Table Of ContentACS SYMPOSIUM SERIES 372
Metal Clusters in Proteins
Lawrence Que, Jr., EDITOR
University of Minnesota
Developed
the Division of Inorganic Chemistry
at the 194th Meeting
of the American Chemical Society,
New Orleans, Louisiana,
August 30-September 4, 1987
American Chemical Society, Washington, DC 1988
In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
Library of Congress Cataloging-ln-Publication Data
Metal clusters in proteins
Lawrence Que, Jr., editor
p. cm.—(ACS symposium series; ISSN 0097-6156; 372).
"Developed from a symposiu Divisio
of Inorganic Chemistry at the
American Chemical Society, Ne
August 30-September 4, 1987."
Includes bibliographies and indexes.
ISBN 0-8412-1487-5
1. Metalloproteins—Structure—Congresses. I. Que,
Lawrence, Jr., 1949- . II. American Chemical Society.
Division of Inorganic Chemistry. III. Series
QP552.M46M46 1988
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In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
ACS Symposium Series
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In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
Foreword
The ACS SYMPOSIU
medium for publishing symposia quickly in book form. The
format of the Series parallels that of the continuing ADVANCES
IN CHEMISTRY SERIES except that, in order to save time, the
papers are not typeset but are reproduced as they are submitted
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In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
Preface
E FIELD OF BIOINORGANIC CHEMISTRY HAS EXPERIENCED a great
explosion of activity over the past 20 years due to the efforts of
biochemists, spectroscopists, crystallographers, molecular biologists, and
inorganic and organic chemists
been found to play vital role
biological processes.
Plans for the symposium from which this book was developed were
made because of the increasing number of metalloproteins that have
active sites consisting of more than one metal center. The presence of at
least one other metal center confers magnetic, spectroscopic, and
chemical properties to the active site not normally observed for a single
metal; thus, these proteins constitute a subset with unique
characteristics. The aim of the symposium was to bring specialists in the
biochemistry and spectroscopy of proteins together with chemists
involved in the design and synthesis of structural and functional models
for the active sites and to provide for a lively exchange of ideas among
these scientists. Associated with the symposium was a tutorial in which
design principles for the synthesis of analogues and the underlying basis
for several spectroscopic techniques were discussed as an introduction to
the field.
The chapters in this book, reflecting material derived from the
symposium and the tutorial, should serve as an up-to-date summary of
some important developments in this area. Metal Clusters in Proteins
may also serve as a starting point for classroom discussions on
bioinorganic chemistry. I am grateful to the authors for the time and
effort they expended in assembling and presenting their views of the
field. I also thank the Division of Inorganic Chemistry of the ACS for
sponsoring the symposium; the Petroleum Research Fund for paying the
travel expenses of the overseas speakers; and E. I. du Pont de Nemours
and Company, Exxon Research and Engineering Company, and the
Division of Biological Chemistry of the ACS for their financial support.
LAWRENCE QUE, JR. April 5,1988
University of Minnesota
Minneapolis, MN 55455
ix
In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
Chapter 1
Metalloprotein Crystallography
Survey of Recent Results and
Relationships to Model Studies
William H. Armstrong
Department o
Berkeley, CA 94720
The recent literature (1979-1987) pertaining to metalloprotein
crystallography is reviewed. Particular systems discussed in some detail
include "blue" copper proteins, iron-sulfur proteins, sulfite reductase,
cytochromes c, myoglobin, hemoglobin, catalase, cytochrome c
peroxidase, cytochromes P-450, hemerythrin, hemocyanin, superoxide
dismutases, Zn enzymes, metallothionein, lactoferrins, ferritin,
bacteriochlorophyll, and the photosynthetic reaction center. Preliminary
results for nitrogenase, ribonucleotide reductase, protocatechuate 3,4-
dioxygenase, hydrogenase, nitrite reductase, ascorbate oxidase, and
cerulplasmin are also mentioned. The influence of metalloprotein
crystallography on bioinorganic model studies and vice versa is
examined.
The interrelationship between protein crystallography and small molecule model
studies has been and will continue to be a very important factor contributing
toward a fundamental understanding of the chemical, magnetic and spectroscopic
properties of metalloproteins. Metal ions perform many different functions in
biological systems including electron transfer, oxygen binding, activation of oxygen
toward substrate oxidation, oxygen evolution, superoxide dismutation, peroxide
disproportionation, isomerization, hydrolysis, dinitrogen reduction, sulfite and nitrite
reduction, and dehydrogenation. The pursuit of low molecular weight analogs or
models for the metal sites in these biological systems has received a great deal of
attention in recent years. In many cases the results of these endeavors have
significantly sharpened our understanding of the involvement of the metal ions in
the aforementioned reactions. This branch of chemistry, commonly referred to as
bioinorganic chemistry or inorganic biochemistry, was reviewed in articles
appearing in 1976 and 1980 (1,2). In the present paper emphasis will be placed on
recent (between 1979 and 1987) protein X-ray crystallographic results and the
impact these results have had and will have on bioinorganic modelling. Rather than
restricting the scope of this survey to metal aggregates in proteins, mononuclear
centers will be examined as well, because important lessons can be learned from
both structural types. This paper presents a collection of representative examples
rather than an exhaustive review of the literature.
0097-6156/88/0372-0001$07.75/0
° 1988 American Chemical Society
In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
2 METAL CLUSTERS IN PROTEINS
Hill defined speculative models as those compounds which are intended to
mimic a protein metal site structure that has not been defined by X-ray
crystallography (7). Complexes related to a known structure were said to be
corroborative models. At first glance, it may appear that with metalloprotein X-ray
crystallography becoming more routine that model studies may become obsolete.
That is, if the central questions of the model study are related to the precise
structure of the metal site including the arrangement of metal atoms and the
identity of coordinated prosthetic groups and protein side chain residues, then a
single crystal X-ray structure will answer these questions unequivocally. There are
a number of problems with this oversimplified view of bioinorganic chemistry: [1]
Depending on the size of the protein, X-ray crystallographic refinements may not
yield a sufficiently well-defined picture of the metal site to permit a precise
geometrical description. [2] Even if the structure is determined to a very high
degree of accuracy, a number of important questions remain which potentially can
be addressed by model studies. For example: What is the effect of the protein
environment on the physica chemica propertie f ? Thi
can only be answered if th
protein matrix (for aggregates) g comple prepare
independently. [3] In most cases, crystal structures have been determined for only
the resting state of the protein. Large structural changes may occur during the
course of the catalytic cycle in an enzyme. In cases where enzyme intermediates
can be prepared but not characterized by crystallography, comparison of the
spectroscopic and magnetic properties of the intermediate state to those of model
compounds may provide structural insight. In some cases the isolated form of the
protein is actually a physiologically irrelevant form.
The points discussed above are probably best illustrated by example. Selected
examples of metal site structures in metalloproteins are presented in this chapter
and are summarized in Table I.
"Blue" Copper Proteins (3-10)
The blue or type 1 copper proteins, examples of which include azurin,
plastocyanin, and stellacyanin, function as electron transfer agents (11,12). Among
their properties that have fascinated bioinorganic chemists for years are intense
charge transfer transitions which gives rise to the intense blue color, unusually
positive reduction potentials, and small hyperfine coupling constants in the EPR
spectrum. Both azurin and plastocyanin have been characterized by X-ray
crystallography. Structure A is adapted from a recent report of the high resolution
structure of oxidized azurin from Alcaligenes dentinficons (3). Structure B depicts
the Cu site in oxidized poplar plastocyanin (8). In the former, the geometry around
copper is best described as distorted trigonal planar with distant axial interactions
to a methionine sulfur and a backbone carbonyl oxygen. In the latter, the structure
is closer to a distorted tetrahedral configuration with coordination to two histidines,
a cysteine, and a long bond to a methionine sulfur atom. Model studies prior to
these crystallographic reports were predominantly directed toward obtaining Cu(II)-
thiolate complexes with distorted tetrahedral geometry. While several Cu(II)-thiolate
species have been isolated and characterized by X-ray crystallography (13-15),
none of these approach tetrahedral geometry. In one case (16), in situ generation of
a Cu(II) thiolate at low temperature produced a species with a visible spectrum
similar to that of the protein. Discovery of the trigonal planar structure for the
azurin copper site presents a difficult challenge to the bioinorganic chemist.
Synthesis of a ligand with the proper donors that will maintain a three-coordinate
geometry, with perhaps one or two distant axial ligands, will be a demanding task.
In order to demonstrate that the unusual coordination geometry is imposed upon
In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
1. ARMSTRONG Metalloprotein Crystallography 3
Tabic I. Selected Metalloprotein Metal Coordination Site Structure
Based on X-Ray Crystallography
Protein Coordination Sphere Reference
Azurin [Cu(N-His)(S-Cys)(S-Met)(0-peptide)] 3
2
Plastocyanin [Cu(N-His)(S-Cys)(S-Met)] 8
2
Rubredoxin [Fc(S-Cys)] 17
4
Superoxide Dismutase, Mn [Mn(N-His)(0-Asp)(0H)] 89
3 2
Lactoferrin [Fe(N-His)(0-tyr)(0-Asp)(OH)(C02-)l 120
2 2 3
Carboxypeptidase [Zn(N-His)(0-Glu)(OH)] 103
2 2
Carbonic Anhydrase [Zn(N-His)(OH)] 113
2 2
Insulin [Zn(N-His)(OH)] 108
2 2
Thermolysin
Liver Alcohol Dehydrogenase
Cytochromes c [Fe(N-porphyrin)(N-His)(S-Met)] 38
4
Cytochrome c* [Fe(N-porphyrin)(N-His)] 34
4
Myoblobin, Hemoglobin, Oxy [Fe(N-porphyrin)(N-His)(0)] 39
4 2
Deoxy [Fe(N-porphyrin)(N-His)] 39
4
Met [Fe(N-porphyrin)(N-His)] 39
4
MetX [Fe(N-porphyrin)(N-His)(X)] 39
4
CO [Fe(N-porphyrin)(N-His)(CO)] 39
4
Catalase [Fe(N-porphyrin)(0-Tyr)(OH)] 59
4 2
Cytochrome P (Fe(N-porphyrin)(S-Cys)(OH)] 67
450 4 2
Cytochrome P450 [Fe(N-porphyrin)(S-Cys]"'camphor 66
cam 4
Cytochrome c Peroxidase [Fe(N-porphyrin)(N-His)(OH)] 64
4 2
Hemerythrin, Met [(N-His)Fe0i-O)(M-O-Asp)0i-O-Glu)Fe(N-His)] 76,77
3 2
Azido [(N-His)Fe0i-O)0i-O-Asp)(M-O-Glu)Fe(N-His)(N)] 72,75
3 2 3
Oxy [(N-His)Fe0i-O)0x-O-Asp)0i-O-Glu)Fe(N-His)(OH)] 74
3 2 2
Deoxy [(N-His)Fe0i-OH)0i-O-Asp)0i-O-Glu)Fe(N-His)] 74
3 2
Hemocyanin [(N-His)Cu"'Cu(N-His)] 86
3 3
Superoxide Dismutase, CuZn [(N-His)Cu0i-N-His)Zn(N-His)(O-Asp)] 94,95
3 2
Metallothionein; Cd,Zn [ZnCdOx-S-Cys)(S-Cys)],[CdOi-S-Cys)(S-Cys)] 115
2 3 6 4 6 6
Ferredoxin, 2 Fe [(S-Cys)FeGi-S)Fe(S-Cys)] 19
2 2 2
Ferredoxin, 3 Fe [Fe0*-S)(S-Cys)(OH)] 25
3 3 5 2
Ferredoxin, 4 Fe [Fe0x-S)(S-Cys)] 25
4 3 4 4
High Potential Iron Protein [Fe(p-S)(S-Cys)] 22
4 3 4 4
Sulfite Reductase [Fe(N-isobacteriochlorin)(At-S-Cys)Fe(/i-S)(S-Cys)] 30
4 4 3 3
Ferritin Up to 4500 Fe atoms as an oxo/hydroxo oligomer 122
Photosystem I Reaction Center 4 bacteriochlorophylls, 2 bacteriopheophytins 131
and a non-heme ferrous center: [Fe(N-His)(0-Glu)]
4
In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
4 METAL CLUSTERS IN PROTEINS
the copper atom, the structure of apoplastocyanin was solved to high resolution.
Indeed, it was found that the ligating side chain residue positions for the
apoprotein are almost identical to those of the holoprotein (9). The crystal structure
of reduced (Cu(I)) poplar plastocyanin has been analyzed for six pH values in a
recent report (6). Near physiological pH the Cu(I) coordination environment is very
similar to that of the Cu(II) form. Thus the condition of minimal structural
rearrangement for efficient electron transfer is satisfied.
Iron-Sulfur Proteins (17-27)
Iron-sulfur proteins serve predominantly as electron carriers (28,29). The best
understood examples are those proteins with lFe, 2Fe, and 4Fe centers. The
environment of the mononuclear iron center, rubredoxin, is shown in structure C
(17). It consists of a distorted tetrahedral array of sulfur atoms from cysteine
residues at nearly equal distances from the iron atom. Crystal structures are
available for 2Fe-2S ferredoxins from Spirulina plantensis (19) and Aphanothece
sacrum (20). A representatio
The 2Fe-2S core is anchore
atoms, yielding distorted tetrahedral geometry for both iron atoms. Crystal
structures of 4Fe-4S and 8Fe-8S ferredoxins have been carried out and yield a
detailed picture of the cubane-like aggregate shown in structure E. The intensive
efforts of Holm and coworkers (29) have produced analogs for each of these
iron-sulfur protein structures. In the case of the 2Fe-2S centers, isolation of model
compounds preceded the X-ray crystal structure determination. The binuclear and
tetranuclear complexes afford an excellent opportunity to examine the effects of the
protein milieu on the properties, especially redox behavior, of the metal centers.
Important questions relating to this are how the polypeptide fine-tunes the
reduction potential of an iron-sulfur site and how the high potential iron proteins
(HiPIP) can access a stable oxidized (4Fe-4S34) cluster level whereas the
ferredoxins can not. Hydrogen bonding interactions and control of ligand
orientation may play key roles. From a comparison of the crystal structures of
Chromatium HiPIP and Pseudomonas aerogenes, it has been postulated (22) that the
distinct differences between the properties of these two proteins arise because the
two 4Fe-4S centers are in nearly diasteriomeric environments with respect to each
other.
A newly emerging class of iron sulfur proteins are those with 3Fe-XS centers.
Aerobically isolated, inactive beef heart aconitase has a 3Fe-4S center which has
been characterized by X-ray crystallography (24). The diffraction data are
consistent with the presence of three Fe sites separated from each other by < 3A.
Thus, structure F represents a possible geometry for this 3Fe-4S unit. It is thought
that the iron-sulfur cluster in aconitase is directly involved in substrate binding
and transformation, a rare example of an iron-sulfur protein having a non-redox
function (see Chapter 17 of this volume). Structure G shows another type of three
iron-center; that characterized by X-ray crystallography for the 7Fe ferredoxin of
Azotobacter vinelandii (25-27). The structure consists of a puckered six-membered
ring with distorted tetrahedral geometry for each of the iron atoms. The single
non-sulfur ligand is thought to be an oxygen atom from OH or OH". Recent re
2
examination of the 7Fe protein from A. vinelandii by two groups reveals that
structural type F, rather than G, is correct for the 3Fe center (27b,c). Three-iron
aggregates of type F or G have not yet been synthesized in vitro, and as such
remain important targets for bioinorganic chemists in this field.
In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
ARMSTRONG Metalloprotein Crystallography
Gly
Cys
Structure B
Structure C
In Metal Clusters in Proteins; Que, L.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
