAVID W. CHRISTIANSON

Hanas, J. S., Hazuda, D. J., Bogenhagen, D. F., Wu, F. Y.-H., and Wu, C.-W. (1983).J.Biol. Chem. 258,14120-14125. Handel, T., and DeGrado, W. F. (199O).J.Am. Chem. SOC.112,6710-671 1 . Hanson, J. E., Kaplan, A. P., and Bartlett, P. A. (1989). Biochemistry 28,6294-6305. Hard, T., Kellenbach, E., Boelens, R., Kaptein, R., Dahlman, K., Carlstedt-Duke, J., Freedman, L. P., Maler, B. A,, Hyde, E. I., Gustafsson, J.-h;., and Yamamoto, K. R. (1990a). Biochemistry 29,9015-9023. Hard, T., Kellenbach, E., Boelens, R., Maler, B. A., Dahlman, K., Freedman, L. P., Carlstedt-Duke, J., Yamamoto, K. R., Gustafsson, J.-h;., and Kaptein, R. (1990b). Science 249, 157-160. Hencher, J. L., Khan, M. A., Said, F. F., and Tuck, D. G. (1985). Polyhedron 4, 1263-1267. Henkens, R. W., and Sturtevant, J. M. (1968).J.Am. Chem. SOC.90,2669-2676. Henkens, R. W., Watt, G. D., and Sturtevant, J. M. (1969). Biochemistly 8, 1874-1878. Higaki, J. N., Haymore, B. L., Chen, S., Fletterick, R. J., and Craik, C. S. (1990).Biochemistry 29,8582-8586. Hilvert, D., Gardell, S. J., Rutter, W. J., and Kaiser, E. T. (1986).J. Am. Chem. SOC. 108, 5298-5304. Hoffman, S. J., Chu, S. S.-T., Lee, H.-H., Kaiser, E. T., and Carey, P. R. (1983).J.Am. Chem. Sac. 105,6971-6973. Hol, W. G . J., van Duiljnen, P. T., and Berendsen, H. J. C. (1978). Nature (London) 273, 443-446. Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., and Kim, S.-H. (1977). Nucleic Acidr Res. 4, 281 1-2820. Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., and Matthews, B. W. (1987). Biochemistry 26,8542-8553. Hollenberg, S . M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Lebo, R., Thompson, E. B., Rosenfeld, M. G., and Evans, R. M. (1985).Nature (London) 318,635-641. Hollenberg, S . M., Giguere, V., Segui, P., and Evans, R. M. (1987). Cell 49, 39-46. Holmes, M. A., and Matthews, B. W. (1981).Biochemistry 20,6912-6920. Holmes, M. A., and Matthews, B. W. (1982).J. Mol. BEol. 160,623-639. Honzatko, R. B., Crawford, J. L., Monaco, H. L., Ladner, J . E., Edwards, B. F. P., Evans, D. R., Warren, S. G., Wiley, D. C., Ladner, R. C., and Lipscomb, W. N. (1982).J. Mol. Biol. 160,219-263. Huber, R. E., and Criddle, R. S. (1967). Arch. Biochem. Biophys. 122, 164-173. Ippolito, J. A., Alexander, R. S., and Christianson, D. W. (199O).J.Mol. Biol. 215,457-471. Iverson, B. L., Iverson, S. A., Roberts, V. A., Getzoff, E. D., Tainer, J. A., Benkovic, S. J., and Lerner, R. A. (1990). Science 249,659-662. Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., and Klug, A. (1977).J. Mol. Bzol. 111, 315-328. Jacob, 0.. Cordenas, R., and Tapia, 0. (199O).J.Am. Chem. SOC. 112,8692-8705. Jacobsen, N. E., and Bartlett, P. A. (1981).J.Am. Chem. SOC.103,654-657. Johnston, M. (1987).Nature (London) 328,353-355. Kagi, J. H. R., and Kojima, Y., eds. (1987). “Metallothionein 11. Proceedings of the Second International Meeting, Zurich, Switzerland.” Birkhauser-Verlag, Basel, Switzerland. Kagi, J. H. R., and Schaffer, A. (1988). Biochemistry 27,8509-8515. Kagi, J. H. R., and Vallee, B. L. (1960).J.Biol. Chem. 235,3460-3465. Kannan, K. K., Petef, M., Fridborg, K., Cid-Dresdner, H., and Lovgren, S. (1977).FEBS Lett. 73, 115-1 19. Karin, M. (1985). Cell 41,9-10. Keegan, L., Gill, G., and Ptashne, M. (1986). Science 231,699-704.

STRUCTURAL BIOLOGY OF ZINC

353

Kester, W. R., and Matthews, B. W. (1977).J.Biol. Chem. 252,7704-7710. Khalifah, R. G. (1971).J.Biol. Chem. 246, 2561-2573. Kim, H., and Lipscomb, W. N. (1990). Biochemistry 29,5546-5555. Kim, H., and Lipscomb, W. N. (1991). Biochemistry, in press. Kim, E. E., and Wyckoff, H. W. (1989). Clin. Chim. Acta 186,175-188. Krebs, J., Fierke, C. A., Alexander, R. S., and Christianson, D. W. (1991). Biochemistry, in press. Kuo, L. C., and Makinen, M. W. (1982).J. Biol. Chem. 257,24-27. Kutzelnigg, W. (1977). Pure Appl. Chem. 49,981-1000. Lee, M. S., Gippert, G. P., Soman, K. V., Case, D. A,, and Wright, P. E. (1989). Science 245, 635-637. Lerch, K., Ammer, D., and Olafson, R. W. (1982).J.Biol. Chem. 257,2420-2426. Lewis, C. A., and Wolfenden, R. (1977). Biochemistry 16,4886-4890. Li, Y., and Houk, K. N. (1989).J. Am. Chem. SOC. 111,4505-4507. Liang, J.-Y., and Lipscomb, W. N. (1987). Biochemistry 26,5293-5301. Liang, J.-Y., and Lipscomb, W. N. (1988). Biochemistry 27,8676-8682. Liang, J.-Y., and Lipscomb, W. N. (1989). Int. J. Quantum Chem. 36,299-312. Liang, J.-Y., and Lipscomb, W. N. (1990). Proc. Nutl. Acud. Sci. U.S.A. 87,3675-3679. Lienhard, G . E. (1972). Annu. Rep. Med. Chem. 7, 249-258. Lindskog, S. (1983). In “Zinc Enzymes” (T. G. Spiro, ed.), pp. 78-121. Wiley, New York. Lindskog, S. (1986). In “Zinc Enzymes” (I. Bertini, C. Luchinat, W. Maret, and M. Zeppezauer, eds.), pp. 307-3 16. Birkhauser, Cambridge, Massachusetts. Lindskog, S., and Coleman, J. E. (1973). Proc. Natl. Acad. Sci. U.S.A. 70,2505-2508. Lipscomb, W. N., Hartsuck, J. A., Reeke, G. N., Quiocho, F. A., Bethge, P. H., Ludwig, M. L., Steitz, T. A., Muirhead, H., and Coppola, J. C. (1968).Brookhaven Symp. Biol. 21, 24-90. Makinen, M. W., Kuo, L. C., Dymowski, J. J., and Jaffer, S. (1979).J. Biol. Chem. 254, 356-366. Margoshes, M., and Vallee, B. L. (1957).J.Am. Chem. SOC.79,4813-4814. Matthews, B. W. (1988).Acc. Chem. Res. 21,333-340. McGregor, M. J., Islam, S. A., and Sternberg, M. J. E. (1987).J.Mol. Biol. 198,295-310. Merz, K. M. (1991).J.Am. Chem. SOC.113,406-411. Merz, K. M. (199O).J.Mol. Biol. 214, 799-802. Merz, K. M., Hoffmann, R.,and Dewar, M. J. S. (1989).J.Am. Chem. SOC.111,5636-5649. Miller, J., McLachlan, A. D., and Klug, A. (1985).E M B O J . 4, 1609-1614 Mock, W. L. (1975). Bioorg. Chem. 4,270-278. Monzingo, A. F., and Matthews, B. W. (1984). Biochemistry 23,5724-5729. Nair, S. K., and Christianson, D. W. (1991).J.Am. Chem. SOC.,in press. Nakagawa, S., Umeyama, H., Kitaura, K., and Morokuma, K. (1981).Chem. Phurm. Bull. 29, 1-6.

Ollis, D. L., Brick, P., Hamlin, R., Xuong, N. G., and Steitz, T. A. (1985). Nature (London) 313,762-766. Omichinski, J. G., Clore, G. M., Appella, E., Sakauchi, K., and Gronenborn, A. M. (1990). Biochemistry 29,9324-9334. Otvos, J. D., and Armitage, I. M. (1980). Proc. Nutl. Acud. Scz. U.S.A. 77, 7094-7098. Pan, T., and Coleman, J. E. (1989). Proc. Nutl. Acad. Sci. U.S.A. 86,3145-3149. Pan, T., and Coleman, J. E. (1990a). Biochemistry 29,3023-3029. Pan, T., and Coleman, J. E. (1990b).Proc. Natl. Acud. Sci. U.S.A. 87,2077-2081. Pan, T., and Coleman, J. E. (1991). Biochemistry 30,4212-4222. Pan, T., Freedman, L. P.. and Coleman, J. E. (1990). Biochemistry 29,9218-9225.

354

DAVID W. CHRISTIANSON

Pantoliano, M. W., Whitlow, M., Wood, J. F., Rollence, M. L., Finzel, B. C., Gilliland, G. L., Poulos, T. L., and Bryan, P. N. (1988).Biochemistry 27,831 1-8317. Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I., and Laskowski, M. (1982).J. Mol. Biol. 158,515-537. PArraga, G., Horvath, S., Eisen, A., Taylor, W. E., Hood, L., Young E. T., and Klevit, R. E. (1988). Science 241, 1489-1492. PArraga, G., Horvath, S. J., Hood, L., Young, E. T.,and Klevit, R. E. (1990).Proc. Natl. Acad. Sci. U.S.A. 87, 137-141. Pauling, L. (1948). Nature (London) 161,707-709. Pavletich, N. P., and Pabo, C. 0. (1991). Science 252,809-817. Pearson, R. G. (1963).J. Am. Chem. Sac. 85,3533-3539. Perutz, M. F., Gronenborn, A. M., Clore, G. M., Fogg, J. H., and Shih, D. T.-B. (1985). J . Mol. Biol. 183,491-498. Peterson, M. R., and Csizmadia, I. G. (1979).J. Am. Chem. Sac. 101, 1076-1079. Phillips, M. A., Fletterick, R., and Rutter, W. J. (199O).J. Biol. Chem. 265,20692-20698. Pitts, J. E. (1990).Nature (London) 346, 113-1 14. Pocker, Y., and Deits, T. L. (1983).J. Am. Chem. Sac. 105,980-986. Pocker, Y., and Sarkanen, S. (1978). Adv. Enzymol. 47, 149-274. Rees, D. C., and Lipscomb, W. N. (1982).J. Mol. Biol. 160,475-498. Rees, D. C., and Lipscomb, W. N. (1983). Proc. Natl. Acad. Sci. U.S.A. 80,7151-7154. Rees, D. C., Lewis, M., and Lipscomb, W. N. (1983).J. Mol. Biol. 168,367-387. Regan, L., and Clarke, N. D. (1990).Biochemistry 29,10878-10883. Reynolds, W. F., Peat, I. R., Freedman, M. H., and Lyerla, J. R. (1973).J. Am. Chem. Sac. 95, 328-331. Riepe, M. E., and Wang, J. H. (1968).J. Biol. Chem. 243,2779-2787. Riordan, J. F. (1973). Biochemistry 12, 3915-3923. Robbins, A. H., McRee, D. E., Williamson, M., Collett, S. A., Xuong, N. H., Furey, W. F., Wang, B. C., and Stout, C. D. (199O).J. Mol. Biol. (submitted). Roberts, V. A., Iverson, B. L., Iverson, S. A., Benkovic, S. J., Lerner, R. A., Getzoff, E. D., and Tainer, J. A. (1990).Proc. Natl. Acad. Sci. U.S.A. 87,6654-6658. Rosenfield, R. E., Parthasarathy, R., and Dunitz, J. D. (1977).J. Am. Chem. SOC.99,48604862. Rosenfield, R. E., Jr., Swanson, S. M., Meyer, E. F., Carrell, H. L., and Murray-Rust, P. (1984).J . Mol. Graphics 2,43-46. Ruan, F., Chen, Y., and Hopkins, P. B. (199O).J. Am. Chem. SOC. 112,9403-9404. Russell, A. J., and Fersht, A. R. (1987). Nature (London) 328,496-500. Russell, A. J., Thomas, P. G., and Fersht, A. R. (1987).J. Mol. Biol. 193,803-813. $ah, D., Bycroft, M., and Fersht, A. (1988).Nature (London) 335,740-743. Sander, M. E., and Witzel, H. (1985). Biochem. Biophys. Res. Commun. 132,681-687. Schena, M., Freedman, L. P., and Yamamoto, K. R. (1989). Genes Dev. 3, 1590-1601. Schepartz, A., and Breslow, R. (1987).J. Am. Chem. Sac. 109, 1814-1826. Schultze, P., Worgotter, E., Braun, W., Wagner, G., VaSAk, M., Kagi, J. H. R., and Wiithrich, K. (1988).J. Mol. Biol. 203, 251-268. Schwabe, J. W. R., Neuhaus, D., and Rhodes, D. (1990). Nature (London) 348,458-461. Severne, Y., Wieland, S., Schaffner, W., and Rusconi, S. (1988). E M B O J . 7,2503-2508. Shang, Z., Liao, Y.-D., Wu, F. Y.-H., and Wu, C.-W. (1989). Biochemistry 28,9790-9795. Sharma, S. K., and Reed, M. D. (1976).J. Inorg. Nucl. Chem. 38, 1971-1972. Shoham, G., Rees, D. C., and Lipscomb, W. N. (1984). Proc. Natl. Acad. Sci. U.S.A. 81, 7767-7771.

STRUCTURAL BIOLOGY OF ZINC

355

Shoham, G., Christianson, D. W., and Oren, D. A. (1988).Proc. Natl. Acad. Sci. U.S.A. 85, 684-688. Silverman, D. N., and Lindskog, S. (1988). Acc. Chem. Res. 21,30-36. Silverman, D. N., and Vincent, S. H. (1983). CRC Crit. Rev. Biochem. 14,207-255. Smulevich, G., Mauro, J. M., Fishel, L. A., English, A. M., Kraut, J., and Spiro, T. G. (1988). Biochemistry 27,5477-5485. Steiner, H., Jonsson, B.-H., and Lindskog, S. (1975).Eur.J. Biochem. 59, 253-259. St. John, T. P., and Davis, R. W. (19Sl).J. Mol. Biol. 152, 285-315. Suh, J., Cho, W., and Chung, S. (1985).J. Am. Chem. Sac. 107,4530-4535. Summers, M. F., South, T. L., Kim, B., and Hare, D. R. (1990). Biochemistry 29,329-340. Tainer, J . A., and Roberts, V. A. (1990).Nature (London) 348, 589. Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982). J . Mol. Biol. 160, 181-217. Thanki, N., Thornton, J. M., and Goodfellow, J. M. (19SS).J. Mol. Biol. 202, 637-657. Thomas, P. G., Russell, A. J., and Fersht, A. R. (1985). Nature (London)318,375-376. Thompson, G. A., and Carpenter, F. H. (1976).J. B i d . Chem. 251, 1618-1624. Tronrud, D. E., Holden, H. M., and Matthews, B. W. (1987). Science 235,571-574. T u , C., Silverman, D. N., Forsman, C., Jonsson, B.-H., and Lindskog, S. (1989).Biochemistry 28,7913-7918. Ueyama, N., Sugawara, T., Sasaki, K., Nakamura, A., Yamashita, S., Wakatsuki, Y., Yamazaki, H., and Yasuoka, N. (1988).Znorg. Chem. 27,741-747. Vallee, B. L., and Auld, D. S. (1990). Biochemistry 29,5647-5659. Vallee, B. L., and Galdes, A. (1984). Adv. Enzymol Relat. Areas Mol. Biol. 56, 283-430. Vallee, B. L., Galdes, A., Auld, D. S., and Riordan, J. F. (1983). In “Metal Ions in Biology” (T. G. Spiro, ed.), Vol. 5, pp. 25-75. Wiley, New York. Van Wart, H. E., and Vallee, B. L. (1978). Biochemistry 17, 3385-3394. Vedani, A., and Dunitz, J. D. (1985).J. Am. Chem. SOC. 107,7653-7658. Vedani, A., and Huhta, D. W. (199O).J. Am. Chem. SOC.112,4759-4767. Vincent, A. (1986). Nucleic Acidc Res. 14, 4385-4391. Wagner, G., Neuhaus, D., Worgotter, E., VaSak, M., Kagi,J. H. R., and Wiithrich, K. (1986). J . Mol. Biol. 187, 131-135. Wallmeier, H., and Kutzelnigg, W. (1979).J. Am. Chem. SOC. 101,2804-2814. Wiberg, K. B., and Laidig, K. E. (1987).J. Am. Chem. SOC.109,5935-5943. Winge, D. R., Nielson, K. B., Zeikus, R. D., and Gray, W. R. (1984).J. Biol. Chem. 259, 11419-1 1425. Wolfenden, R. (1972). Acc. Chem. Res. 5, 10-18. Woolley, P. (1975). Nature (London) 258,677-682.

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AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed.

A Aasa, R., 156, 194 Acharya, K. R., 100, 106,140 Ackrell, B., 252,277 Ackress, B., 221,275 Adams, A., 292,351 Adams, M.,218,219,221,223,252,254, 272,274 Adams, M. J., 49,66 Adman, E., 112,140, 208,215,272,273, 285,300,309,310,349 Adman, E. T., 39,42,66, 146, 147, 148, 153, 161, 166, 167, 186, 190,192, 193, 196, 203, 215, 219, 222, 223, 253, 254,272,276 Agarwal, R. C., 53, 70 Agrawal, B. B. L., 108,140 Ahrland, S., 5,67 Ainscough, E. W., 157,192 Ainsworth, C. F., 108,142 Aisen, P., 209, 237,272,274 Akeson, A., 216, 217,274 Alard, P., 50, 73 Albertsson, J., 82, 140 Alden, R. A., 42,69, 203, 208,273 Alexander, R.S., 285,288, 290, 292, 295, 297, 300, 307, 309, 315,317, 318, 321,330,339,349,350,352,353 Alfred, A. L., 4, 67 Allen, F. H., 17, 21,32,67, 281,349 Allen, J. P., 202,217,235, 236,272,279 Allen, P., 229, 235,274 Allendorf, M. D., 183,192, 196 Allerhand, A., 39.69 Almassey, R. J., 150, 192 Ambler, R. P., 160, 192 Amma, E. L., 83,88, 134, 144 Ammer, D., 333,353

Anderegg, G, 285,349 Andersen, L., 78,143 Anderson, B. F., 39,41,42, 72, 74, 153, 195,202,210,229,237,272,273 Anderson, G., 218, 219,257,272 Anderson, L., 33 1,349 Anfinsen, C. B., 85, 105,141 Antanaitis, B., 209,274 Antonio, M., 260,272 Appella, E., 50, 72, 340,353 Arciero, D., 205,229, 233, 234,272,276 Argos, P., 285, 307,322,337, 338,350, 351 Arima, K., 146, 185, 194 Armitage, I. M., 334,353 Armstrong, W. H., 39,42,67 Aro, H., 229,277 Arseniev, A., 334,350 Arvidsson, R. H. A., 156, 192 Asahara, H., 252,274 Ascione, R.,292,351 Asher, S. A., 171,197 Askew, B., 30,73 Atkin, C., 245, 247,273 Auf der Heyde, T. P. E., 17,45,67 Auld, D. S., 38,45,50, 75, 286, 324, 326, 329,350,351,355 Auk, J., 82, 142 Auric, P., 208,277 Averill, B., 260, 272 Averill, B. A., 185,193, 256,275 Avigliano, L., 146, 194 Axcell, B. C., 229,273

Babu, Y. S., 53,67, 87, 88,140, 141 Bachovchin, W. W., 27, 74 Backes, G., 247,248,278 357

358

AUTHOR INDEX

Bacon, G. E., 20,67 Bailey, S., 202, 237,273 Bajorath, J., 84, 92, 99, 135, 141 Bak, H. J., 42,69 Baker, E. N., 25,39,41,42,49,66, 67, 72,74, 110, 111, 112, 120-121, 137, 141, 151, 153, 157, 192, 195, 202, 210,229,237,238,272,273 Baker, H. M., 202, 210,237,238,272 Baldwin, M., 164, 196 Ballan, D., 270,276 Ballou, D., 233,270,273,275,279 Bank, J., 252,274 Banner, D., 161,192 Banner, D. W., 161, 186,195 Bannister, J. W., 170, 192 Barber, M. J., 268,278 Bare, R., 261,279 Barker, W. C., 251,275 Barnes, C. L., 38, 70 Baron, L. A., 286,287,301,351 Bartels, K. S., 43, 75 Bartlett, P. A., 330, 331,350,351, 352 Bartsch, H. H., 159,193 Bartsch, R. G., 203,273 Bartunik, H. D., 159, 193 Barynin, V. V., 43, 75 Bass, S., 349,351 Batie, C., 270,275, 276,279 Bauman, R. A,, 178,196 Baur, W. H., 17,20, 24, 67, 76 Bayley, P. M., 133, I43 Bazer, F., 239,273 Beal, R. B., 57,68 Becker, J. W., 108,141 Beem, K. M., 55,75, 168,196, 298, 309, 355 Beevers, C. A., 20,67 Behravan, G., 321,351 Beinert, H., 204, 2 19, 222,223,262, 263, 264, 265,267,273,275,276,278, 2 79 Beintema, J. J., 42, 69 Bell, G. I., 168, 169, 170, 193 Bellamy, H. D., 267,273 Bellard, S., 17,21, 32, 67 Beltramini, M., 173, 195 Bengtsson, U., 49, 71 Benkovic, S. J., 345,347, 348,352,354 Benson, B. A., 38, 70

Beppu,T., 146,161, 166, 185, 186,192, 193,194,195,197 Bereman, R. D., 153, I93 Berendsen, H. J. C., 306,352 Berg, J., 205, 206, 219,273 Berg, J. M., 50,67, 284, 285, 309, 310, 338,340,341,342,343,350 Bergsten, P.-C., 49, 71 Bergstrom, J., 156, 195 Bergstrom, R.,87, 144 Berhardt, F., 229,273 Berliner, L. J., 85, 106,143 Bernardo, M., 164,196 Bernstein, F. C., 39,67, 78, 81, 141, 210, 273,281,350 Berridge, M. J., 78, 141 Bertini, I., 314, 350 Bethge, P. H., 322,353 Betzel, C., 83.99, 134, 142 Biellmann, J.-F., 33, 50,68 Bier, M., 97,141 Bieth, J., 84, 142 Bill, E., 229, 273 Billiald, P., 172, 194 Bingham, A. G., 157,192 Blankenship, R. E., 146, 149, 164, I96 Blomstrom, D. C., 256,273 Blow, D. M., 50, 70 Blundell, T. L., 38,49,66,67, 224,273, 296,350 Bode, W., 53,69, 82,92,98, 141, 340, 354 Boel, E., 53,67, 113,141 Boelens, R., 343,352 Bogenhagen, D. F., 337,338-339, 352 Boiwe, T., 2 16,2 17,274 Boldingh, J., 229,274 Bolognesi, M., 179, 194 Bonam, D., 252,256,278 Bonaventura, J., 172,194 Bonsen, P. P. M., 85,141 Bordas, J., 49,67 Borisov, V. V., 43,75 Bott, R. R., 53, 74 Bouwman, E., 174, 176,196 Braaksura, A., 208,274 Bradbury, J. H., 307,350 Brady, L., 53,67, 113,141 Bramson, R., 161,192

AUTHOR INDEX

Branden, G I . , 216, 217, 224,273, 274, 285,307,322,350 Braun, W., 56, 74, 334,350,354 Breslow, E., 28, 67 Breslow, R., 322, 324,325,327,350,354 Brew, K., 106,141 Brice, M. D., 17,21, 32,39,67, 78, 81, 141,210,273, 281,350 Brick, P., 290,353 Bricogne, G., 53, 72 Briedigkeit, L., 84, 141 Briggman, B., 82,141 Brill, W., 260, 278 Brill, W. J., 259, 280 Bringle, K., 261, 262,278 Briving, C., 183, 192 Brock, W. H., 2, 71 Brodie, A. M., 157,192 Brodin, P., 133,143 Brown, I. D., 4, 8,9, 11, 12, 21, 22,24, 67,68, 82, 112,141 Brown, R. S., 292, 337, 338,350,351,352 Bruice, T. C., 42,68 Bruschi, M., 42, 66, 69, 203, 251,272, 273,274 Bryan, P. N., 83,98, 134,143, 306,350, 353 Brzozowski, A. M., 53,67, 113,141 Buchbinder, J. L., 5, 68 Buck, M., 260,275 Buckel, W., 252,278 Buckingham, D. A., 8,68 Bugg, C. E.,52,53,67, 69, 78,82, 87, 88, 108, 111, 115, 118, 133, 134, 140, 141,142,144, 295,351 Buisson, G., 174, 192 Bull, C., 233,273 Buoscio, B. W., 65, 74, 134, I44 Burger, A. R., 56,65, 75 Burger, V., 33,50,68 Burgess, B., 260,261,274 Burgess, B. K., 217,252,255,256,273,276 Biirgi, H.-B., 17, 68, 72 Burgmayer, S., 261,279 Burley, S. K., 284, 331, 332,350 Burns, A., 259,279 Burnstein, E. A., 106, I43 Burris, R., 261,276 Burroughs, A. K., 184,196 Bycroft, M., 307,354

359 C

Calabrese, L., 184-185, 185, 192 Calderone, T., 315, 317,351 Cammack, R., 208,248,273,279 Campbell, H., 239,273 Campbell, J., 108, 141 Candlen, R., 36, 68 Canters, G. W., 155, 156, 157, 164, 192, 193,195 Capon, D. J., 146,196 Carbonaro, M., 184-185, 185, I92 Carey, P. R., 329,352 Carlbom, U., 49, 71 Carlioz, A., 202,273 Carlstedt-Duke, J., 343,352 Carney, M. J., 208,273 Carpenter, F. H., 331,355 Carrell, A. B., 14, 71 Carrell, C. J., 17, 68, 82, 118, 124, 141, 204,273, 288,289,290,292,330,350 Carrell, H. L., 14, 17,29, 32, 33,42, 50, 68, 71, 73, 82, 118, 124,141, 204, 273,288,289,290,292,330,350,354 Carter, C. W., Jr., 42,68, 69, 203, 208, 2 73 Carter, D. C., 56, 72 Cartwright, B. A., 17, 21, 32, 67 Carver, J. A., 307,350 Case, D. A., 159,194, 338, 339,353 Cash, V. L., 217,255, 257,275,276 Cecchini, G., 221, 252,275,277 Cedergren-Zeppezauer, E., 49, 74 Cerny, R., 261,275 Cetorelli, J., 239,273 Chafouleas, J. G., 87, 143 Chakrabarti, P., 118, 120, 121, 124, 141, 203,258,275, 290, 295,296, 297, 299,300,301,303,348,350 Chambers, J. L., 53, 71 Chandrasekhar, K., 17,68 Chang, C. L., 258,276 Chatt, J., 5 , 6 7 Chazin, W. J., 91,144, 159, 194 Chen, E. Y., 98,144 Chen, S., 37, 70, 346, 347,352 Chen, T., 239,273 Chen, V., 229,273 Chen, Y., 344,354 Chiba, K., 91, I41

360

AUTHOR INDEX

Chipman, D. M., 37, 73 Chirlian, L. E., 220,292, 295,349 Chishnell, J., 260,278 Cho, W., 328,355 Choi, C., 307, 344,351 Christianson, D. W., 285, 286, 288, 290, 297,300,307, 309,315,317, 318, 321, 322, 324, 325, 326,327, 328, 329, 330,332, 333, 339,349,350, 351,352,353,354 Christner, J. A., 259,268,273,275 Chu, S. S.-T., 329,352 Chuknyisky, P. P., 57, 68 Chung, S., 328,355 Church, G. M., 292,352 Church, W. B., 159,192 Cid-Dresdner, H., 321, 333,352 Claessens, M., 50, 73 Clancy, L. L., 56,69, 334,351 Clark, I. E., 242,273 Clarke, N. D., 50, 73,345,354 Classen, H. G., 35, 74 Cliffe, S. G. R., 8, 141 Cline, J., 270,273 Cline, J. F., 268,273 Clingeleffer, D. J., 159, 193 Clore, G. M., 50, 72, 307, 340,353,354 Codd, R., 166,193 Cohen, G. H., 53,74 Cole, G., 92, 143 Coleman, J. E., 302, 312, 313,317,324, 341-342,342,351,353 Collett, S. A., 334,354 Collyer, C. A., 50, 70, 160, 192 Colman, P. M., 146, 148,192 Conradson, S., 260, 261,274 Cook, W. J., 53,67, 87,88, 140, 141 Coppola, J. C., 322,353 Cordes, F., 159,193 Cotton, F. A., 52,68, 105,141, 283, 285, 351 Coucouvanis, D., 260,274 Cox, D., 233,274 Cox, J. A., 84, 144 Cox, J. M., 42, 73 Coyle, C. L., 187-188, 190,192, 195 Craik, C. S., 37, 70, 327, 346, 347,351, 352 Cramer, S., 260,274 Crawford, J. L., 217,275, 334,336,352

Crelli, G., 342-343,352 Criddle, R. S., 303,352 Crouch, T. H., 87,142 Csizmadia, I. G., 30, 73, 288,354 Cuatrecasas, P., 85, 105,141 Cuff, M. E., 174,193, 195 Cunningham, B. A., 108,141,144 Cunningham, B. C., 349,351 Cunningham, R., 252,274 Cupp, J., 222,274 Curry, N. A., 20,67

Dahlrnan, K., 343,352 Dale, D. H., 29, 72 Dalgarno, D. C., 91, 141 Dalton, H., 239,248,275,279 Dameron, C. T., 184,193 Dauter, Z., 83,99, 134,142, 161, 164,195 David, P. R., 331, 332, 350 Davidson, V. L., 164, 165,194, 195 Davies, D. R., 42, 53, 71, 74, 166, 195 Davies, N. R., 5, 67 Davis, K., 252,274 Davis, L. C., 258,276 Davis, R., 257,275 Davis, R. W., 340,355 Dawson, J., 242,274 Dawson, J. H., 148,196 Day, E., 269, 270,274 Day, E. P., 208,276 De Haas, G. H., 85, 106,141,144 de Ley, M., 173,196 Dean, D., 257,258,261,262,275,278, 279 Dean, D. R., 217,255,276 Dean, P. A. W., 286,331 Debrunner, P., 244,277 Debus, R. J., 236,274 Dege, J., 239,248,249,274 DeGrado, W. F., 344-345,346,348,352 deGroot, J., 229,274 Deisenhofer, J., 202, 235,274 Deits, T. L., 319,354 Delange, R. J., 98, 143 Demaille, J. G., 91, 142 Dent-Glasser, L. S., 12,68 Derancout, J., 91, 242 Derewenda, Z., 53,67, 108, 113,141

36 1

AUTHOR INDEX

DerVartanian, D. V., 303,351 Deutsch, E., 20,69 DeVito, V. L., 171,197 Devlin, F., 243,278 Dewar, M. J. S., 32 1,353 Diakun, G. P., 337,351 Dickerson, R. E., 38,42,68, 71, 150,192 Diets, T. L., 258,274 Dijkman, R., 106,144 Dijkstra, B. W., 53,69, 106, 141, 142, 144 Dimicoli,J. L., 84, 142 Dixon, R., 261,277 Dobler, M., 36, 75 Dobson, B., 174, 176,196 Dodson, E. J., 108,141, 202, 210, 237, 2 72 Dodson, G. G., 49,53,66,67, 113,141 Doi, K., 209,274 Dooley, D. M., 148, 185, 190,193, 195, 196 Dorfman, J. R., 153,193 Doubleday, A., 17, 21, 32, 67 Drendel, W., 87,143 Drenth, J., 53,69, 106, 134, 141, 142, 144, 164,196 Dreyer, J., 262, 263,275,276 Dreyer, J. L., 219, 262,273 Dreyer, J.-L., 252,274 Duee, E., 174,192 Duff, R. J., 30, 73 Duine, J. A,, 164, 171,196 Dunford, H. B., 185,194 Dunham, W., 267,269,270,273,274 Dunham, W. R., 267,279 Dunitz, J. D., 17, 20, 36, 72, 73, 75, 297, 302,303,354,355 Durham, W., 208,274 Dutscho, R., 252,278 Dwulet, F. E., 178, 196 Dymowski, J. J., 328,353

E Eady, R. R., 259,275 Eaton, D., 146,196 Eaton, R., 229,276 Eccleston, E., 221, 223, 254,274 Edelman, G. M., 108,141,144 Edwards, B. F. P., 53, 71, 217,275, 334, 336,352

Edwards, S. L., 43,69 Ehrenberg, A., 244,245,247,277,278 Eichhorn, G. L., 57,68 Eidness, M., 260,274 Eidsness, M. K., 303,351 Eigen, M., 312,351 Einspahr, H., 52,69, 78, 82, 108, 111, 115, 118, 133, 134,142,144, 295,351 Eisen, A,, 338,354 Eisenberg, D., 34,38,68, 76, 124,144, 215,280 Eisenman, G., 34,76, 124,215,280,1144 Eklund, H., 49,50, 72, 74, 202, 210, 216, 217,221,245,246,247,274,277 Elder, R. C., 20,69 Eliasson, R., 248,274 Ellis, F., 242, 243,280 Ellis, W. R., 157, 192 Empie, M. W., 340,354 Emptage, M., 219, 252, 262, 263,264, 265,270,273,274,275,276 Engeseth, H. R., 155,193 English, A. M., 309,355 Ensign, S. A., 252,256,278 Epp, O., 92,143,202,235,274 Epstein, O., 184, 196 Eriksson, A. E., 312, 314, 318, 321,351 Erkelens, C., 156, 157, 164,193 Erlebacher, J., 17, 68, 82, 118, 124, 142, 204,273, 288, 289, 290, 292, 330,350 Ernst, R. G., 334,351 Estell, D. A., 98, 144 Evans, D. R., 217,275,334,336,352 Evans, R. M., 342-343,352 Evans, R. W., 202,237,273 Eventoff, W., 285,307,322,350

F Faggiani, R., 12, 68 Fairall, L., 337,351 Fajans, K., 4 , 6 9 Falke, J. J., 65, 74, 134, 144 Farber, G. K., 50,69 Farr-Jones, S., 27, 74 Farrelly, D., 206, 215,278 Fauman, E. B., 230,277 Fee, J., 223, 269, 270,273,274, 275,276, 277 Fee, J. A., 147, 148,193, 202,273,278

362

AUTHOR INDEX

Feher, G., 202,229,235,236,272,274, 277 Fehlhammer, H., 53,69 Feiters, M. C., 155, 174, 176,193, 196 Feldman, R. J., 172,194 Fenderson, F. F., 190,193 Fermi, G., 42,73 Ferrari, E., 98,144 Fersht, A., 307,354 Fersht, A. R., 304, 305,306,354,355 Fielden, E. M., 146,193 Fields, B. A., 159, I93 Fierke, C . A., 315, 317, 321,351,353 Finazzi-Agro, A., 179,194 Findling, K., 269, 270,274 Fine, R., 171, 195 Finer-Moore, J. S., 230,277 Finzel, B. C., 44, 73, 83, 98, 134,143, 306, 351,353 Fish, W. W., 146, 195 Fishel, L. A., 309,355 Fita, I., 43, 75 Flank, A., 260,274 Fleming, G. R., 155, 195 Fletterick, R., 324, 328,354 Fletterick, R. J., 37, 70, 346, 347,352 Flint, D., 252, 270,274 Flohe, L., 170, 196 Flood, A., 260,274 Fogg, J. H., 307,354 Foner, S., 215,276 Fontecave, M., 248,274 Forsen, S., 91, 133, 143, 144 Forsman, C., 321,351,355 Fox, B., 239,248,249,274,275 Frank, JZn, J., 164,196 Frankel, R., 208,273 Frankel, R. B., 215,276 Freedman, L. P., 302, 343, 352,353,354 Freedman, M. H., 298,354 Freeman, H. C., 36, 39,41,42,69, 70, 146, 148, 158, 159, 160, 161, 164, 166,192,193 Freeman, J. C., 146, 149, 164,196 Freer, S. T., 42,69, 203,208,273 Freisner, M. J., 53, 70 Fresco, J. R., 292,351 Frey, M., 42,69, 203,274 Frey, M. H., 334,351 Fridborg, K., 49, 71, 321,333,352

Fridovich, I., 229,280 Froland, W., 239,248, 249,274 Frolik, C., 229,273 Frommel, C., 9, 84, 141, 142 Fuchs, S., 85, 105, 141 Fuh, G., 349,351 Fujimori, K., 87, 142 Fujisawa, K., 176, 194 Fukui, K., 17, 69 Fukuyama, K., 42,69, 75, 150, 193, 203, 251,254,274,279 Fullmer, C. S., 91,141, 144 Fung, C . H., 39,69 Furey, W. F., 56,69, 334,351, 354 Furlong, C. E., 107, 142 Furst, P., 147, 193

Gaillard, J., 208,277 Galdes, A., 326,329,350,351, 355 Gandour, R. D., 30, 69, 124,142, 287, 288,351 Gandvik, E.-K., 183, 192 Garavito, R. M., 285, 307, 322, 350 Gardell, S. J., 327, 351,352 Garner, C., 260,274 Garner, C. D., 147,193 Garratt, R. C., 202, 237,273 Garrett, T. P. J., 159, 193 Garssen, G., 229,274 Gatti, D., 270, 275 Gatti, G., 179, 194 Gaykema, W. P. J., 42,69 Geary, P. J., 229,273 Gelin, B. R., 43, 70 Geoghegan, K. F., 326,329,350,351 George, D. G., 166,194,251,275 Georgiadis, M. M., 203, 258,275 Gergely, J., 87, 133, 142 Getzoff, E. D., 55, 75, 168, 169, 170, 171, 193, 196, 298, 309, 345, 347, 348, 352, 354,355 Ghadiri, M. R., 307,344,351 Giannousis, P. P., 331,351 Gibson, D. T., 229,279 Gibson, J. F., 207,275 Gibson, T. J., 338, 351 Gielens, C., 146, 172, 173, 174, 195 Giguere, V., 342-343,352

AUTHOR INDEX

363

Gilliland, G. L., 83,98, 134, 143, 306,353 Gusalus, R.,22 1,275 Gillis, J. M., 91, 142 Cuss, J. M., 39,41, 70, 146, 148, 158, 159, Giniger, E., 340,352 160, 161, 164,192,193 Giocometti, G., 173, 195 Gustafsson, J. A., 343,352 Gippert, G. P., 159, 194, 338, 339,353 Girerd, J. J., 208,277 Glusker, J. P., 14, 17,24,29,33, 38,42, 50,68, 70, 71, 72, 82, 118, 124,141, Haaker, H., 208,259,274,275 204,273,288,289,290,292,330,350 Hackert, R., 147,193 Goaman, L. C . G., 42, 73 Hagen, W., 208,274 Gocometti, G. M., 173,195 Hagen, W. R.,259,275 Godden, J. W., 186,193 Hahn, J., 219, 262,273 Goldschmidt, V. M., 8, 70 Haiech, J., 84, 91, 142 Goldstein, I. J., 108, 140 Halaban, R.,177, 193 Golomb, M. L., 38, 69 Hall, 0. A., 207,275 Goode, C . A., 184,195 Hallewell, R. A., 168, 169, 170,193 Goodfellow,J. M., 120, 137, 144, 295, ?55 Hamer, D., 147, I93 Gordon, W. E., 30,73 Hamlin, R.,203, 21 1, 252, 266,276, 290, Gorinsky, B., 202,237,273 353 Gormal, C., 260,275 Hamlin, R.C., 43,69 Gotzmann, D. J., 185,197 Harnmes, G. G., 312,351 Gouet, P., 174, 192 Han, J., 166,193 Grabarek, Z., 87, 133,142 Hanas, J. S., 337,338-339,352 Grandoni, J., 252, 256,275 Handel, T., 344-345,346,348,352 Grant, D. A. W., 84,141 Hansen, R., 269,278 Graslund, A., 244, 245, 247,277,278 Hanson, J. E., 330,331,352 Gray, H., 242,274 Hanzl, M. T., 259,280 Gray, H. B., 42, 72, 148, 157,192, 196 Harada, Y., 146, 164,196 Gray, W. R.,333,355 Hard, T., 343,352 Greaser, M., 87, 143, 144 Warding, M. M., 36,49,66, 68 Grebenko, A. I., 43, 75 Hardman, K. D., 53, 70, 108,142 Green, J., 248,275 Hardy, F., 230,277 Greenhough, T. J., 53,67, 87,140 Hare, D. R.,340,34 1,355 Gregely, J., 87,143 Hare, J. W., 148,196 Grewe, H., 49, 67 Harel, M., 203,279 Griffin, R. G., 27, 74 Harioka, T., 203,279 Groendijk, H., 164,196 Harkins, R. N., 146,196 Groeneveld, C. M., 155, 156, 157, 164, Harpel, M., 229,273 193 Harris, E. D., 184, 193 Groh, S., 260,272 Harrison, S. C., 53, 72 Gronenborn, A. M., 50,72, 307,340,353, Harrowell, P. R., 39,42, 70, 158, 193 354 Hart, R. G., 42, 71 Gros, P., 83,99, 113, 134, 135,142 Hartmann, H. J., 146,196 Groves,J., 250,277 Hartsuck, J. A,, 322,353 Groves, J. T., 286, 287, 301,351 Hase, T., 42, 69, 75, 150,193, 203, 251, Grundstrom, T., 133,143 254,274,279 Guerlesquin, F., 25 1,273 Hasegawa, Y., 50, 75 Gunsalus, I. C., 44,73 Haser, R.,42,69, 203,274 Gurbiel, R.,270,275 Hasnain, S., 202, 237,273 Curd, F. R. N., 3, 70 Hasnain, S. S., 147, 155, 193

364

AUTHOR INDEX

Hatchinkian, E., 252,277 Hatefi, Y., 252,274,275 Hausinger, R.,221,275 Hausinger, R. P., 222,223, 257,275 Hausmann, E., 229,275 Havel, T. F., 159, 194 Hawkes, T., 260,275 Hayes, R.,261,275 Haymore, B. L., 37,70, 346,347,352 Hazen, E. E., Jr., 52,68, 105, 141 Hazuda, D. J., 337, 338-339,352 He, F.-C., 13, 70 Hearshen, D., 269, 270,274 Hedrnan, B., 161, 164,193, 261,274 Heeg, M. J., 20,69 Helbig, J., 35, 74 Helliwell,J. R., 108, 141 Hencher, J. L., 286,352 Henderson, R., 106,142 Hendrich, M., 249,275 Hendrickson, W. A., 38,44, 74, 75, 174, 193,195, 202,225, 240,278 Henkens, R. W., 285,352 Henley, D., 292,351 Henner, D. J., 98,144 Henrick, K., 50, 70 Henzl, M. T., 259,275 Herriott, J. R.,42, 70 Herskovitz, T., 172,193, 256,275 Herzberg, O., 50, 53, 70, 82.83, 87, 88, 110, 127, 135,142 Higaki, J. N., 37, 70, 346,347,352 Higgs, H., 17, 21, 32, 67 Hikichi, K., 50, 75 Hilewicz-Grabska, M., 184, 193 Hill, R. L., 106, 141 Hille, R., 269, 270,274 Hilvert, D., 327,351,352 Hinrichs, W., 84,92,99, 135,141 Hng, L., 166,193 Hoard, J. L., 43, 70 Hoch, M. H. J., 49,67 Hodges, R.S., 131,143 Hodgkin, D. C., 49,66 Hodgson, K., 219, 260, 261,262,273,274 Hoenig, H., 242,274 Hoffman, B., 260, 261,263, 264, 270, 273,275,279 Hoffman, B. M., 268,273 Hoffman, S. J., 329,352

Hoffmann, R.,32 1,353 Hohne, W. E., 99,142 Hol, R.,205,206,219,273 Hol, W. G. J., 42,53,69, 83,99, 106, 130, 134, 142, 164, 174, 176, 177, 196, 306,352 Holbrook, S. R.,292,352 Holden, H. M.,330,352,355 Hollenberg, S. M., 342-343,352 Holm, R., 208, 220, 261,273,274,279 Holm, R. H., 42, 70, 215,256,275,276 Holmes, M. A., 45, 57, 70, 100, 105, 142, 297,310,322,329,347,352 Holmquist, B., 324,326,329,350 Homrna, K., 42, 75 Homquist, B., 326,351 Honig, B., 171,195 Honzatko, R. B., 217,275, 334, 336,352 Hood, L., 338,354 Hoover, T., 260,261,275 Hopkins, P. B., 344,354 Horsburgh, C., 202,237,273 Horvath, S., 338,354 Horvath, S. J., 338,354 Hossain, M. B., 38, 70, 75 Houk, K. N., 288,353 Houseman, A., 265,279 Howard, A,, 44, 73 Howard, A. J., 306,351 Howard, J., 221,223,254,274 Howard, J. B., 218,219, 221, 222, 223, 238,257, 258, 260, 263, 265,272, 274,275,276,277,278,279 Howell, P. L., 202,279 Howell, S. F., 108, 144 Hu, S., 147,193 Hubbard, R. E., 25,67, 110, 111, 112, 120-121, 137,141 Huber, F., 183, 187,194 Huber, M., 178,193 Huber, R.,53,69, 156, 179, 183,193, 194,195,202,235,274,340,354 Huber, R.E., 303,352 Hughes, M. N., 2, 3, 7, 27,60, 70 Huheey, J. E., 11, 70 Huhta, D. W., 17, 36, 75, 297, 301, 321, 355 Huitema, F., 164, 196 Hulse, C. L., 185, 193 Hummelink, T., 17,21, 32,67

365

AUTHOR INDEX

Hummelink-Peters, B. G., 17, 21, 32, 67 Hunt, L. T., 166,194,251,275 Hurley, T. J., 50,68 Husain, M., 164, 165,194, 195 Huynh, B., 208,277 Huynh, B.-H., 205, 229, 239,259, 261, 262,272,275,276 Hyde, E. I., 343,352

I Ibers, J. A., 256,275 Imperial, J., 260, 261,275 Ioanvidis, I., 260,275 Ippolito, J. A., 288, 300,309, 318,352 Irving, H., 26-27, 71 Islam, N., 30, 73 Islam, S. A., 301,353 Ito, N., 171,194 Iverson, B. L., 345, 347, 348,352,354 Iverson, S. A., 345,348,352 Iwasaki, H., 185, 194

J Jack, A., 292,352 Jacobsen, B. L., 83,85, 100, 117, 134, I44 Jacobsen, N. E., 330,352 Jaffer, S., 328,353 Jahn, H. A., 20, 71 Jakob, W., 187-188,192 Jalal, M. A. F., 38, 70, 75 James, M. N. G., 50,52, 53, 70, 74, 78, 82, 83, 87, 88, 92, 98, 107, 109, 110, 111, 117, 125, 127, 135,142, 143, 144 Janick, P. A., 268,273 Janin, J., 50, 73 Jansonium, J. N., 134,142 Jarup, 49, 71 Jaurez-Garcia, C., 218, 245, 247,276 Jenkins, J., 50, 75 Jensen, G. M., 217,255,276 Jensen, J. H.;112,140, 203, 219,222, 223,254,276 Jensen, K. A., 2.71 Jensen, L., 242, 243,278 Jensen, L. H., 39,42,44,66, 70, 74, 75, 148,192, 202, 203, 208, 215, 219,

240, 253, 254,272,273,278,279, 285,300,309,310,349 Jensen, V. J., 53,67, 113, 141 Jhoti, H., 202, 237,273 Jin, H., 261,279 Job, R., 42, 68 Johansson, C., 133,143 Johnson, C., 242,277 Johnson, C. K., 29, 71 Johnson, L. N., 38,67, 224,273 Johnson, M., 252,277 Johnson, M. K., 221,275 Johnston, M., 341,352 Jolles, J., 172, 194 Jolles, P., 172, 194 Jones, T. A., 53, 71, 224,273, 312,314, 318,321,351 Jongejan, J. A., 164, 171, I96 Jonsson, B.-H., 312, 321,351,355 Jorgensen, 2, 71

K Kabsch, W., 109,142 KBgi, J. H. R., 56, 74, 333, 334, 336,350, 351,352,354,355 Kaiser, E. T., 327, 329,352 Kakudo, M., 42,69,75,203,274 Kakutani, T., 146, 185,194 Kalb (Gilboa), A. J., 108, 141, 143 Kalb, A. J., 53, 71, 85, 108, 142, 144 Kalinichencko, L. P., 106, 143 Kalk, K. H., 53,69, 106, 141, 142, 144, 164, 174, 176,196 Kannan, K. K., 49,71,321,333,352 Kanyo, Z. F., 292, 295, 330,349 Kaplan, A. P., 330, 33 1,352 Kaptein, R., 343,352 Karin, M., 334,352 Karipedes, A., 82,142 Karlin, K. D., 148, 194 Karlson, B. G., 156,194 Karplus, M., 43, 70 Kashem, M. A., 185,194 Kato, I., 340,354 Katsube, Y., 42, 69, 75, 150, 193, 203, 251,254,274,279 Kauffman, G. B., 2, 71 Kaufman, E., 229,275

366

AUTHOR INDEX

Kaufman, S., 229,275 Kauzlarich, S., 260,272 Kay, L. M., 53, 71 Kearney, E., 252,277 Keck, R., 146,196 Keen, J. N., 171,194 Kellenbach, E., 343,352 Kendrew, J. C., 42, 71 Kennard, O., 17,21,32, 39,67, 78,81, 141, 210,273, 281,349,350 Kennedy, M. C., 222,223,262,263,264, 265,273,275,276,278,279 Kennelly, P. J., 161, 194 Kenney, W. C., 266,278 Kent, H., 260,275 Kent, T., 208,229, 231, 262,263, 265, 269,270,274,275,276,277,279 Kent, T. A., 205,208,229,268,272,273, 276 Kester, W. R., 57, 72, 322,353 Keyser, P. K., 229,276 Keyt, B., 146, 196 Khalifah, R. G., 312,353 Khan, M. A., 286,352 Killoran, M., 30, 73 Kim, B., 340,341,355 Kim, E. E., 290,353 Kim, H., 330,331,353 Kim, S.-H., 292,352 Kindon, N., 261,269,276 Kissinger, C. R., 203,219, 222, 223, 254, 276 Kitajima, N., 176, 194 Kitaura, K., 299, 308, 309,353 Kivirikko, K., 229,277 Klee, C. B., 84,87, 142 Klevit, R. E., 338,354 Klippenstein, G. L., 44, 75 Klopman, G., 6, 71 Klotz, I., 239, 240, 242,276, 277 Klug, A., 50, 71, 292,337,351,352 Knight, E., Jr., 256,273 Knowles, P. F., 146, 171, 194 Koekoek, R., 134,142 Koetzle, T. F., 39, 67, 78, 81, 141, 210, 273, 281,350 Kojima, Y., 333,352 Kollman, P. A., 168, 170, 171, 193 Komiya, H., 202,235,272 Komoroski, R., 39,69

Kopka, M. L., 38,68 Korner, H., 187-188,192 Kosman, D. J., 171,194 Kossiakoff, A. A., 53, 72 Kowal, A,, 221,275 Krajewski, T., 184,193 Kraut, J., 42,43,44,69, 73, 203, 208, 273,309,355 Krebs, B., 49,67 Krebs, J., 321,353 Kredich, N. M., 252, 268,269,277 Kretsinger, R. H., 52,53, 71, 72, 78, 83, 88,91, 108, 134,142, 143, 144, 216, 277 Kroneck, D. M. H., 187-188,192 Kroneck, P. M. H., 146, 185, 188, 190, 195,197 Krueger, R. J., 268,278 Kruger, G. J., 20, 71 Kuila, D., 270,276 Kuklinska, E., 177, 193 Kumar, S., 190, 193 Kumar, V. D., 53, 71 Kuo, L. C., 328,353 Kurtz, D., 220, 240, 242,276, 279 Kurtz, P., 244,277 Kusunoki, M., 203,279 Kutzelnigg, W., 295, 330,353,355 Kuwahara, J., 229,279 Kwon, B., 177,195 Kwon, B. S., 177,193 Kylsten, P. M., 314, 318,351

L Ladenstein, R., 179, 194 Ladner, J. E., 217,275,292,334,336,352 Ladner, R. C., 217,275, 334,336,352 Laidig, K. E., 288,355 Lamy, J., 172, 174,192, 194 Lamy, J.-N., 174, 192 Lang, G., 245,247,273 Laskowski, E. J., 215,276 Laskowski, M., 340,354 Lasters, I., 50, 73 Lattaire, E., 270,273 Lattman, E. E., 52, 72, 83, 100, 105, 129, 130,143 Lawn, R. M., 146,196 Leavis, P. C., 87,143

.4UTHOR INDEX

Lebioda, L., 50, 71 Lebo, R.,342-343,352 Lee, A. W.-M., 43,70 Lee, H.-H., 329,352 Lee, L., 53, 71 Lee, M. S., 338,339,353 Lee, Y. J., 43, 73 Lefevre, I., 91, 142 LeGall, J., 42,69, 146, 160, 185, 186, 190, 193, 194, 196, 203,204, 208, 219, 221, 222, 223, 239, 252, 254, 255, 272,274,275,276,277,279, 303,351 LeGall, L., 42, 66 Legg, M. J., 52,68, 105,141 Lehnert, R.,204,276 Leohr, T., 247,248,278 Leone, C., 65, 76 Lerch, K., 178,193,194, 333,353 Lerner, A. B., 177,193 Lerner, R. A., 345,347,348,352,354 Leszcynski,J. F., 97, 109,143 Levine, B. A., 91, 141, 143 Levitski, A., 85, 108,142, 144 Levy, M., 260,277 Lewandoski, D. A., 42,74 Lewin, R.,170,194 Lewinski, K., 50, 71 Lewis, C. A., 326,353 Lewis, G. N., 4, 71 Lewis, M., 45,48, 73, 100, 106, 110, 121, 137,140,143,322,329,354 Li, X.-Y., 13, 70 Li, Y., 288,353 Liang, J., 261,276 Liang, J.-Y, 314, 315, 316, 321,353 Liang, Z.-W., 321,351 Liao, D., 14, 71 Liao, Y.-D., 337,354 Liddington, R.C., 42, 73 Lidqvist, O., 78, 143 Lienhard, G. E., 326,353 Liljas, A., 49, 71, 312,314, 318, 321,351 Liljas, L., 53, 71 Lim, L., 266, 267,273,277 Lim, L. W., 203, 21 1, 252, 266,276 Lim, L. W. G., 164,194 Lin, S.-L., 53, 71 Lindahl, P., 260,272 Lindahl, P. A., 208,259,276 Lindahl, T., 292, ?51

367

Linder, M. C., 184,195 Lindley, P. F., 202, 237,273 Lindskog, S., 311, 312, 314,315, 317, 321, 333,351,35?, 354,355 Linse, S., 133, 143 Lippard, S., 240,276 Lippard, S. J.. 56,65, 75 Lipscomb, J., 205, 206, 208, 229, 231, 233, 234, 239, 248, 249,272,273, 274,275,276,277,279 Lipscomb, J. D., 202, 230,277 Lipscomb, W. N.,45,48, 73, 110, 121, 137,143, 217,275, 286, 290, 297, 308,310, 314, 315, 316, 318, 321, 322, 324, 325, 326, 327, 328, 329, 330,331, 332,333,334, 336,350, 351,352,353,354 Lipson, H., 20,67 Listowsky, I., 237,272 Liu, L.-B., 13, 70 Liu, M.-C., 146, 160,194 Liu, M.-Y., 146, 160, 185, 186, 190,193, 194,196 Liu, S.-M., 252,280 Liu, T. N., 229,279 Ljones, T., 146,194 Ljungdahl, L., 252,280 LoBrutto, R., 270,279 Loehr, T., 242,244,245,277,278 Loehr,T. M., 157, 165, 166,192,193, I95 Loewenstein, W. R., 78, 143 Loll, P. J., 52, 72, 83, 100, 105, 129, 130, 143 Lornmen, A., 164,195 Longworth, J. W., 155,195 Lontie, R., 147, 173, 178,194, 196 Lovenberg, W., 42, 70, 204,252,276 Lovenberg, W. L., 42, 72 Lovgren, S., 49, 71, 321, 333,352 Lowery, R. G., 258,276,277 Lowry, M., 164,196 Luchinat, C., 31,350 Ludden, P., 252,256,260,261, 269,275, 276,278 Ludden, P. W., 258,276,277,278 Ludwig, M., 202,278 Ludwig, M. L., 45, 74, 202, 230,273,276, 322,353 Luisi, B., 42, 73

368

AUTHOR INDEX

Lundberg, L. G., 156,192,194,195 Lusty, C., 252,276 Lyerla, J. R., 298,354 Lynch, J., 218, 245, 247,276

MacDonald, J., 208,279 Machinist, J., 252,276 MacNeela, J., 331,349 Madden, M., 260,261,269,275,276 Magliozzo, R. S., 173,195 Magneson, J., 260,276 Magnusson, S., 78, 143 Makaroff, C., 252,256,275 Makinen, M. W., 328,353 Maler, B. A., 343,352 Maley, F., 230,277 Maley, G. F., 230,277 Malkin, R., 145, 194 Malmstrom, B. G., 145, 156,194,195 Manfre, F., 33, 50, 68 Mann, S., 77,143 Mantsch, H. H., 155, 196 Marchesini, A., 179,194 Margoshes, M., 334,353 Marlowe, C. K., 330, 331,350 Maroney, M., 245,278 Maroney, M. J., 242,276 Marsden, B. J., 131,143 Marshall, L., 30, 73 Martin, A. E., 217,255,276 Martin, R.G., 82, 111, 133, 143 Martin, S. R., 133, 143 Martinelli, R. A., 326, 329,350,351 Martinkus, K., 161,194 Massey, V., 252,276 Masuko, M., 185,194 Mateescu, M. A., 184-185.192 Mathews, F. S., 164,194, 266,267,273, 277 Matsubara, H., 42,69, 75, 150, 193, 203, 25 1,254,274,279 Matsubata, H., 221,280 Matsumoto, S., 42, 69, 203,274 Matthews, B. W., 45,57, 70, 72, 100, 105, 142, 286,290, 297,310,322, 324, 327, 328,329,330, 332,333,347, 352,353,355 Matthews, F. S., 42, 73

Mattyssens, G., 50, 73 Mauk, A. G., 42,72 Mauro, J. M., 309,355 May, H., 261,262,278 McCallum, J., 242, 243,278 McCormisk, J., 243,278 McCracken, J., 252, 257, 258,274,277 McDonnell, P. J., 56,65, 75 McGandy, E. L., 42, 73 McGregor, M. J., 301,353 McIntire, W., 266,278 McKenna, M.-C., 252,256,258,276,278 McLachlan, A. D., 337,353 McLean, P., 260,261,275,277 McLean, P. A., 259,277 McManus, J. D., 146, 149, 164,196 McMillin, D. R., 155,193 McPhalen, C. A., 92,98,143 McPherson, M. J., 171,194 McRee, D. E., 42, 72,268,269,277, 334, 354 Means, A. R., 53,67, 87,140,143 Meinhardt, S., 270,275 Melik-Adamyan, W. R., 43, 75 Melis, K. A., 56, 72 Merkle, D. L., 284,350 Merkle, H., 263, 265,275,276 Merritt, E. A., 161, 164, 193 Merz, K. M., 302,310,315,316,321,353 Messerschmidt, A., 156, 179, 183, 187, 194,195 Metzger, A. L., 230,276 Meyer, E., 92,143 Meyer, E. F., 32,39,67, 73, 289,354 Meyer, E. F., Jr., 78,81,141, 210,273, 281,350 Meyer, J., 208,277 Meyer, S. A,, 91,144 Michel, H., 202,235,274 Michelson, A. M., 170, 196 Miki, K., 202, 235,274 Mildvan, A. S., 5, 35, 39, 50,52,69, 72 Miller, B. E., 252, 268, 269,277 Miller, J., 337,353 Miller, K. I., 174, 195 Mims, W., 270,273 Mims, W. B., 257,258,277 Mizushima, M., 203,279 Mock, W. L., 327,353 Moellmann, G., 177, 193

369

AUTHOR INDEX

Moews, P. C., 53, 72, 216,277 Moffat, K., 53, 74, 88, 144 Mohri, T., 91, 141 Moldenhauer, B., 257,275 Monaco, H. L., 217,275, 334,336,352 Mondovi, B., 146, 184-185,192,194 Monnanni, R., 314,350 Montfort, W. R., 230,277 Monti, C. T., 24, 72 Monzingo, A. F., 290,322,324, 327,330, 352,353 Moog, R. S., 185,193 Moore,J. M., 159, I94 Moratal, J. M., 314,350 Morgan, T., 261,279 Morgan, T. V., 257,258,277 Morningstar,J., 221, 252,275,277 Moro-oka, Y., 176,194 Morozova, L. A,, 106,143 Mortenson, L., 220, 260,274,279 Mortenson, L. E., 257,258, 259,277, 280 Moshkov, K. A., 184,195 Mothenvell, W. D. S., 17,21, 32, 67 Moulis, J.-M., 208,277 Moult,J., 87, 108, 133,142, 143 Moura, I., 204, 208, 22 1,239, 255,275, 276,277,279,303,351 Moura, J., 204,221,239, 252,255,275, 276,277,279 Moura,J. J., 208,277 Moura, J. J. G., 303,351 Muirhead, H., 42, 73,322,353 Muller, G., 177,195 Munck, E., 204,205,208,218,229,231, 245, 247, 248, 249,252, 255, 259, 260, 261,262,263, 265, 268,269, 270,272,273,274,275,276,277, 278,279,280 Munck,J., 255,277 Munro, I., 156,195 Murakami, K., 85,143 Murata, M., 39,42,70, 146, 148, 158, 161, 164,192, 193 Murray, T., 250,277 Murray-Rust, P., 17, 24, 32, 72, 73, 289, 354 Murrell, S. A., 258,277,278 Musci, G., 106,143, 185,192 Mud, D., 98, 141

Musselman, R., 243,278 Mydin, A., 202, 237,273

N Nagahara, Y.,150,193, 203, 251,254, 274 Nagasawa, T., 229,279 Nair, S. K., 315,317,349 Nakagawa, S., 299,308,309,353 Nakahara, A., 185,194 Nakamura, A., 303,355 Nar, H., 156,195 Nassimbeni, L. R., 17,45,67 Neidhart, D. J., 98, 143 Neifakh, S. A., 184, 195 Nelson, M., 260, 261,272,277,279 Nemeth, D., 30, 73 Neuhaus, D., 334,336,343,351,354,355 Newton, D. L., 84,142 Newton, W., 260,261,262,274,278 Ni, W. C., 84,142 Nielson, K. B., 333,355 Nishikawa, Y., 203,279 Nocek, J., 242,244,276,277 Nockolds, C. E., 52, 71 Noodleman, L., 208,277 Nord, F. F., 97,141 Nordling, M., 156,192 Nordlund, P., 50, 72, 202,210, 221, 245, 246,247,277 Nordstrom, B., 216,217,274 Norris, G. E., 39,41,42, 72, 74, 153, 157, 192,195, 202,210,237,238,272 Norris, V. A., 39,42, 70, 146, 148, 158, 192,193 Nuttleman, P., 239,277 Nyman, P.-O., 183,192

0 O'Brian, D. P., 146, 196 O'Connell, E., 264,278 O'Connor, B. H., 29,72 Oewerling, M. C., 156, 157, 164,193 Ogura, K., 50, 75 Ohkawa,J., 179,195 Ohlendorf, D. H., 202,230,277 Ohlsson, I., 216, 217,274 Ohnishi, T., 270,275,279

370

AUTHOR INDEX

Okada, N., 179,195 Okamura, M., 239,242,277 Okamura, M. Y., 229,235,236,274,277 Olafson, R.W., 97,143, 333,353 Olaitan, S. A., 98, 143 Oliver, M., 221,275 Ollis, D. L., 290,353 Olson, A. J., 53, 72 Olsson, G., 78,143 Omichinski, J. G., 50, 72, 340,353 Ong, E. S., 342-343,352 Oppermann, H., 146,196 Oren, D. A., 326,327,354 Orena, S. J., 184, 195 Orgel, L. E., 11, 72 Orme-Johnson, M. R.,268,278 Orme-Johnson, W., 260, 261,272,275, 277,278,279 Orme-Johnson, W. H., 208,252, 256,257, 258,259,268,275,276,277,278,280 Oro, A., 342-343,352 Ortel, T. L., 178,196 Orville, A., 229, 231, 234,273, 276,277, 279 Oskarsson, A., 82, 140, 141 Ostrowski, J., 252, 268, 269,277 Ostsaka, S., 233,273 Otting, F., 170, 196 Otvos, J. D., 334,353

P Padlan, E. A., 53, 74 Palmer, G., 205,223,270,277,279 Palmer, S., 245,278 Pan, T., 302,341-342,342,353 Pankalainen, M., 229,277 Pantoliano, M. W.. 56,65, 75, 83,98, 134, 143,306,351,353 Papaefthymiou, G. C., 208,215,273,276 Papaefthymiou, V., 208,255, 259,276, 277 Papamokos, E., 98,141, 340,354 Papiz, M. Z., 108,141 Park, C. H., 78,144 Park, J., 221, 223, 254,274 Parker, M. W., 170,192 Phrraga, G., 338,354 Parris, K., 30, 73 Parthasarathy, R.,20, 73, 302,303,354

Pascher, T., 156,195 Patil, D., 248,279 Pattridge, K. A., 45, 74, 202, 230,276, 2 78 Pauling, L., 8, 22,23, 72, 73, 283, 326, 354 Paustian, T., 260,276 Payan, F., 42,69, 203,274 Payne, M. D., 20,69 Payne, W. J., 146, 160, 185, 186, 190,193, 194,196 Pearce, L., 231, 242,276,279 Pearson, R.G., 5, 73, 282, 283,354 Peat, I. R.,298,354 Pechere, J.-F., 91, 142 Pecht, I., 156,195 Peck, H. D., 303,351 Peisach, J., 173,195, 252,257,258,274, 277 Penfield, K. W., 148, 153, 173, 175,196 Penner-Hahn, J., 270,279 Pepe, G., 42,69, 203,274 Permyakov, E. A., 85, 106,143 Perry, K. M., 230,277 Perutz, M. F., 42, 73, 307,354 Petef, M., 49, 71, 321, 333,352 Petering, D., 223,277 Petersen, S. B., 53,67, 113, 141 Petersen, T., 78, 143 Peterson, M. R.,30, 73, 288,354 Peterson, L., 244,245, 247,277 Petratos, K., 161, 164, 186, 192, 195, 196 Petrich, J. W., 155,195 Petruzzeli, R.,179, 194 Petsko, G. A., 50,69, 98,143, 202,279, 284,350 Phillips, D. C., 100, 106, 140 Phillips, M. A., 324, 328,354 Phillips, S. E. V., 171, 194 Phillips, W. D., 256,273,275 Phizackerley, R. P., 161, 164, 193 Pieterson, W. A., 85, 141 Pietrobon, D., 78, 143 Pitts, J. E., 296,350,354 Pitts, 0. M., 146, 195 Plank, D., 263,277 Plank, D. W., 222,223,263,265,278 Plonka, A., 184,193 Plowman, J. E., 157, 192 Pocker, Y., 21 1,319,354

AUTHOR INDEX

Pomerantz, S. H., 177,193 Pope, M. P., 258,278 Posnjak, E., 8, 76 Postma, J. P. M., 338,351 Potter, J. D., 84, 87, 144 Potter, J. J., 159,192 Poulos, T. L., 44, 73, 83, 98, 134, 143, 306,353 Powers, L., 175,197 Powers, T. B., 202,278 Powls, R., 159, 194 Pozzan, T., 78,143 Preaux,G., 146, 172, 173, 174,195 Preston, R. M., 24, 72 Prewitt, C. T., 8, 10, 74 Prickril, B., 239, 276 Prickril, B. C., 303, 351 Priest, D. G., 146, 195 Ptashne, M., 340,342,351 Puget, K., 170,196 Pujar, B. G., 229,276 Putnam, F. W., 178,196 Pyzalska, D., 87, 143

Que, L., 204,206, 207, 218, 231,233, 240, 241,242,243,245,247,274, 276,278,279 Quiocho, F. A., 45, 73, 83,85, 100, 107, 109, 117, 125, 134, 137,144,322,353

Rabinowitz, J., 256,278 Radahakrishnan, R., 92,143 Raghunathan, S., 99, 135, 141 Ramakrishna, B. L., 146, 149, 164,196 Ramshaw, J. A. M., 146, 148,192 Randall, Sir John, F. R. S., 49,67 Rao, S. T., 87, 143, 144 Rasrnussen, H., 1, 73 Rawlings, J., 259,260,278,280 Rea, T., 161,194 Read, R. J., 92,143 Rebek, J., Jr., 30, 73 Reed, A. J., 82,142 Reed, G. H., 65,68 Reed, M. D., 286,354 Reedijk, J., 155, 174, 176,193,196

37 1

Reeke, G. N., 322,353 Reeke, G. N., Jr., 108, I41 Reem, R., 243,278 Rees, D. C., 45,48, 73, 110, 121, 137,143, 202, 203, 206, 215, 229, 235, 258, 272,274,275,278, 308, 310, 322, 324,327,329,354 Regan, L., 50, 73, 345,354 Reichard, P., 239,244, 245,247, 248,273, 274,277,278,279 Reinach, F. C., 87, 142 Reinhammer, B., 146,195 Ren, X., 321,351 Renetseder, R., 106, 142 Rey, F., 50, 75 Reynhardt, E.C., 20,71 Reynolds, J. G., 215,276 Reynolds, W. F., 298,354 Rhodes, D., 50, 71, 292, 343,352,354 Ribbons, P. W., 229,276 Rice, D. W., 202,272 Richards, F. M., 215,278 Richardson, D., 243,278 Richardson, D. C., 42,55, 72, 75, 166, 168, 170, 171,193, 195, 196, 268, 269,277,298,309,355 Richardson, J. S., 42,55, 72, 75, 110, 130, 143, 166, 168, 170, 171,193, 195, 196, 209,268,269 277,278, 298,309, 355 Richman, P. G., 87, 142 Riepe, M. E., 314,354 Rieske, J., 269,278 Riester, J., 146, 188, 190, 195 Rifkind, J. M., 57,68 Rimmer, B., 49,66 Ringe, D., 50,69 Ringe, D. M., 202,279 Riordan, J. F., 324, 326,354, 355 Robb, D. A,, 146, 177,195 Robbins, A. H., 56, 69, 203, 263,278, 334,351,354 Roberts, G., 260,276 Roberts, R., 239,273,277 Roberts, V. A., 345, 347, 348,352,354, 355 Robertson, A., 261,275 Robson, R., 258,278 Roche, R. S., 85, 105,144 Rochow, E. G., 4,67

372

AUTHOR INDEX

Rodgers, J. R., 17, 21,32, 39,67, 78, 81, 141,210,273,281,350 Rodriguez, H., 146, 196 Roe, A., 245,261,274,278 Roelens, S., 314,350 Rogers, S. J., 159, 193 Rollence, M. L., 83, 98, 134, 143, 306,353 Rose, B., 78, 143 Rose, G. D., 97, 109,143 Rose, I., 264,278 Rosenfeld, M. G., 342-343,352 Rosenfield, R. E., 20, 73, 302, 303,354 Rosenfield, R. E., Jr., 32, 73, 289,354 Rossi, A., 179, 194 Rossman, G., 242,274 Rossmann, M. G., 43,53, 74, 75, 259,280, 285,307,322,350 Rotblat, F., 146, 196 Rotilio, G., 146,193 Roychowdhury, P., 87,144 Royer, W. E., 174,195 Ruan, F., 344,354 Rubin, B. H., 50, 68 Rueger, D. C., 252,268,269,277 Rueger, M. J., 268,278 Rumball, S., 229,237,273 Rumball, S. V., 202, 210,237, 238,272 Ruppert S., 177,195 Rusconi, S., 343,354 Russell, A. J., 304, 305, 306,354,355 Rutter, W. J., 324, 327,327 351, 328,352, 354 Rydel, T. J., 78, 144 Ryden, L., 146, 166, 178, 183, 184, 191, 195

Sack,J . S., 53,67, 87, 140 Sage, J., 244,277 Sahlin, M., 247,248,278 Said, F. F., 286,352 Saike, T., 252,280 St. John, T. P., 340,355 Saitsev, V. N., 184, 195 Sakaguchi, K., 50, 72 Sakauchi, K., 340,353 Sakurai, T., 185,194 Salerno, J. C., 268,273

Sali, D., 307,354 Salowe, S., 245,278 Salvato, B., 173,195 Sander, C., 109,142,337,350 Sander, M. E., 328,354 Sanders-Loehr, J., 44, 74, 165, 166,193, 195, 240, 242, 243, 244,247,248, 277,278,280 Sands, R. H., 267,279 Sandstrom, J., 321,351 Sanger, W., 84,92,99, 135,141 Santos, H., 221,275 Sarkanen, S., 21 1,354 Sarra, R., 202,237,273 Sasaki, K., 303,355 Satyshur, K. A., 87, 143 Scarrow, R., 245,278 Schaffer, A., 333,352 Schaffner, W., 343,354 Scheidt, W. R., 37,43, 73 Schena, M., 343,354 Schepartz, A., 322, 324, 327,354 Schmidbaur, H., 35, 74 Schneider, G., 49, 74 Scholes, C., 252,274 Schoonover, J., 270,276 Schredder, J., 242,274 Schultze, P., 56, 74, 334,350, 354 Schutz, G., 177,195 Schwabe,J. W. R., 343,354 Schwager, P., 82,92,141 Schwarzenbach, G., 5, 74 Schweiger, G., 252,278 Scott, D., 261,262,278 Scott, R., 219, 262,273 Scott, R. A., 42, 72, 190,195, 303,351 Scrutton, M. C., 39,69 Seel, F., 204,276 Segui, P., 342-343,352 Sekharudu, Y. C., 131,143 Serpersu, E. H., 35,52, 72 Severne, Y., 343,354 Shaanan, B., 42, 73 Shah, V., 260,261,269,276,278 Shah, V. K., 259,260,261,275,278,280 Shamala, N., 203,211,252,266,276 Shang, Z., 337,354 Shannon, R. D., 8, 10, 24,68, 74 Sharma, K. D., 165,195 Sharma, S. K., 286,354

AUTHOR INDEX

Sharp, K., 171,195 Shavlovski, M. M., 184, 195 Sheet, S., 49,66 Shelling, J. G., 84, 143 Shepard, W., 153,195 Shepard, W. E. B., 42,74 Sheriff, S., 38,44, 53, 74, 202,225, 240, 2 78 Shiemke, A., 242,243,244,277,278,280 Shih, D. T.-B., 307,354 Shimanouchi, T., 39,67, 78, 81,141, 210, 273,281,350 Shimura, F., 91, 144 Shinmyo, A., 179,195 Shoham, G., 326,327,329,354 Shoham, M., 108,143,203,279 Siecker[sic], L. C., 44, 74 Siegel, L. M., 42, 72, 252, 268,269,273, 277,278 Sieker, L., 203, 242, 243,274,278 Sieker, L. C., 39, 42, 66, 70, 75, 148, 186, 192, 196, 202,203, 208, 219, 222, 223, 240,253,254,272,273,276, 278,279 Silva, A. M.,53, 74 Silverman, D. N., 311, 314, 317, 321, 333, 354,355 Silverton, E. W., 166,195 Simons, K., 229,277 Simptin, D., 270,279 Singer, T., 252,276 Singh, T. P., 267,279 Sivaraja, M., 270,275 Sizaret, P. Y., 172, 194 Sjoberg, B.-M., 50, 72, 202, 210,221,244, 245,246,247,248,277,278 Sjolin, L., 78, 143 Skelton, N. J., 91, 144 Skotland, T., 146,194 Skowron, A,, 4,68 Skrzypczak-Jankun, E., 78, I44 Stnillie, L. B., 97, I43 Smith, B., 260,261,274,275,277 Smith, B. E., 259,275 Smith, E. L., 98,143 Smith, J. L., 38,44, 74,202,225,240, 278 Smith, S. O., 27, 74 Smulevich, G., 309,355 Snyder, E. E., 65,74, 134,144

373

Soderberg, B.-O., 216,217,274 Soderlund, G., 216, 217,274 Soeter, N. M., 42, 69 Soloman, E., 243,278 Solomon, E. I., 148, 153, 164, 168, 173, 175,177, 183,192,195,196,197 Soman, K. V., 338,339,353 Sorensen, 0. W., 334,351 Sorenson, M., 87,142 Sottrup-Jensen, L., 78, 143 South, T. L., 340, 341,355 Spartalian, K., 208,273 Spira, D. J., 183,192 Spira-Solomon, D. J., 183,196 Spiro, T., 204,278 Spiro, T. G., 175,197,309,355 Srai, S. K., 184, 196 Stabie, J., 245,278 S t a h g s , W. C., 24,45, 72, 74, 202, 230, 273,276,278 Steenkamp, D. J., 203, 21 1, 252, 266,276, 2 78 Steffens, G. J., 170, 196 Stein, E. A., 84,144 Steiner, H., 312,355 Steinman, H. M., 170, 196, 202,273 Steitz, T. A., 290, 322,353 Stempien, M. A., 168, 169, 170,193 Stenkamp, R., 242, 243,278 Stenkamp, R. E., 39, 44, 66, 74, 148, 192, 202,240,278 Stephens, P. J., 148,196, 217, 255, 258, 276 Stern, E., 242, 243,280 Stern, E. A., 53, 71 Sternberg, M. J. E., 301,353 Stevens, C., 171,194 Stevens, P., 243,252, 256,278 Stevenson, R.C., 267,279 Stiefel, E., 261,279 Stoddard, B. L., 202,279 Stout, C. D., 56, 72, 203, 217, 219, 254, 255,263,276,278,279,334,351,354 Stout, D. C., 56, 69 Stout, G. H., 219, 254,279 Strandberg, B., 49, 71 Strandberg, B. E., 42, 71 Strasburg, G., 87,144 Strassburger, W., 170, I96 Strong, R. K., 45, 74

374

AUTHOR INDEX

Strothkamp, K. G., 65, 76 Stroud, R. M., 53, 71, 230,277 Stryer, L., 156,196 Strynadka, N. C. J., 52, 74, 78, 87, 107, 109,111, 117,125, 127,144 Stuart, D. L., 100, 106,140 Stubbe, J., 239, 244,279 Sturtevant, J. M., 285,352 Subramanian, E., 53, 74 Subramanian, V., 229,279 Sugawara, T., 303,355 Sugimura, Y., 160, 192 Sugiura, Y., 229,279 Suguna, K., 53,74 Suh, J., 328,355 Summers, M. F., 340, 341,355 Sumner, J. B., 108,144 Sundaralingam, M., 87, 131, 143, 144 Surerus, K., 204,229, 248,249,252, 255, 273,274,279 Surewicz, W. K., 155,196 Sussman, J. L., 108, 143, 203,279, 292, 352 Suzuli, S., 185, 194 Svensson, C., 82,140 Swain, A. L., 83,88, 134,144 Swanson, S. M., 32, 73, 289,354 Swift, H., 53, 67, 113, 141 Switzer, R., 252, 256,275 Sykes, B. D., 84, 131,143 Szabo, A. G., 155,196 Szebenyi, D. M. E., 53, 74, 88, I44

T Tahon, J. P., 173,196 Tainer, J. A., 55, 75, 168, 169, 170, 171, 193, 196, 298, 309, 345, 347, 348, 352,354,355 Takahashi, N., 178,196 Takano, M., 179,195 Tamura, A., 177,193 Tan, R.-Y., 87, 133,142 Tanaka, N., 42,69,203,274,279 Tanake, N., 42, 75 Tao, T., 87, 133,142 Tapia, O., 216, 217,274 Tarien, E., 57,68 Tarr, G., 269,270,274

Tash, J. S., 87,143 Tasumi, M., 39,67, 78, 81,141, 210,273, 281,350 Taylor, A., 331,332,350 Taylor, A. N., 91, 144 Taylor, R., 28 1,349 Taylor, W. E., 338,354 Teler, E., 20, 71 Teller, D. C., 186, 193 Telser, J., 270,279 Tennent, D. L., 259,279 Teo, B.-K., 260,272 Tesler, J., 263, 264,275 Thanki, N., 120, 137,144,295,355 Thelander, L., 239, 244, 245, 247,273, 279 Thim, L., 53,67, 113,141 Thomann, H., 164,196,261,279 Thomas, E., 184, 196 Thomas, K. A., 166,195 Thomas, P. G., 304,354,355 Thomason, P., 91,142 Thompson, E. B., 342-343,352 Thompson, G. A., 331,355 Thomson, A., 219,262,273 Thornley, J. H., 207,275 Thornton, J. M., 120, 137,144,295,355 Thulin, E., 133, 143 Thunnissen, M. M. G. M., 106, 144 Tickle, 1. J., 296,350 Tiedje, J. M., 185, I93 Timkovitch, R., 161,194 Tobari, J., 146, 160, 164,192, 196 Toparceanu, F., 184,196 Touati, D., 202,273 Traub, W., 108, 143 Trautwein, 229,273 Tritsch, D., 33, 50, 68 Trkula, M., 20,69 Tronrud, D. E., 330,352,355 Trost, J. T., 146, 149, 164,196 True, A., 204,206,207,231,240,241, 243,260,261,270,275,278,279 Tsai, A.-L., 270,279 Tsang, H.-T., 270,279 Tsernoglou, D., 161,192 Tsernoglous, D., 161, 186,195 Tsuda, S., 50, 75 Tsukihara, T., 42,69, 75, 150,193, 203, 251,254,274,279

375

AUTHOR INDEX

Tsutsui, T., 203,279 Tu, C., 321,355 Tuck, D. G., 286,352 Tuddenham, E. G. D., 146,196 Tulinsky, A., 78,144 Turley, D. C., 186,193 Turley, S., 161, 186,192, 196, 219, 254, 279 Tzagoloff, A., 270,275

Ueyama, N., 303,355 Umeyama, H., 299,308,309,353 Underwood, E. J., 145,196 Uozuni, T., 161,197 Upchurch, R., 220,279 Urdea, M. S., 327,351

V

Vickery, L., 222,274 Vickery, L. E., 146, 148,192 Viebrock, A., 190,196 Vijayan, M., 49,66 Vilegenhart, J., 229,274 Vincent, A., 338,355 Vincent, M. G., 174, 176,196 Vincent, S. H., 321,333,354 Vinckier, C., 173, 196 Virgilio, F., 78,143 Vittal, J. J., 286, 351 Voet, D., 3, 75 Voet, J. G., 3, 75 Volbeda, A., 42,69, 174, 176, 177, 196 von Beemen, J., 164,195 Voordoux, G., 85, 105,144 Vyas, M. N., 83, 85, 100, 107, 109, 117, 125, 134, 137,144 Vyas, N. K., 107, 109, 125, 137,144

W Vagin, A. A., 43, 75 Vainshtein, B. K., 43, 75 Valentine, J. S., 56, 65, 75 Vallee, B. L., 38,45,50,66, 75, 286, 324, 326,329,334,350,351,352,353,355 Van Deenen, L. L. M., 85,141 van der Helm, D., 38,70, 75 Van der Meer, R. A,, 171,196 van Duijnen, P. T., 306,352 van Holde, K. E., 174,195 Van Hoof, D., 173,196 van Rijn, J., 155,193 Van Wart, H. E., 324,355 Vanaman, T. C., 106,141 Vannghd, T., 156,195 Varnum, J., 38,68 Varnum, S. M., 340,342,351 VaSiik, M., 56, 74, 334, 336,350, 351, 354,355 Vaude Kamp, M., 156,195 Vaughn, S., 261,274 Vedani, A., 17,36,75,297,301,321,355 Vehar, G. A., 146,196 Veldink, G., 229,274 Vellieux, F. M. D., 164, 196 Venkatappa, M. P., 146, 148,192 Venters, R., 260,261,279 Vereijken, J. M., 42,69 Verheij, H. M., 106,144

Waara, I., 49, 71 Wada, K., 42,69,203,274 Wagner, G., 56, 74, 334, 336,350,351, 354,355 Wagner, G. C., 44, 73 Wakaboyashi, S., 221,280 Wakatsuki, Y., 303,355 Walker, N. P. C., 100, 106,140 Wallgren, K., 321,351 Wallmeier, H., 295, 330,355 Wan, T., 108,141 Wang, B. C., 56,69,87,144,334,354 Wang, C.-C., 178,196 Wang, J., 87, 133,142 Wang, J. H., 314,354 Wang, J. L., 108,141, 144 Wang, R., 242,274 Ward, K. B., 44,75 Warren, S. G., 217,275, 334, 336,352 Warrent, R. W., 292,352 Wasastjerna, J. A., 8, 75 Wasserman, R.H., 91,141, 144 Wassink, H., 259,275 Watanabe, H., 146, 161, 185, 192, 194 Watenpaugh, K. D., 42,66, 75, 112,140, 203,208,215,272,279, 285, 300, 309,310,349 Waters, J. M., 202, 210, 237,272

376

AUTHOR INDEX

Watley, F. R.,207,275 Watson, D. G., 17, 21, 32,67 Watson, J . L., 202,273 Watt, G., 208,279 Watt, G. D., 259,279, 285,352 Weaver, L. H., 57, 72, 330,352 Webb, J., 242,273 Webb, L. E., 42, 73 Weber, E., 340,354 Weber, P. C., 202,230,277 Weiher, J . F., 256,273,275 Weinberger, C., 342-343,352 Weiner, P. K., 168, 170, 171,193 Weininger, M. S., 259,280 Wells, J. A,, 98,144, 349,351 Werner, A., 8,75 Wernick, D. L., 325,350 Werst, M., 263,264,275,279 Weser, U., 146,196 Wesson, L., 34, 76, 124,144, 215,280 Whangbo, W.-H., 153,193 Whitlow, M., 83,98, 134,143, 306,353 Whittaker, J., 206,229,231,234,276,279 Whittaker, J. W., 171,197 Whittaker, M.M., 171,197 Wiberg, K. B., 288,355 Wieland, S., 343,354 Wilcox, D. E., 148, 153, 173, 175, 196 Wilcox, P. E., 3, 70 Wild, W. I., 146,196 Wiley, D. C., 217,275, 334, 336,352 Wilkinson, G., 283, 285,351 Williams, G. J. B., 39,67, 78, 81, 141, 210, 273, 281,350 Williams,J . C., 217, 236,279 Williams, R.,242,277 Williams, R.J. P., 2,26-27,59,61,66, 71, 75, 76, 91, 133,141, 143, 144 Williamson, M., 334,354 Willing, A., 258,279 Wilson, K. S., 83,99, 134,142, 161, 164, 195 Winge, B. C., 334,351 Winge, D. R.,56,69, 72, 333,355 Winkler, M., 175,197 Winter, M., 242,277 Wittaker, K., 221,275 Witters, R. B., 173,196 Witzel, H., 328,354 Wnuk, W., 84, 144 Wodak, S., 50, 73

Wogotter, E., 334,350 Wolak, R., 30, 73 Woldike, H. F., 53,67, 113,141 Wolfenden, R.,326, 331,349,355 Wolgel, S., 229,234,276 Wolle, D., 258,279 Wong, G. B., 220,279 Wood, J . F., 83,98, 134,143, 306,353 Wood, S. P., 296,350 Woodland, M., 239,248,279 Woodruff, W., 270,276 Woolery, G. L., 175,197 Woolley, P., 286,355 Word, B., 184,196 Worgotter, E., 56, 74, 334, 336,351,354, 355 Wright, P. E., 159,194, 338,339,353 WU,C.-W., 296,337,338-339,350,352, 354 WU,F. Y.-H., 337,338-339,352,354 WU,J.-Y., 252,268,269,277 Wiithrich, K., 56, 74, 334,336,350,351, 354,355 Wyckoff, H. W., 290,353 Wyckoff, R.W. G., 8, 76

X Xavier, A., 208,221,239, 252,275,276, 277 Xia, Y.-M., 244,277 Xuong, N. G., 290,353 Xuong, N. H., 43,69,203,211,252,266, 276, 334,354 Xuong, N.-H., 203,273

Y Yadav, K.D. S., 146, 171,194 Yagi, K., 50, 75 Yamada, H., 229,279 Yamamoto, I., 252,280 Yamamoto, K., 161,197 Yamamoto, K. R., 343,352,354 Yamane, T., 259,280 Yamashita, M., 34, 76 Yamashita, M. M., 124,144,215,280 Yamashita, S., 303,355 Yamazaki, H., 303,355 Yao, Y., 221,280 Yariv, J., 108,141, 144

AUTHOR INDEX

Yarrnolenko, V. V., 106,143 Yasuoka, N., 303,355 Yeates, T. O., 202,235,272 Yeh, L. S., 166,194 Yeh, L.-S., 251,275 Yonath, A., 108,143 Yong, G . , 65,76 Yoshida, T., 269,270,274 Yoshizaki, F., 160,192 Yost, F., 229,280 Young, E. T., 338,354 Yu, C., 221,280 Yu, L., 221,280

z Zachariasen, W. H., 8, 24, 76 Zahrobsky, R. F., 20, 76

377

Zalkin, H., 252,256,275 Zangg, W., 269,278 Zeikus, R. D., 333,355 Zell, A., 82, I44 Zeppezauer, E., 216,217,274 Zeppezauer, M., 49, 74 Zerner, B., 239,273 Zgirski, A., 184, 193 Zhang, K., 242,243,280 Zhang, Y., 53,71 Zilio, F., 173, 195 Zimmermann, R., 259,260,275,278, 280 Zot, A. S., 84,87,144 Zubieta, J., 148, 194 Zurnft, W. G., 146, 185, 187-188, 190, 192,195,196, I97

SUBJECT INDEX

A Achromobacter nitrite reductase, 185-186 Aconitase composition, 262-263 environment of iron-sulfur center, 264 Fe:S cluster substitutions, 265 iron cluster role in catalysis, 263-265 iron ligands in, 203 polypeptide fold and metal centers, 214 structure, 263 Actin:DNase I, X-ray crystal structure, 80 Alcaligenes A . denztrzfcans azurin, 153-154 apoazurin, 155 titration behavior of His-35, NMR, 157 A . faecalis nitrite reductase, 185-186 pseudoazurin gene isolation, 161 Alcohol dehydrogenase, zinc binding, 49 Alkali metals, locations of binding, 32 Alkaline earth metals, locations of binding, 32 Alpha helices macrodipoles, metal binding and, 306-307 peptides, engineering of de n o w metal-binding site, 344 Amicyanin classification, 164 crystals for, 164 function, 146 ligands, 149, 164-165 source, 146, 149 spectral characteristics, 149, 164- 165 Amino acid analyses, iron center components, 219 Amino acid sequences ceruloplasmin, 178

homology of sulfite reductase /3 subunit and nitrite reductase, 269 identification of metal ligands with, 221-222 Rieske-type Fe:S proteins, 271 Amino acid side chains, metal-binding, 3-4 a-Amylase Ca2+-binding ligands, 53, 55 X-ray crystal structure, 8 1 Androctonus australie, hemocyanin crystals, 174 Anions binding, transferrin coupling between iron and, 237 Lewis base strength, and coordination numbers, 12 zinc-anion interactions, 329-33 1 Antibodies, engineering of zinc-binding sites, 345-346 anti conformation, 30-32 Antiferromagnetic coupling, irons in binuclear clusters, 206-207 Apocarbonic anhydrase, zinc-binding, kinetics and thermodynamics, 285 Ascorbate oxidase copper binding, 179 domain 1 backbone, 180 copper site, 180 topology, 180 domain 2 backbone, 181 copper site, 181 topology, 181 domain 3 backbone, 182 copper sites in, 182 topology, 182 domains, 179 378

379

SUBJECT INDEX

function, 146 sequence alignments with laccase and ceruloplasmin, 183 source, 146 structure determination, 179 trinuclear copper site, 18 1, 183 Asparagine, hydrogen-bonding interactions, 127 Aspartate carboxylate-zinc interactions, 287-290 Aspartate residues, predominance in Ca2+-bindingsites, 113 Asx turns, in Ca*+-binding sites, 110-1 11 Asymmetry, metal ions, 20-21 Auracyanin classification, 164 ligands, 149, 164 source, 149 spectral characteristics, 149, 164 visible spectrum, 164 Atotobacter vinelandii Fe protein, 255, 257-258 ferredoxin, 203,213 Fe:S clusters, 253-254 nitrogenase iron protein, 203 Azurin backbone, 152 bond lengths, 151 coordination, 151, 153 copper site in, 152 Cu-ligand geometry, 151, 154 electron transfer, 156 folding topology, 152 function, 146 gene for (P. aeruginosu), 156 lifetimes of tryptophan fluorescence, 155- 156 ligands, 149 mutants, 156 protein folding, 151 reduced, refined structure (A. denitrijicuns), 153- 154 secondary structure, 155 self-exchange (P. aemginosa), 156-157 source, 146, 149 spectral characteristics, 149 titration behavior of His-35 ( A . denitrificans), 157 Azurin Cu(II), 39-41 stereo view and atomic designations, 40

B B . thermoprotolyticus, 203, 213, 254 Barium(II), minimum metal-oxygen distance in carboxylates, 19 Beta strands, in helix-loop-helix sites, 109 Bidentate glutamate ligands, 114-1 16 Binuclear clusters, iron centers antiferromagnetic coupling, 206-207 oxidation states, 207 Binuclear octahedral iron proteins (see also specific protein) common properties, 239 oxygen activation by, 249-25 1 table of, 239 Blood plasma, metal ions in, 2 Blue copper proteins (see also Cupredoxins) Cu(I)/Cu(II) redox system, 39-41 electron transfer in, 58-59 Bond angles, among iron-sulfur structures, 205 Bond valence C-F bonds, 24 of metal-ligand interactions, 22 model of, 24 in sodium compounds, 26

C Cadmium, Cu, Zn, Cd liganding, 56 Cadmium(II), direct bonding, 33 CalbindingK Ca2+-binding sites, 91 Asx turns, 112 characteristics, 84 continuous coordinating peptides, 108 lack of interaction with helix dipoles, 130-13 1 stereo view of site 1, 89 modulation by Ca2+,86 X-ray crystal structure, 79 Calcium carbonyl oxygen-Ca2+ interactions, 119-121 coordination number in small organic crystals, 52 in enzymatic catalysis, 86 homeostasis in eukaryotic cells, 78 modulation of protein action, 86

380

SUBJECT INDEX

preferred Ca2+-ligandtypes, ligand geometry, and distances, 82 stabilization of protein structure, 86 syn, anti and bidentate binding modes with carboxylate groups, 124 synltrans and antilcis conformations, 122 Zn, Ca liganding, 57 Calcium(I1) direct bonding, 33 minimum metal-oxygen distance in carboxylates, 19 site specificity, 65 Calcium-binding proteins, 38 (see also specific protein) Ca-binding ligands in various proteins, 55 y-carboxyglutamic acid-containing proteins, 78 EF hands, 52-53 helix-loop-helix motif, 52-53 X-ray crystal structures, 79-81 Calcium-binding sites Asx turns, 110-111, 136 Ca2+-helix dipole lack of interaction, 130-13 1 continuous coordination peptides, 108-109 coordination numbers, 112- 113 discontinuous coordinating peptides, 108-109 distribution of Ca2+ions around ligand oxygen atoms, 119 empty sites, 135-136 hydrogen bonds networks among ligand and binding loop residues, 124- 130, 137 n ton+2, 111-112 ion binding affinity-structure correlations, 131-134 ligands geometry, 115- 118 mean Ca2+-Egand distances, 113-115, 137 types, distribution, 113-115 main-chain conformations, 109- 1 12 prediction and design of, 139-140 predominance of aspartate and glutamate residues, 113 regular sites, 136-138 secondary structural elements, 109- 112

selectivity of, 134-135 semicontinuous coordinating peptides, 108- 109 structure-function relationships, 138- 139 Calcium-ligand interactions, 115 angles defining, 119 hydrogen bond networks, 124-130 mean Ca2+-ligand distances, 113-1 15 stereochemistry of, 118-124 Calmodulin Ca2+-binding ligands, 53,55 Ca2+-bindingsites, 87,90 Ca2+-ligand distance, 115 characteristics, 84 lack of interaction with helix dipoles, 130- 131 stereo view of site 3, 89-90 modulation by Ca2+,86 X-ray crystal structure, 79 Calmodulin Ca(II), stereo view and atomic designations, 60 Cambridge Structural Database, 14-15, 17-19 Carbonate dehydratase Zn(I1) stereo view and atomic designations, 49 zinc binding, 49 Carbon dioxide hydration catalysis, of carbonic anhydrase, 313 Carbon-fluorine bonds, bond valence, 24 Carbonic anhydrase I1 active site, 315-317 catalysis of CO2 hydration, 313 as catalyst, 312,332-333 discrete binding site for substrate COP, 314 distribution, 31 1 hydrogen bond network, 318 hydrophobic pocket, 315-317 proton transfers, 318-321 X-ray crystallography, 312-313 Carbonyl groups, metal binding to, 38 Carbonyl oxygen, Ca2+interactions, 119-121 Carbonyl-zinc interactions, 295-297 y-Carboxyglutamic acid-containing proteins, Ca2+-binding, 78 Carboxylate groups anti conformation, 30-32 metal binding, 30-36

SUBJECT INDEX

scatterplots, 34 sites of, 24-26 minimum metal-oxygen distances in, 19 syn conformation, 30-32 Carboxyl groups anti conformation, 30-32 scatterplots of metal ions around, 31-34 syn conformation, 30-32 Carboxypeptidase A active-site residues of, 323 anion-zinc interactions and, 329-33 1 bidentate interaction of tetrahedral intermediate, 330-33 1 characterization, 322 coordination polyhedron of zinc and, 329 inhibition of, 313 intermediate proton donors, 327 peptide hydrolysis catalyzed by, 324-326 water-promoted tetrahedral intermediate, 326-328 X-ray crystallography, 322-324 Carboxypeptidase A Zn(I1) stereo view and atomic designations, 48 zinc binding, 45 Catalase, porphyrin-liganding group, 43-44 Catalysis carbon dioxide hydration, of carbonic an hydrase, 3 13 entatic state, 66 zinc in carbonic anhydrase 11,311-321 carboxypeptidase A, 322-333 metallothionein, 333-337 transcription factor CAL4, 340-343 transcription factor IIIA, 337-340 Cations anti,syn, and direct arrangements, 30-32 coordination in inorganic structures, 24 coordination numbers, 10-1 1 radii of metal ions, 8-9 transition metal Cambridge Structural Database, 17-19 coordination number, 18 coordination polyhedra geometries, 18

38 1

electronic configurations, 10, 18 high- and low-spin, radii and electronic configurations, 10 radii, 10 Cerium(IV), direct bonding, 33 Ceruloplasmin amino acid sequence, 178 functions, 146, 178, 184 from goose serum, 184 hypothetical domain arrangement, 187, 189 nonequivalence of copper sites, 185 sequence alignments with ascorbate oxidase and laccase, 183 from sheep plasma, 184-185 source, 146 structural data, 184 Charge-charge interactions, in protein-zinc recognitions, 282-283 Chemical modification, for identification of metal ligands, 220-223 Chromatium, iron protein from, 42 Chromium(I1) coordination number, 18 electronic configuration, 10, 18 polyhedron geometry, 18 radii, 10 Chromium(II), manganese(II1) electronic configuration, 10 radii, 10 Chromium(II1) coordination number, 18 electronic configuration, 18 polyhedron geometry, 18 Circular dichroism Cu(A) site in N20reductase and cytochrome oxidase, 190 hemerythrin, 243 non-heme iron protein metal centers, 228 Clostridium pasteurianum, rubredoxin from, 42-43 Cobalt(I) coordination number, 18 electronic configuration, 18 minimum metal-oxygen distance in carboxylates, 19 polyhedron geometry, 18 Cobalt(I I) coordination number, 18

382

SUBJECT INDEX

electronic configuration, 10, 18 electron transfer to Co(III), 60 imidazole liganding, 36-37 minimum metal-ligand distances, 19 minimum metal-oxygen distance in carboxylates, 19 polyhedron geometry, 18 radii, 10 Cobalt(I I I) coordination number, 18 electronic configuration, 10, 18 electron transfer from Co(II), 60 polyhedron geometry, 18 radii, 10 Cofactor (see Molybdenum iron protein cofactor) Complexation, metal ion selection for, 26-29 Complex-forming properties, of metal ions, 7 Concanavalin A Ca2+-binding ligands in, 55 Ca2+-bindingsites, 108 Ca2+-ligand distance, 115 characteristics, 85 continuous coordinating peptides, 108 w loops, 110 Ca2+-binding without EF hands, 53 Ca2+-Mn2+ binding site, 104 modulation by Ca2+,86, 108 saccharide-binding site, 108 stereo view and atomic designations, 62 X-ray crystal structure, 8 1 Coordination numbers, 2 Ca"-binding site, 112-1 13 carboxylate groups, 24-26 of cations, 10-1 1 coordination polyhedra for, 12-13, 16-17 effective cation radii and, 8-9 Lewis base strength of anion and, 12 radii ratios for, 1 1- 13 transition metal cations, 18 for various metal ions, 14-15 zinc, 285-286 Coordination polyhedra hemerythrin, 241-242 ligands around metal ions, 12-13, 16 octahedral/ trigonal bipyramidal iron centers, 204 transition metal cations, 18

for various coordination numbers, 12-13, 16-17 zinc, 286 zinc in carboxypeptidase A, 329 Copper binding in proteins, 39-42 Cu, Zn, Cd liganding, 56 Copper(I) characterization, 146-147 coordination number, 18 Cu(I)/Cu(II) redox system, 39-41 electronic configuration, 18 minimum metal-oxygen distance in carboxylates, 19 polyhedron geometry, 18 proteins azurin, 39-40 plastocyanin, 41-42 Copper(I I) characterization, 147 coordination number, 18 Cu(I)/Cu(II) redox system, 39-41 electronic configuration, 10, 18 minimum metal-oxygen distance in carboxylates, 19 polyhedron geometry, 18 radii, 10 relative affinity for protein side chains, 28 Copper(III), characterization, 147 Copper-containing proteins (see also Cupredoxins) function and source, table, 146 Copper sulfate pentahydrate, Jahn-Teller effect on bond lengths, 2 1 Copper sulfate trihydrate, Jahn-Teller effect on bond lengths, 2 1 Copper thionein function, 146 source, 146 Copper,zinc-superoxide dismutase characterization, 168 liganding, 55-56 stereo view and atomic designations, 63 Crystallographic analysis Fe protein tertiary structure, 258 hemerythrin, 224-226 molybdenum iron protein (MoFe protein), 259 structure of non-heme iron proteins, 224-226

SUBJECT INDEX

Cucumber basic blue protein backbone, 163 copper site in, 163 function, 146, 161 ligands, 149 source, 146, 149 spectral characteristics, 149 structure, 161- 162 topology, 163 Cupredoxins (see also specific copper-containing protein) P-barrel structure, 165-166 electronic character of type I site, 150 electron transfer, 167- 168 evolution of multi-copper-binding proteins, 191 features common to all blue sites, 150-151 geometry of copper center, 148 and immunoglobulin folding topology, 165, 167 subclassifications,table, 149 type I copper-binding, 148-151 type I1 copper-binding, 168-172 type 111 copper-binding, 172- 177 visible spectra, 148 Cysteine ligands to Fe:S clusters, identification, 212-213 as zinc ligand, 300-301 Cytochrome b g , electron transfer in, 58-59 Cytochrome c, electron transfer in, 58-59 Cytochrome c peroxidase, porphyrin-liganding group, 43-44 Cytochrome oxidase Cu(A)-type site in, 190 electron spin-echo spectra, 190 function, 146 Greek key domains, 191 source, 146 Cytochrome P-450 Fe(I1) porphyrin-liganding group, 43-44 stereo view and atomic designations, 46

D Dactylium dendroides, galactose oxidase, 171-172 Denitrifying bacteria, nitrite reductase in, 185

383

Desulfouibrio D. gigm ferredoxin, 254,256 rubredoxin, 42-43 D. vulgaris, rubredoxin, 42-43 Dihydrofolate reductase, X-ray crystal structure, 81 Disulfide bonds, in stellacyanin, 162- 164 DNase I, X-ray crystal structure, 80 Dopamine P-monooxygenase function, 146 source. 146

E91Q azurin mutant, 156 EF hands, 52-53 types of, 53 in vitamin D-dependent calcium-binding protein, 53 E form, 30-32 Elastase Ca2+-bindingsites, 93 Asx turns, 111 characteristics, table, 84 continuous coordinating peptides, 108 coordination number, 112 w loops, 110 X-ray crystal structure, 79 Electron density maps histidine side chain, 298-299 ribonucleotide reductase B2 subunit, 245 trimethylamine dehydrogenase, 266 Electronic configurations high- and low-spin transition metal cations, 10 transition metal cations, 18 Electronic coupling, multinuclear iron centers, 208 Electronic spectra, non-heme iron protein metal centers, 228 Electron nuclear double resonance aconitase, 263-264 non-heme iron protein metal centers, 228 Rieske-type Fe:S proteins, 270 Electron paramagnetic resonance Cu(A)-type site in, 190 cupredoxins, 148 Fe protein tertiary structure, 258

384

SUBJECT INDEX

galactose oxidase, 171 nitrite reductase, 185 nitrous reductase, 190 nonequivalence of copper sites in ceruloplasmin (sheep plasma), 185 non-heme iron protein metal centers, 228 Rieske-type Fe:S proteins, 269-270 type I1 copper in ceruloplasmin (chicken plasma), 185 Electron spin-echo spectra cytochrome oxidase, 190 Rieske-type Fe:S proteins, 270-27 1 Electron transfer azurins, 156 cupredoxins, 167-168 from Fe protein to MoFe protein, 256-258 in metalloproteins, 58-65 plastocyanin, 160, 191 role of photosynthetic reaction center, 235-236 trimethylamine dehydrogenase, 266-268 Electrostatic effects long-range, in protein-zinc interactions, 304-307 in protein-zinc recognition, 282-284 Enhancer-binding protein, zinc finger domain, 340 Entatic state, 66 Entennorpha prolijkra plastocyanin, X-ray diffraction, 160 Entropy effects, in protein-zinc recognition, 284-285 Enzymes entatic state, 66 Irving-Williams order, 27 modification using metal affinity to imidazoles, 37 Escherichia coli, sulfate reductase, 42 Extended X-ray absorption fine structure spectroscopy hemerythrin, 241-242 non-heme iron protein metal centers, 228 3,4-proteocatechuate, 230-23 1 ribonucleotide reductase B2 subunit, 245 Rieske-type Fe:S proteins, 270 transcription factor IIIA, 337-338

Extradiol dioxygenases oxygen activation by, 233-234 reaction mechanisms, 233 Extrusion technique, for Fe:S cluster identification, 2 19-220

F F114A azurin mutant, 156 Factor V function, 146 source, 146 Factor VIII function, 146 source, 146 Favin, X-ray crystal structure, 8 1 Fe protein (see Iron protein) Ferredoxin Fe(II), 42 electron transfer in, 58-59 stereo view and atomic designations, 44 Ferredoxins amino acid sequence comparisons (P. furosiu), 221-222 evolution, 251-253 2Fe:2S A. sacrum,203 H . mcarismortii, 203 iron ligands in, 203 polypeptide fold and metal centers, 212 S . platensis, 203,2 12 3Fe:4S A. vinelandii, 213,253-254 D. gigas, 254,256 ligands, 203 polypeptide fold and metal centers, 212-213 4Fe:4S A. vinelandii, 203,213,253-255 B. thmoprotolyticw, 213,254 ligands, 203 P. aerogenes, 203, 212, 253-254 polypeptide fold and metal centers, 2 12-213 history of metal centers, 256 role of 3Fe clusters, 255-256 structures, crystallographic determination, 253 Ferrichromes, Fe(1II)-binding, 38-39 Ferric iron, intradiol deoxygenase, substrate activation by, 232-233

SUBJECT INDEX

Fe-superoxide dismutase iron ligands in, 202 polypeptide fold and metal centers, 2 10 Flavin-binding domain, polypeptide fold and metal centers, 2 14 Frontier orbitals, 6-7

G D-Galactose-bindingprotein Ca2+-bindingsites, 107-108 Asx turns, 111 bidentate glutamate in, 117 characteristics, 85 semicontinuous coordinating peptides, 109 stereo view, 104 stabilization by Ca2+,87, 107 X-ray crystal structure, 80 Galactose oxidase domain structure, 171-172 function, 146 ligands for copper, 172 source, 146 structure (D. dendroides), 171-172 topology, 172 type I1 copper site, 171 Gamma turns, in helix-loop-helix sites, 109 Gene duplication, EF hands and, 52 Gene transfer, in superoxide dismutase, 170 Glutamate carboxylate-zinc interactions, 287-290 Glutamate residues, predominance in Ca2+-binding sites, 113 Glutamine, hydrogen-bonding interactions, 127 Glycine chelates-zinc interactions, 296-297 Gold(I), binding to sulfhydryl groups, 38 Greek key folds cytochrome oxidase, 191 need for, 191 nitrite reductase, 186 nitrous reductase, 190-191 origin of term, 168

H Hard acids, 5 Hard bases, 5

385

Helix dipoles, lack of interaction with Ca2+, 130-132 Helix-loop-helix proteins Ca2+-binding, 87-91 elbow arrangement, 107 formal charge balance for Ca2+, 131 hydrogen bonding within, 127-129 main-chain conformations, 109-1 12 motif of, 52-53 secondary structural elements, 109- 112 types of ligands in, 114 HelLrpomatia hemocyanin, 173-174 Hemerythrin (see a6So Myohemerythrin) coordination of iron sites, 241-242 crystal structure, 240 environment of binuclear irons, 241 iron center properties, 242-244 iron liganding, 44-45 iron oxidation, 240 ligands, 240-241 oxygen binding, 240,242-244 structure, 240 superoxide intermediate, 243-244 Hemocyanin arthropod, 174 arthropodan, 172 backbone, 176 bridging ligands, 173, 175-1 76 copper binding, 42 copper site, 172-173, 176 geometry of, 175-176 crystallography, 174 domains of protein folds, 175 function, 146 hexameric heterogeneity (L. polyphaw), 172 ligand classes, 173 ligand interactions, 177 ligand orientation, 176- 177 molluscan, 172- 173 nitrite reactions, 173 oxidation states, 173 packing defect regions, 175 source, 146 X-ray diffraction studies, 173-1 74 Hemoglobin, iron liganding, 42,45 Highest occupied molecular orbitals, 6-7 High-potential iron protein iron ligands in, 203 polypeptide fold and metal centers, 213

386

SUBJECT INDEX

Histidine side chains, as metal-binding sites, 36-37 as zinc ligand, 297-299 H35K azurin mutant, 156 Human immunodeficiency virus, gag protein P55,340 Hydrogen bonds around Ca2+-bindingloop 4 of troponin C, 126 around Cu-binding site of hemocyanin, 176- 177 carbonic anhydrase I1 network, 318 interactions for histidine zinc ligands, 297-299 networks, among Ca2+-ligand residues, 124-130, 137 networks of zinc ligands, 307-310 nton +2, 111-112 syn-oriented, 307-3 10 Hydrophobicity contrast, metal-binding sites and, 34-35 a-Hydroxycarboxylate, binding of metal ions. 38-39

I Imidazole groups binding to metalloporphyrins, 37 metal binding, 36-37 Immunoglobulin, folding topology, 167 Insulin, zinc binding, 49 Interprotein cluster transfer, 2 19-220 Intestinal calcium-binding protein, calcium-binding ligands in, 55 Intradiol dioxygenases oxygen activation by, 230-233 reaction mechanisms, 233 structure, 230 Ionic radii cation, and coordination numbers, 8-9 coordination number-radius ratio relationship, 11-13 high- and low-spin transition metal cations, 10 radius ratio rule, 11 ratios, 11- 13 Iron binding in proteins, 42-45

non-heme iron proteins (see Non-heme iron proteins) Iron(I I) coordination number, 18 electronic configuration, 10, 18 minimum metal-oxygen distance in carboxylates, 19 polyhedron geometry, 18 radii, 10 Iron(II), cobalt(II1) electronic configuration, 10 radii, 10 Iron(II1) chelating groups binding, 38-39 coordination number, 18 electronic configuration, 10, 18 minimum metal-oxygen distance in carboxylates, 19 polyhedron geometry, 18 radii, 10 Iron protein role in coupling of electron transfer to ATP hydrolysis, 257-258 structure, 257 tertiary structure, 258 Iron-sulfur proteins, 42-45 (see also specific protein; Tetrahedral iron centers) identification clusters by extrusion, 219-220 ''F NMR and exogenous thiolate ligand, 220 labeling schemes for cysteine ligands, 2 12-2 13 iron center location, 2 16-2 17 iron ligands in, 203 with non-thiolate ligands, 269-27 1 with other prosthetic groups, 265-269 sulfite reductase, 268-269 trimethylamine dehydrogenase, 266-268 oxidation states, 207-208 reaction performed by, table, 252 Rieske-type, 269-27 1 Irving-Williams order, 26-27

J Jahn-Teller effect, 20-22 trans effect, 20-22

SUBJECT INDEX

L Laccase distribution, 178 function, 146, 178 sequence alignments with ascorbate oxidase and ceruloplasmin, 183 source, 146 a-Lactalbumin a-carbon backbone trace, stereo view, 103 Ca2+-binding sites, 106-107 a helix, 109 Asx turns, 111 continuous coordinating peptides, 108 coordination number, 112 lack of interaction with helix dipoles, 130- 131 0 loops, 110 stereo view, 103 modulation by Ca2+,86, 106 X-ray crystal structure, 80 Lactoferrin iron center location, 216 iron ligands in, 202 polypeptide fold and metal centers, 21 1 structure, 237 Lactotransferrin folding pattern, 237 iron environment in, 238 Lectin IV, X-ray crystal structure, 81 Leucine aminopeptidase, zinc coordination and, 331 Lewis acid-base theory, 4-5 Lewis bases, anion strength, coordination number and, 12 Ligands ascorbase oxidase, 179.181, 183 bidentate aspartate, 114-1 16 bidentate glutamate, 114-1 16 Ca2+-binding sites, geometry, 115-1 18 cysteine, to Fe:S clusters, identification, 2 12-213 galactose oxidase, 171- 172 hemerythrin, 240-24 1 hemocyanin, 173, 176-177 iron centers octahedral centers, 203-205 tetrahedral centers, 205 trigonal bipyramidal centers, 203-205

387

metal, identification, 220-223 non-thiolate, Rieske-type Fe:S proteins, 269-270 oxygen atom, 113-1 15 syn and anti lone pairs, 124 polarizability, 4-7 Rieske-type Fe:S proteins, 270-271 shape for coordination polyhedra, 12-13, 16 superoxide dismutase, 168-169 types, Ca2+-bindingsites, 113-1 15 tyrosinase, 177 unidentate aspartate, 114-1 16 unidentate glutamate, 114-1 16 water, 114-116 zinc aspartate carboxylate, 287-290 carbonyl, 295-297 cysteine, 300-301 glutamate carboxylate, 287-290 glycine chelates, 296-297 histidine, 297-299 hydrogen bond networks, 307-310 methionine, 302-303 phosphate, 290-295 selenocysteine, 302-303 solvent, 301-302 Limulus polyphemus, hemocyanin, 172 Lowest occupied molecular orbitals, 7 Lysine oxidase function, 146 source, 146

Magnesium, binding in proteins, 50-51 Magnesium(II), minimum metal-oxygen distance in carboxylates, 19 Magnesium citrate decahydrate, structure of hydrated magnesium ion, 28 Magnesium--oxygen bonds average bond length, 19 coordination number, 19 Magnetic circular dichroism, non-heme iron protein metal centers, 228 Magnetic properties, iron centers, 206-209 Manganese binding in proteins, 45 in superoxide dismutase, 45

388

SUBJECT INDEX

Manganese(I I) coordination number, 18 electronic configuration, 18 minimum metal-oxygen distance in carboxylates, 19 polyhedron geometry, 18 Manganese(II), iron(II1) electronic configuration, 10 radii, 10 Mavicyanin ligands, 149 source, 149 spectral characteristics, 149 Mercury(11) binding to sulfhydryl groups, 38 direct bonding, 33 Metal binding to carboxylate groups, 30-36 entatic state, 66 to imidazole groups, 36-37 to main-chain carbonyl groups, 38 metal-binding amino acid side chains, 3-4 metal competition and replacement, 65-66 to sulfur-containing groups, 37-38 to two groups, 38-39 Metal ions asymmetry in shape, 20-2 1 complex-forming properties, 7 coordination numbers, 2 coordination numbers for, 14-15 peptide carbonyl oxygen atom-ion interactions, 118-1 19 polarizability, 4-7 redox processes, 7-8 in seawater and blood plasma, 2 selection for complexation, 26-29 soft, d-orbital electrons, 5-6 Metal-ligand bonds bond valence, 22 competition with protons, 28 hydrophobicity contrast and, 34-35 Jahn-Teller effect, 20 lengths, 20-21 Mg-0, 19 strength of, 21-26 superoxide dismutase, 168- 169 Metal-ligand distances Ca2+-binding sites, 113-1 15

Ca2+-ligand distance vs individual ligand deviation, 116 changes in valence states and, 60 octahedralltrigonal bipyramidal iron centers, 204 in sodium compounds, 26 Metalloantibody engineering, 345-346 Metalloporphyrins, axially bound imidazole groups, 37 Metalloproteins, electron transfer in, 58-65 Metallothionein-I1 characterization, 333-334 half-turn identification, 336 structure, 334 tetrahedral coordination, 334-335 three-metal-thiolate cluster, 336 Metallothioneins metal ligand identification, 220-223 metal liganding, 56 Methane monooxygenase components, 248-249 reactions, 248 Methemocyanin green, formation, 173 production, 173 Methionine, as zinc ligand, 302 MoFe protein (see Molybdenum iron protein) Molecular orbital effects, in protein-zinc recognition, 284 Molybdenum iron protein crystallographic analysis, 259 iron center, 259 structure, 258-259 Molybdenum iron protein cofactor homocitrate component, 261 identification, 260 structure, 260 substrate specificity, 262 Molybdenum(IV), wolfram(1V) coordination number, 18 electronic configuration, 18 polyhedron geometry, 18 Molybdenum(V), wolfram(V) coordination number, 18 electronic configuration, 18 polyhedron geometry, 18 Molybdenum(V1). wolfram(V1) coordination number, 18

SUBJECT INDEX

electronic configuration, 18 polyhedron geometry, 18 Mononuclear octahedral/trigonal bipyramidal iron centers characterization, 204-205 location, 216-217 oxidation states, 206 Mossbauer spectroscopy aconitase, 262-263 hemerythrin, 242 methane monooxygenasease, 248-249 non-heme iron protein metal centers, 228 ribonucleotide reductase B2 subunit, 245,247 Rieske-type Fe:S proteins, 270 Multinuclear clusters, iron centers, electronic coupling, 208 Mutants, azurin, 156 Myoglobin, iron liganding, 42 Myohemerythrin (see also Hemerythrin) environment of binuclear irons in, 241 oxygen-binding model, 243 Myohemerythrin Fe(1) iron ligands in, 202 polypeptide fold and metal centers, 21 1 Myohemerythrin Fe(I1) iron ligands in, 202 polypeptide fold and metal centers, 21 1 stereo view and atomic designations, 47

Nickel(I1) coordination number, 18 electronic configuration, 18 minimum metal-oxygen distance in carboxylates, 19 polyhedron geometry, 18 Nickel(II1) electronic configuration, 10 radii, 10 Nitrite, reaction with molluscan deoxy-hemocyanin, 173 Nitrite reductase copper-binding sites, 186- 187 crystallography, 186 distribution, 185 domain 1 ribbon drawing, 187 topology, 187

389

domain 2 ribbon drawing, 188 topology, 188 function, 146 Greek key P-sheet folding, 186 protein folds, 186 sequence homology with sulfite reductase P subunit, 269 source, 146 trimeric arrangement, 189 Nitrogenase cofactor, 260-262 enzyme system, 256-257 Fe protein, 257-258 MoFe protein, 258-259 Nitrogenase iron protein iron ligands in, 203 polypeptide fold and metal centers, 2 14 Nitrous reductase dimeric nature of, 188 function, 146, 187 Greek key domains, 191 source, 146 Non-heme iron proteins binuclear iron ligands, table, 202 biological roles of, 200 iron centers hydrophobic environment of, 209, 214-2 15 identification chemical modification approaches, 220-223 extrusion technique, 2 19-220 interprotein cluster transfer, 2 19-220 by stoichiometry of cluster components, 218-219 location, 216-217 magnetic properties, 206-209 octahedral, 203-205 polarity of protein environment, 2 15 redox properties, 206-209 side chain effects on conformation, 215 spectroscopic characterization, 226-228 structure, 201-204 trigonal bipyramidal, 203-205 iron ligands nonlocal nature of, 2 16-2 18

SUBJECT INDEX

replacements, 217 table of, 202-203 iron-sulfur proteins (see Iron-sulfur proteins) mononuclear iron ligands, table, 202 oxygen-utilizing enzymes, 230-235 reactions catalyzed by, 229 structure determination, crystallographic methods, 224-226 Nuclear magnetic resonance "F, Fe:S proteins, 220 metallothionein-11,334 non-heme iron protein metal centers, 228 plastocyanin structure ( S . obliquus), 157 titration behavior of His-35 in azurin (A. denitrijicalzs), 157 Xenopus Xfin zinc finger, 338 zinc-binding sites on GAL4 protein, 341-342 Nucleic acid-binding proteins, zinc-binding domains, 340 Nucleophilocity, zinc promotion of, 286

0 Octopus dojieini, hernocyanin crystals, 174 Omega loops, Ca'+-binding site peptides, 109-1 10, 136 Oncomodulin, X-ray crystal structure, 79 Oxidation states binuclear iron centers, 207 multinuclear iron centers, 207-208 Oxygen binding, hemerythrin, 240,242-244 coordination complexes involving, 28 ligands mean Ca2+-ligand distances, 114-115,137 in protein crystal structures, 113 syn and anti lone pairs, 124 Oxygen activation by binuclear octahedral iron proteins, 249-25 1 by extradiol dioxygenases, 233-234 by 3,4-proteocatechuate, 230-233

P Packing region defects, hernocyanin, 175 Panuliris intemptus hernocyanin, 174 Parvalbumin Ca2+-bindingsites, 91 Asx turns, 111-1 12 characteristics, 84 lack of interaction with helix dipoles, 130-131 selectivity of, 134-135 stereo view of site 1, 90 EF hands, 53 modulation by Ca2+,86 X-ray crystal structure, 79 Parvalbumin Ca(II), stereo view and atomic designations, 58 Pea Lectin, X-ray crystal structure, 81 Peptide carbonyl groups, metal binding to, 38 Peptide carbonyl oxygen ligands, 114-1 16 Peptide carbonyl-zinc interactions, 295-296 Peptide hydrolysis, catalyzed by carboxypeptidase A, 324-326 Peptococcus aerogenes ferredoxin cluster-binding sites, 253-254 ligands, 203 iron protein from, 42 Phosphate, zinc interactions, 290-295 Phospholipase AP a-carbon backbone, stereo view, 102 Ca'+-binding ligands, 55 Ca'+-binding sites, 105-106 characteristics, 85 lack of interaction with helix dipoles, 130-13 1 0 loops, 110 semicontinuous coordinating peptides, 109 stereo view, 102 without EF hands, 53 catalytic activity modulated by Ca2+,87, 105 X-ray crystal structure, 80 Photobacterium leignathi superoxide dismutase, 170 Photosynthetic reaction center iron center location, 216

39 1

SUBJECT INDEX

iron environment in, 236 iron ligands in, 202 polypeptide fold and metal centers, 210 role in electron transfer, 235-236 role of iron in structural stabilization, 236 structure, 235 rr bonds formation, 5-6 sulfur-binding metals, 37 Plastocyanin algal, structure, 159-160 apo form, 159 backbone, 158 bond lengths, 157, 159 copper site in, 158 electronic structure, 150 electron transfer, 160, 191 function, 146 high-resolution X-ray diffraction (E. prolifera), 160 ligands, 149 oxidized (poplar), 41-42 plant, 158 reduced structure, pH effects, 159 source, 146, 149 spectral characteristics, 149 stereo view and atomic designations, 4 1 topology, 158 Polarizability, metals and ligands, 4-7 Potassium(I), minimum metal-oxygen distance in carboxylates, 19 Proteinase K Ca2+-bindingsites characteristics, 84 coordination number, 112-1 13 discontinuous coordinating peptides, 109 empty, 135 lack of interaction with helix dipoles, 130- 131 n to n + 2 hydrogen bonds, 112 site 1, 98-99 stereo view of site 1, 95 structure-function relationships, 139 polygon formed by eight ligand atoms, 95 X-ray crystal structure, 80 Protein engineering, zinc-binding sites, 343-348

Protein folding, azurin, 151-152 Protein side chains, affinities of protons, Cu(II), and Zn(I1) for, 28 Prothrombin, structure, 78 3,4-Protocatechuate active-site region, 23 1 oxygen activation, 230-233 reactions catalyzed by, 230-23 1 structure, 230 3,4-Protocatechuate dioxygenase iron center location, 216 iron ligands in, 202 polypeptide fold and metal centers, 210 Proton donors, carboxypeptidase A, 327 Protons anti conformation, 30-32 relative affinity for protein side chains, 28 syn conformation, 30-32 Proton transfers, carbonic anhydrase 11, 318-321 Pseudoazurin backbone, 162 copper site in, 162 function, 146 gene for (A. faecalis), 161 isolation, 160-161 ligands, 149 salient features, 161 source, 146, 149 spectral characteristics, 149 structure, 161 topology, 162 Pseudomonas P . aeruginosa azurin, 39-4 1 gene for, 156 self-exchange, 156-157 P . stutzerei N 2 0 reductase, 187-188 Purple acid phosphatase, identification, 239 Pyrococcw furosia, 22 1-222 Pyruvate kinase, selectivity for metals, 65

R Radii (see Ionic radii) Radius ratio rule, 11 Redox potentials, cupredoxins, 148 Redox processes, of metal ions, 7-8

392

SUBJECT INDEX

Redox properties iron centers, 206-209 ribonucleotide reductase, 244 Resonance Raman spectroscopy non-heme iron protein metal centers, 228 ribonucleotide reductase B2 subunit, 245 Rieske-type Fe:S proteins, 270 R factor, apoazurin, 155 Rhizopus pepsin Ca2+-binding sites, 83 X-ray crystal structure, 80 Ribonucleotide reductase B2 subunit chemical function, 245-247 diferrous, Mossbauer spectrum, 247 iron center of, 244-245 location of tyrosine radical, 247 overall reaction, 248 structure, 245 environment of binuclear iron in, 246 iron ligands in, 202 polypeptide fold and metal centers, 2 1 1 redox state, 244 structure, 244 subunits, 244 Rieske-type Fe:S proteins, 269-271 Rubredoxin iron ligands in, 203 polypeptide fold and metal centers, 212 Rubredoxin Fe(II), 42 stereo view and atomic designations, 43 Rubrerythrin, identification, 239 Rusticyanin ligands, 149 source, 149 spectral characteristics, 149

S S- 100a. calcium-binding ligands in, 55 S . platemis, 2 12 Samarium(III), direct bonding, 33 Satellite tobacco necrosis virus, X-ray crystal structure, 81 Scatterplots construction strategy, 31 a-hydroxycarboxylate binding of metal ions, 39

metal-carboxylate binding, 34 peptide carbonyl-metal ion interactions, 295-296 Scenediesmw obliquus plastocyanin, structure, 159-160 Seawater, metal ions in, 2 Selectivity, of Ca2+-bindingsites, 134- 135 Selenocysteine, as zinc ligand, 302 Self-exchange, azurin (P.a r m ~ n o s a ) , 156-157 Serine protease, addition to second histidine to, 37 Serine proteinases Ca2+-binding sites, 91-99 stabilization by Ca2+,86 subtilisin-like, common Ca2+-binding site, 97-98 Serum transferrin folding pattern, 237 structure, 237 Side chains amino acid, metal-binding, 3-4 histidine, as metal-binding sites, 36-37 protein, affinities of protons, Cu(II), and Zn(I1) for, 28 Siderophores, Fe(II1)-binding, 38-39 a bonds, sulfur-binding metals, 37 Silver(I), binding to sulfhydryl groups, 38 Site-directed mutagenesis addition of second histidine to serine protease, 37 chemical modification with, 220-221 Sodium acetate bond valence, 26 metal-ligand distance, 26 Sodium fluoroacetate bond valence, 26 metal-ligand distance, 26 Sodium(I), minimum metal-oxygen distance in carboxylates, 19 Soft acids, 5 Soft bases, 5 Southern bean mosaic virus, X-ray crystal structure, 81 Spectroscopy, methods for identification of iron centers, 226-228 Spirulznu plantensis ferredoxins, 42 Staphylococcus nuclease Ca2+-bindingligands in, 55 Ca2+-bindingsites, 105

393

SUBJECT INDEX

active site, stereo view, 101 characteristics, 85 lack of interaction with helix dipoles, 130-13 1 n to n + 2 hydrogen bonds, 112 catalytic activity modulated by Ca2+,87, 105 hydrogen bonding network, 129-130 stereochemistry of mechanism of action, 52 X-ray crystal structure, 80 Stellacyanin bond lengths, 164 disulfide bonds in, 162-164 function, 162 ligands, 149 source, 149 spectral characteristics, 149 tryptophan fluorescence quenching, 164 Streptomyces S. grisew trypsin Ca2+-binding site, 94,97 X-ray crystal structure, 79 S. rubiginosus xylose isomerase, metal-binding sites, 33-35 Structure-function relationship, proteins at Ca2+-bindingsites, 138-139 Subtilisin Carlsberg a-carbon backbone trace, 94 Ca2+-bindingsites, 98 Ca2+-0 distance, 120 characteristics, 84 continuous coordinating peptides, 108 coordination number, 112 lack of interaction with helix dipoles, 130- 131 0 loops, 110 selectivity of, 134- 135 semicontinuous coordinating peptides, 108 stereo view of site 1, 94 X-ray crystal structure, 80 Subtilisin Novo, X-ray crystal structure, 79 Sulfate reductase, from E. coli, 42 Sulfhydryl groups, metal binding to, 37-38 Sulfite reductase active site, 268 amino acid sequence homology with nitrite reductase, 269

spectroscopic studies, 268-269 substrate reduction mechanism, 269 Sulfur, C-S-C group, nucleophile vs electrophile approach to, 20-22 Superoxide dismutase backbone, 169 biological function, 170 cabbage, 170 copper and zinc sites in, 169 copper distance in, 171 Cu,Zn-superoxide dismutase characterization, 168 liganding, 55-56 stereo view and atomic designations, 63 electrostatic loop, 170 function, 146 function of conserved residues, 170 gene transfer in, 170 Greek key fold, 170 hydrogen bonding, 168-169 iron center location, 2 16 ligands, 168 metal-ligand side-chain orientations, 168- 169 Mn(II1) in, 45 photobacterium vs eukaryote sequences, 170 source, 146 topology, 169 Superoxide intermediate, hemerythrin, 243-244 syn conformation, 30-32 syn-oriented Glu-106,318, 321 syn-oriented hydrogen bonds, 307-310

T Tetrahedral iron centers (see also Iron-sulfur proteins) arrangements of ligands in 4Fe:4S clusters, 217-218 bond angles and distances, 205 ligands, 205 Thallium(III), direct bonding, 33 Thermitase Ca2+-bindingsites, 99 Asx turns, 111 Ca2+-0 distance, 119-120 characteristics, 84

394

SUBJECT INDEX

continuous coordinating peptides, 108 coordination number, 112 lack of interaction with helix dipoles, 130-13 1 0 loops, 110 selectivity of, 134- 135 semicontinuous coordinating peptides, 108-109 stereo view of site 2, 96 polygon formed by eight ligand atoms, 96 X-ray crystal structure, 80 Thermolysin active-site residues of, 323 Ca2+-binding ligands in, 55 Ca2+-binding sites, 83,99, 105 a helix, 109 Asx turns, 111 characteristics, 85 continuous coordinating peptides, 108 coordination number, 112-1 13 double site, stereo view, 100 lack of interaction with helix dipoles, 130- 131 0 loops, 110 semicontinuous coordinating peptides, 109 stereo view of site 3, 101 lack of interaction between Ca2+and helix dipoles, 132 stabilization by Ca2+,87 X-ray crystal structure, 80 Zn and Ca binding, 57 Thermolysin Zn(II), stereo view and atomic designations, 64 Thioether sulfur atoms, metal binding, 38 Titanium(1I), vanadium( I I I) electronic configuration, 10 radii, 10 Transcription activator GAL4 rnultinuclear protein-zinc interactions in vivo, 342 structure of zinc-binding domain, 340-343 Transcription factor IIIA characterization, 337 sequence analysis, 337-338 zinc fingers, 337-338 trans effect, 20-22

Transferrins conformation changes, 237-238 coupling between iron and anion, 237 structure, 237 types, 237 Transition metal cations Cambridge Structural Database, 17- 19 coordination number, 18 coordination polyhedra geometries, 18 electronic configurations, 10, 18 radii, 10 Trimethylamine dehydrogenase, 266-268 active-site environment in, 267 electron transfer, 266-268 first domain and cofactor-binding sites, 266 iron ligands in, 203 polypeptide fold and metal centers, 214 structure, 266 Troponin C Ca2+-binding ligands in, 55 Ca2+-binding sites, 87, 127-129 Asx turns, 110- 111 characteristics, 84 empty, 135 hydrogen bonding around, 126 lack of interaction with helix dipoles, 130-131 structure-function relationships, 139 EF hands, 53 hydrogen bonding network, 127- 129 magnesium binding in calcium sites, 50 modulation by Ca2+,86 X-ray crystal structure, 79 Troponin C Ca(Il), stereo view and atomic designations, 56 Trypsin bovine a-carbon backbone trace, stereo view, 92 Ca2+-binding sites, 97 Asx turns, 111 characteristics, 84 continuous coordinating peptides, 108 coordination number, 112 0 loops, 110 stereo view, 92 Ca2+-binding ligands in, 55

395

SUBJECT INDEX

regulatory metal-binding site engineering, 346-347

S.griseus Ca2+-binding sites, 94 discontinuous coordinating peptides, 109 lack of interaction with helix dipoles, 130- 131 X-ray crystal structure, 79 P-Trypsin, X-ray crystal structure, 79 Trypsinogen, calcium binding without EF hands, 53 Tryptophan fluorescence lifetimes azurins, 155-156 stellacyanin, 164 Tyrosinase Cu(II1)-binding site, 177-178 function, 146 source, 146 Tyrosine radical, in ribonucleotide reductase 8 2 subunit, 246-247

U Unidentate aspartate ligands, 114-1 16 Unidentate glutamate ligands, 114-1 16 Unidentate side chains, stereochemistry of Ca2+ interaction with, 122 Uranium(VI), direct bonding, 33 Uricase function, 146 source, 146

Valence bond (see Bond valence) change of states, metal-ligand distances and, 60 Valence models ball-and-stick, 21-22 bond valence, 24 Vanadium(II), chromium(Il1) electronic configuration, 10 radii, 10 Vanadium( IV) electronic configuration, 10 radii, 10 Vitamin D-dependent calcium-binding protein, calcium binding in, 53

W Water ligands, 114- 116 as zinc ligand, 301-302

X X-ray absorption fine structure spectroscopy azurin ( P . aeruginosa), 155 copper proteins, 147 Cu(A) site in N 2 0 reductase and cytochrome oxidase, 190 non-heme iron protein metal centers, 228 X-ray crystallography carbonic anhydrase I I , 3 12-3 13 carboxypeptidase A, 322-324 metal-ligand bond distances, 17- 19 X-ray diffraction azurin and plastocyanin, 39-42 copper proteins, 147 hemocyanin, 173- 174 plastocyanin (E. prolifera), 160 Xylose isomerase, metal-binding sites, 33-35 D-Xylose isomerase, stereo view and atomic designations, 5 1 D-Xylose isomerase Mn(II), magnesium binding, 50

Z form, 30-32 Zinc binding in proteins, 45-50 binding to apocarbonic anhydrase, kinetics and thermodynamics, 285 in catalysis and regulation carbonic anhydrase 11,311-321 carboxypeptidase A, 322-333 thermolysin, 322-333 zinc proteases, 322-333 coordination numbers, 285-286 coordination polyhedron, 329 liganding Cu, Zn, Cd, 56 Zn, Ca, 57 promotion of nucleophilocity, 286

396

SUBJECT INDEX

protein-metal interactions, long-range electrostatic effects, 304-307 hydrogen bond networks, 307-310 protein-metal recognition, 282-285 charge-charge interactions, 282-283 electrostatic effects, 282-284 entropy effects, 284-285 molecular orbital effects, 284 role in protein-DNA recognition, 339 Zinc(I I) coordination number, 18 electronic configuration, 10, 18 imidazole liganding, 36-37 minimum metal-ligand distances, 19 polyhedron geometry, 18 radii, 10 relative affinity for protein side chains, 28 Zinc-binding sites

in antibodies, 345-346 protein engineering of, 343-348 Zinc fingers, 50 enhancer-binding protein, 340 transcription factor IIIA, 337-338 Xenopus Xfin protein, 338-339 Zinc-ligand interactions amino acid chelates, 295-297 aspartate carboxylates, 287-290 carbonyl, 295-297 cysteine, 300-301 glutamate carboxylates, 287-290 glycine chelates, 295-297 histidine, 297-299 methionine, 302 phosphate, 290-295 selenocysteine, 302 solvent, 301-302 Zinc proteases, as catalysts, 332-333

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