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Journal of Inorganic Biochemistry 71 (1998) 115±127

Coordination geometries of selected transition metal ions (Co2‡, Ni2‡,


Cu2‡ , Zn2‡, Cd2‡ , and Hg2‡) in metalloproteins
Lubomõr Rulõsek *, Jirõ Vondr
asek
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n
am. 2, 16610 Prague 6, Czech Republic
Received 5 January 1998; received in revised form 18 June 1998; accepted 7 July 1998

Abstract

In order to determine preferred coordination geometries of six divalent cations (Co2‡ , Ni2‡ , Cu2‡ , Zn2‡ , Cd2‡ , and Hg2‡ ), two
sources of experimental data were exploited: Protein Data Bank and Cambridge Structural Database. Metal-binding sites of ap-
proximately 100 metalloproteins and 3000 smaller transition metal complexes were analyzed and classi®ed. The correlation between
the geometries of small-molecule crystal structures and the metal-binding sites in metalloproteins was investigated. The abundance
of amino acid residues participating in coordination metal-protein bonds of metalloproteins was evaluated. From the performed
analysis it follows that the octahedral arrangement is preferred by Co2‡ and Ni2‡ , tetrahedral by Zn2‡ , square planar by Cu2‡ , and
linear by Hg2‡ . Cadmium (II) cation tends to bind in both tetrahedral and octahedral arrangements and single coordination ge-
ometry cannot be unambiguously ascribed to it. Ó 1998 Elsevier Science Inc. All rights reserved.

Keywords: Metal-binding site; Coordination geometry; Metalloproteins; Metal-binding residue

1. Introduction aware of any recent study summarizing all publicly


available structural information about the geometries of
Interactions of metals with biomolecules belong to transition metal binding sites.
one of the most studied ®elds in inorganic biochemistry. Therefore, we have carried out a comprehensive
The areas of application of new knowledge or infor- analysis of the known structures of metalloproteins
mation may range from medicinal chemistry [1] through containing selected transition metals to ®nd the corre-
``classical'' organometallic chemistry to environment lation between the small-molecule crystal structures of
protection (metal-binding biomass [2]). speci®ed metals and their coordination in metallopro-
The role of metal ions in the structure and function of teins.
proteins, nucleic acids, and peptide hormones is funda- Our work is intended to draw nearer the facts known
mental, yet often unknown at the molecular level. The from basic inorganic chemistry textbooks [13] and the
hypotheses about principles governing selectivity and facts known about the structure and function of me-
speci®city of macromolecules for a given metal are often talloproteins. We endeavor to investigate in a purely
based on semiempirical and qualitative theories, such as empirical and statistical way if the principles and
the HSAB (hard and soft acids and bases) principle of mechanisms determining the structures of small-mole-
Parr and Pearson, [3,4] and Irving±Williams series of cule complexes (such as ligand-®eld stabilization energy
stability constants for divalent ions [5,6]. and charge transfer from ligands to metal) remain un-
A plethora of experimental and theoretical material changed when describing the complex metal-binding
about the interactions of metals with biological macro- sites in metalloproteins. This can be proved by the de-
molecules is compiled in several excellent reviews [7±10]. gree of correlation found between the distribution of
Besides, there are papers dealing with structural aspects coordination geometries among small-molecule com-
of metal binding in biomolecules [11,12]. We are not plexes and metalloproteins. The other questions are
whether single coordination geometry could be identi-
®ed as preferred for a particular metal and to what ex-
tent does a given metal prefer coordination sites with
*
Corresponding author. E-mail: lubos@uochb.cas.cz this geometry.

0162-0134/98/$ ± see front matter Ó 1998 Elsevier Science Inc. All rights reserved.
PII: S 0 1 6 2 - 0 1 3 4 ( 9 8 ) 1 0 0 4 2 - 9
116 L. RulõÂsek, J. Vondr
asek / J. Inorg. Biochem. 71 (1998) 115±127

The transition metal elements analyzed here were metal center and determined the coordination ge-
selected because of two reasons. First, their metallo- ometry. 1
protein complexes belong to the most abundant. Sec- In certain cases, the coordination geometry deviated
ond, these metals are the major pollutants of the signi®cantly from the ideal coordination arrangements.
environment. Cadmium and mercury are highly toxic The structures with the bond angles at the metal center
heavy metals. Nickel, cobalt, and copper, as represen- deviating less than ‹10° from ideal geometric arrange-
tatives of the ®rst-row transition metals, are undesirable ments and with fully occupied coordination spheres
elements in the environment as well. The same holds were accepted as unperturbed. The structures with the
true for zinc (biogenic element) which is toxic in higher bond angles within the above limits but containing one
concentrations. The development of methods for selec- or two empty sites were designated with the pre®x
tive binding of these metals may ultimately lead to their pseudo-. The structures with a conserved coordination
successful removal from the environment (see for ex- number but with the bond angles out of the above limits
ample Ref. [2]). The molecular design of such speci®c were designated as distorted.
sites should necessarily start with the type of structural Binding modes of the residues bound to a metal ion
information we are trying to provide here. We presume were classi®ed as well. Most of the residues bind through
that it cannot be obtained by a purely theoretical ap- a single donor atom (oxygen, nitrogen, sulfur). In some
proach, represented by ab initio calculations, but can be cases (e.g., aspartate and glutamate functional groups),
revealed by careful analysis of the experimental struc- they bind through two atoms simultaneously, and oc-
tures. cupy either one or two coordination sites. Such binding
mode is referred to as bidentate. If ®ve-, six-, or seven-
member chelate ring is formed upon binding of the
protein to the metal ion, this mode is referred to as
2. Methods chelated.
If a metalloprotein contains a multinuclear metal
2.1. Metalloproteins center that can be separated into two or more mono-
nuclear centers with regular coordination, they are
The Protein Data Bank (PDB) [14] was searched. We treated as two independent centers. Otherwise, the site is
accepted the following entries: designated as (metal±metal)n bridged. Such binding sites
1. Protein contains divalent (2+) cation of Co, Ni, Cu, have not been analyzed in depth. The binding in those
Zn, Cd, or Hg. sites depends on the properties of the metal±metal
2. Metal cation is coordinated to at least three donor at- bonds, which vary even within the set of cations inves-
oms of protein or coordinated in linear fashion to tigated here. A detailed analysis of metal±metal bridged
only two donor atoms of protein (it excludes entries sites is beyond the scope of this work.
where metal is bound to the surface of a protein
mainly by non-covalent interactions while the rest 2.2. Small-molecule structures
of the coordination sphere is occupied by solvent
molecules; such binding modes cannot be denoted The Cambridge Structural Database [16] (CSD, ver-
as true metal-binding sites). sion 5.12 [17]) has been used. At the time of search, it
3. Metal is not coordinated to DNA fragments in pro- contained 181 309 entries, mainly crystal structures of
tein-DNA complexes. The accepted entries were an- smaller molecules. The accepted entries met the follow-
alyzed with the Insightâ II molecular modeling ing criteria:
system. 1. no disorder in crystal structure;
The residues with a donor atom lying within the 2. no errors;
 from the metal center for the ®rst row
distance of 2.40 A 3. R-factor less than 0.05;
transition metals and 2.70 A  for Cd2‡ and Hg2‡ were 4. only mononuclear metal center;
classi®ed as the binding residues. The residues with a 5. each coordination site occupied by either nitrogen,
donor atom lying in the distance between 2.40 and 3.00 oxygen or sulfur;
 from the metal center (2.70±3.30 A
A  for Cd2‡ and 6. divalency of metal cation explicitly stated in the title

Hg , respectively) as weakly bound. If sulfur partici- of the corresponding paper.
pated in the coordination bond, the above limits were The structures were classi®ed according to the bond
increased by 0.2 A  to account for its greater van der angles at the metal center with the allowed deviation of
Waals radius. These bonding limits are about 15% ‹10% from the ideal geometrical arrangements. In the
higher than the mean metal±ligand bond lengths in co-
ordination compounds of the studied cations [15]. The 1
The total number of binding sites does not have to be equal to the
same criteria were applied to ligands other than amino
number of entries for a given metal ion since a single protein can
acids, but only structural water molecules were evalu- contain several metal-binding sites. If a protein contained two or more
ated (other ligands included some inhibitors bound in di€erent binding sites, these sites were treated separately (i.e., as if they
the metal site, small inorganic molecules, such as carbon belonged to two di€erent proteins). In the case of proteins composed
dioxide, sulfate anion, and molecular oxygen). Subse- from identical subunits (e.g., homodimers), they were counted only
quently, we have calculated all the bond angles at the once.
L. RulõÂsek, J. Vondr
asek / J. Inorg. Biochem. 71 (1998) 115±127 117

case of low abundance of a given coordination number, found to exist in tetrahedral arrangement. Therefore, we
further subdivision was not done. It should be noted assume that square planar coordination is preferred by
that accurate distinction between square pyramidal and Cu2‡ cation. There is also a considerable number of
trigonal bipyramidal complexes (C4v and D3h symme- small-molecule crystal structures containing copper (II)
tries) of penta-coordinated species would require deeper bound in trigonal bipyramidal geometry.
analysis based on the use of Berry pseudo-rotation co- Zn2‡ can be found almost exclusively in tetrahedral
ordinates [18]. arrangement in metalloproteins. Even though there are
Very similar criteria for allowed deviations in bond more small-molecule crystals of Zn2‡ in octahedral ar-
angles were used for both PDB and CSD entries, even rangement, the dominance of tetrahedral metal-binding
though the latter structures (small-molecule crystals) are sites in proteins is clearly demonstrating its preference
almost by an order of magnitude more accurate. How- for tetrahedral geometry.
ever, in PDB search the allowed deviation limits served Cd2‡ can be found in tetrahedral and octahedral
only for the purpose of terminology (all entries were arrangements and preferred coordination cannot be
analyzed). On the other hand, in CSD search, the entries decided. There are slightly more metalloproteins con-
with greater deviations were excluded from the subsets taining tetrahedral binding sites, but octahedral arrange-
with the assigned coordination geometry. They consti- ment prevails in small complexes.
tute the di€erence between the number of compounds Hg2‡ prefers linear arrangement of two coordinating
with the given coordination number and the sum of residues as can be seen both from PDB and CSD ana-
compounds with the coordination geometries associated lyses.
with this coordination number. The results for Co2‡ and Ni2‡ must be examined with
greater care since it is well known that the coordination
geometry of their compounds depends on the spin state
3. Results and discussion of the metal. Both ions can exist in low- and high-spin
states and they can alter between them depending on the
The results are summarized in eight tables. Tables 1±6 ligand ®eld strength. The information about the spin
contain the detailed results of the search in PDB, in- state is not supplied with the crystal structures deposited
cluding the codes of the analyzed entries, the resolu- in PDB and CSD and the entries are not classi®ed with
tions, and list of the binding residues for each metal- respect to it. However, we presume that the metallo-
binding site. Table 7 contains the distribution of coor- proteins analyzed and classi®ed here are the high-spin
dination geometries of speci®ed metals in metallopro- complexes. The typical ligands (such as carbonyls and
teins together with the total abundance of the binding cyanates) with a high ligand ®eld strength are not
residues. The results of CSD search are then in Table 8. present in metalloproteins. Moreover, the loops between
Tables 1±6 are accessible via world wide web (http:// the binding residues in most of the analyzed metallo-
milenka.uochb.cas.cz/metalgeom) in both graphical and proteins consist of several residues. It implicates their
text forms. rather independent chemical behavior. As a conse-
The preferred coordination geometries. We have at- quence, the spin state of the central metal should be the
tempted to show the preference of certain metals for same as its spin state in the free Me(Yi )c‡ n complex,
speci®c coordination environment by classifying most of where Yi is the functional group representing the bind-
publicly available experimental structural data. One of ing amino acid residue. Both nickel (II) and cobalt (II)
the aims of this paper has been to select one unique ions will be preferably in high-spin state in such com-
coordination for each transition metal ion that might be plexes.
considered as the preferred. For the sake of clarity, the A di€erent situation occurs when analyzing CSD
results are discussed separately for each atom. The results. The metal in small-molecule crystal complexes
preferred coordination geometry is depicted in Fig. 1 for can be in both high- and low-spin states. Since the in-
each metal ion separately. formation about spin state is not directly available in
Co2‡ ion prefers octahedral arrangement, as can be most of the structures in CSD, the results represent
seen both from PDB and CSD data. It prevails over ``spin-averaged'' information. It can be still examined if
tetrahedral coordination. proper chemical intuition is applied. For example, the
Ni2‡ also prefers octahedral arrangement, though typical arrangement in low-spin nickel complexes is
approximately the same number of small-molecule square planar coordination that is approximately as
crystal complexes can be found in square planar geom- abundant in CSD as the octahedral coordination pre-
etries. Owing to total absence of square planar coordi- ferred in high-spin complexes. But the square planar
nation among the nickel metalloproteins, octahedral coordination is completely absent among the nickel
arrangement should be considered as favorable. We metalloproteins found in PDB, which corroborates the
presume (as will be discussed further) that the com- above hypothesis about the nature of Ni2‡ spin state in
pounds with square planar coordination of nickel (II) metalloproteins. Among cobalt (II) complexes found in
are mainly the low-spin Ni2‡ complexes. CSD, the octahedral arrangement prevails and since it
Cu2‡ in metalloproteins tends to bind mainly in tet- correlates well with the results obtained from PDB, we
rahedral and square planar modes of coordination. presume that most of the complexes of cobalt (II) ana-
However, no small-molecule crystal structures has been lyzed here are high-spin complexes.
118

Table 1
The metalloproteins containing the coordinated cobalt (II) ion

Compound PDB code 


Resolution (A) Coordination geometry Binding Residues

Carbonic anhydrase II (native Zn replaced by Co) complexed with 1cah 1.88 Distorted octahedral His 3 (2 Ne, 1 Nd), H2 O 1, CO2ÿ
3 1 (bidentate)
bicarbonate
Superoxide dismutase (Co substituted) 1cob 2.0 Tetrahedral His 3 (Nd), Asp 1
Astacin with Zn replaced by Co 1iab 1.79 Trigonal bipyramidal His 3 (Ne), Tyr 1, H2 O 1
Thermolysin complexed with Co 1lna 1.9 (Distorted octahedral; (His 2 (Ne), H2 O 2, Glu (biden.); H2 O 3, Asp 2, Gln 1
octahedral) a (amide O))
Methionine aminopeptidase 1mat 2.4 (Co)2 bridged Asp 2 (2 biden.), Glu 2 (1 biden.), His 1 (Ne)
Carbonic anhydrase II (native Zn replaced by Co) at pH ˆ 6.0 1rzb 1.8 Tetrahedral His 3 (2 Ne, 1 Nd), SO2ÿ4 1
Concanavalin A (Co substituted for Mg) 1scs 1.6 Octahedral Asp 2, H2 O 2, Glu 1, His 1 (Ne)
D -xylose isomerase complex with xylitol-Co 1xim 2.2 Octahedral Asp 2, Glu 2, xylitol 2
L -fuculose 1-phosphate aldolase crystal form T with Co 2fua 2.0 Highly distorted trigonal His 3 (Ne), Glu 1 (biden.)
Bipyramidal
L. RulõÂsek, J. Vondr

a
Round brackets indicate that more than one metal binding site was found and classi®ed in the metalloprotein. Their coordination geometries and the binding residues are separated by
semicolon.

Table 2
The metalloproteins containing the coordinated nickel (II) ion

Compound PDB 
Resolution [A] Coordination geometry Binding residues
code

Ni±Fe hydrogenase 1frv 2.85 2 ´ (Ni-Fe) bridged, highly distorted tetrahedral Cys 4
Astacin with Zn replaced by Ni 1iae 1.83 Octahedral His 3 (Ne), H2 O 2, Tyr 1
asek / J. Inorg. Biochem. 71 (1998) 115±127

Klebsiella aerogenes urease 1kau 2.2 (Ni)2 bridge with pseudooctahedral and pseudotetrahedral (His 2 (Nd, Ne), CBX (carbamylated Lys); His 2 (Ne), Asp, H2 O,
arrangements of ligands at Ni CBX 1)
Azurin (W48M mutant) 1nzr 2.2 Distorted tetrahedral His 2 (Nd), Cys 1, peptide O (Gly) 1
Carbonic anhydrase II (with Zn 1rze 1.9 Octahedral His 3 (2 Ne, 1 Nd), H2 O 2, SO2ÿ
4 1
replaced by Ni)
Concanavalin A (Ni substituted 1scr 2.0 Octahedral H2 O 2, Asp 2, Glu 1, His 1 (Ne)
for Mg)
Ecotin 1slw 2.0 Pseudooctahedral (two empty sites) His 2 (Ne), H2 O, His (Ne) weakly bound
Phosphoglucomutase 1vkl not applicable (Distorted tetrahedral; square pyramidal) (Asp 3, Sep (phosphonoserin) 1; Asp 3, Sep 1, H2 O 1)
Table 3
The metalloproteins containing the coordinated copper (II) ion

Compound PDB code 


Resolution [A] Coordination geometry Binding residues

Amicyanin 1aac 1.31 Distorted tetrahedral His 2 (Nd), Cys, Met


Ascorbate oxidase 1aoz 1.9 (Square planar with empty site; (His 2 (Ne), (Cu±O±Cu) linkage; His 3 (2 Ne,
2 ´ tetrahedral; trigonal pyramidal; linear) Nd), (Cu±O±Cu) linkage; His 3 (Ne), (Cu±O±Cu)
linkage; His 2 (Nd), Cys, Met; His 2 (Ne))
Azurin II 1arn 2.1 Trigonal bipyramidal His 2 (Nd), Cys, peptide O (Gly), Met
Galactose oxidase 1gof 1.7 Square planar His 2 (Ne), Tyr, acetate (carboxyl O)
L. RulõÂsek, J. Vondr

Astacin with Zn replaced by Cu 1iaa 1.9 Trigonal bipyramid His 3 (Ne), Tyr, H2 O
Cucumber stellacyanin 1jer 1.6 Tetrahedral His 2 (Nd), Cys, Gln (amide O)
Ceruloplasmin 1kcw 3.0 (Tetrahedral; trigonal) (His 3 (Ne), (Cu±O±Cu) linkage; His 2 (Nd), Cys)
Lactoferrin 1l® 2.1 (Square planar; distorted octahedral) (Asp, His (Ne), Tyr, CO2ÿ
3 ; Asp, His (Ne), Tyr 2 ,
CO2ÿ3 (bidentate))
Nitrite reductase 1nib 2.7 (Tetrahedral; tetrahedral) (His 2 (Nd), Cys, Met; His 3 (Ne), O)
Oxygenated hemocyanin 1nol 2.4 (2 ´ irregular (®ve-coordinated)) (His 6 (Ne), (Cu±O2 ±Cu) linkage)
copper amine oxidase 1oac 2.0 tetrahedral His 3 (2 Ne, Nd) trihydroxyphenylalanin(O)
Cytochrome C oxidase 1occ 2.8 (2 ´ irregular (four-coord.); trigonal) (His 2 (Nd), Glu, Met, (Cu-S-Cu) linkage 2; His 3
(Ne))
Pseudo-azurin 1paz 1.55 Tetrahedral His 2 (Nd), Cys, Met
Plastocyanin 1plb not applicable Irregular His 2 (Nd), Cys
Carbonic anhydrase II (Zn replaced by Cu) 1rzc 1.9 Trigonal bipyramidal His 3 (2 Nd, Ne), O2 , H2 O
Cu, Zn superoxide dismutase 1sda 2.5 Square planar His 4 (3 Ne, Nd)
Ecotin 1slv 2.3 Tetrahedral (highly distorted) His 3 (Ne), H2 O
asek / J. Inorg. Biochem. 71 (1998) 115±127

Alcohol dehydrogenase 2oxi 2.1 Trigonal Cys 2, His (Ne)


Thioredoxin 2trx 1.68 Square planar terminal N (Ser), Peptide N (Asp), Asp (bicyclic
5-membered chelate), H2 O (weakly bound)
Azurin 4azu 1.9 Trigonal His 2 (Nd), Cys
119
120

Table 4
The metalloproteins containing the coordinated zinc (II) ion

Compound PDB code 


Resolution [A] Coordination geometry Binding residues

Adenosine deaminase 1add 2.4 Trigonal bipyramidal His 3 (Ne), Asp, H2 O


N-ADA 10 1adn not applicable Tetrahedral Cys 4
Alkaline protease 1akl 2.0 Tetrahedral His 3 (Ne), H2 O
Alkaline phosphatase 1ali 2.2 (Tetrahedral; bridged to Zn1) (Asp 2, His (Ne), Ser; Asp, PO3ÿ
4 )
Aminopeptidase 1amp 1.8 (Tetrahedral; trahedral) Asp 3 (3 bidentate), His 2 (Ne), H2 O
Astacin 1ast 1.8 Tetrahedral His 3 (Ne), H2 O
Aspartate carbamoyl transferase 1at1 2.8 Tetrahedral Cys 4
Carbonic anhydrase I 1azm 2.0 Tetrahedral His 3 (2 Ne, Nd), Azm (inhib., bound by N)
Carbonic anhydrase II 1bcd 1.9 Tetrahedral His 3 (2 Ne, Nd), Fms (inhib., bound by N)
Leucine aminopeptidase 1bll 2.4 Complex structure (three chelate rings) Asp 3 (2 bridging), Lys, peptide N,O (Leu
L. RulõÂsek, J. Vondr

5-mem. chelate), Asp (7-mem. chelate, peptide O,


Od)
Metallo-beta-lactamase 1bmc 2.5 Tetrahedral (1 empty site) His 3 (2 Ne, Nd)
Verotoxin-1 1bov 2.2 Tetrahedral (1 empty site) Thr, terminal N (Thr), H2 O
Barnase 1brk 2.0 Tetrahedral (1 empty site) Lys, Glu, H2 O
B nerve growth factor 1btg 2.5 Trigonal bipyramidal (1 empty site) H2 O 2, His (Ne), Asp
Alcohol dehydrogenase 1cdo 2.1 (Tetrahedral; tetrahedral (1 empty site)) (Cys 4; Cys 2, His (Ne))
Collagenase 1cgl 2.4 (Tetrahedral; tetrahedral) (His 3 (2 Ne, Nd), Asp; His 3 (Ne), ABU (bound
by O))
Carboxypeptidase A 1cps 2.25 Tetrahedral His 2 (Nd), Glu (biden.), sulfodiimine (N)
Avian cysteine rich protein 1ctl not applicable (Tetrahedral; Tetrahedral) (Cys 4; Cys 3, His (Ne))
Cytidine deaminase 1ctt 2.2 Tetrahedral Cys 2, His (Nd), H2 O
Murine mitoch. carbonic anhydrase V 1dmx 2.45 Tetrahedral His 3 (2 Ne, Nd), H2 O
Atrolysin C 1dth 2.0 Tetrahedral His 3 (Ne), Batimastat (O)
Concanavalin A 1enr 1.8 Octahedral Asp 2, H2 O 2, Glu, His (Ne)
asek / J. Inorg. Biochem. 71 (1998) 115±127

Elastase 1ezm 1.5 Tetrahedral His 2 (Ne), Glu, H2 O


Nuclear factor XNF 7 1fre not applicable Tetrahedral Cys 2, His 2 (Ne)
L-fuculose 1-phosphate aldolase 1fua 1.92 Tetrahedral (distorted) His 3 (Ne), Glu (bidentate)
Glucocorticoid receptor 1gdc not applicable Tetrahedral Cys 4
Core GP32: DNA binding protein 1gpc 2.2 Tetrahedral (one empty site) Cys 3
6-pyruvol tetrahydropherin synthase 1gtq 2.3 Tetrahedral (one empty site) His 3 (Ne)
Fibroplast collagenase 1hfc 1.56 (Tetrahedral; octahedral (one empty site)) (His 3 (2 Ne, Nd), Asp; His 3 (Ne), HAP (5-m.
chelate))
Retinoic acid receptor 1hra not applicable (Trigonal bipyramidal (distorted); (Cys 4; Cys 4)
tetrahedral (distorted))
Hexose-1-phosphate uridyltransferase 1hxp 1.8 Tetrahedral (Cys 2, His 2 (Nd))
Meprin (a-chain) 1iaf not applicable Trigonal bipyramidal His 3 (Ne), Tyr, H2 O
Adamalysin II 1iag 2.0 Tetrahedral (distorted) His 3 (Ne), H2 O
Lysozyme 1lba 2.2 Tetrahedral His 2 (Nd), Cys, H2 O
Thermolysin: complex with Zn 1lnd 1.7 (Tetrahedral; trigonal) (His 2 (Ne), Glu, H2 O; Tyr, His (Ne), H2 O)
Methionyl-tRNA synthetase 1mea not applicable Tetrahedral Cys 4
Matrix metalloproteinase-8 1mmb 2.1 (Tetrahedral; tetrahedral) (His 3 (Ne), Batimastat (O); His 3 (2 Ne, Nd),
Asp)
Matrilysin 1mmq 1.9 (Tetrahedral; square pyramid) (His 3 (2 Ne, Nd), Asp; His 3 (Ne), RRS (5-m.
chelate))
Neutrophil collagenase 1mnc 2.1 (Tetrahedral; square planar) (His 3(2 Ne, Nd), Asp; His 3 (Ne), PLU (5-m.
chelate))
HIV1-P7 nucleocapsid protein 1ncp not applicable Tetrahedral Cys 3, His (Ne)
Stromelysin-1 1slm 1.9 (Tetrahedral; tetrahedral) (His 3 (Ne), Cys; His 3 (2 Ne, Nd), Asp)
Serratia protease 1srp 2.0 Tetrahedral His 3 (Ne), H2 O
Staphylococcal enterotoxin C2 1ste 2.0 Trigonal His 2 (Nd, Ne), H2 O
D-xylose isomerase complexed with Zn 1xll 2.5 (Tetrahedral (distorted); octahedral (Asp 2, Glu 2; Asp 2 (1 bidentate), Glu, His (Ne),
(distorted)) H2 O)
LAsp-1 1zfo not applicable Tetrahedral (Cys 3, His (Nd)
L. RulõÂsek, J. Vondr

Cu, Zn superoxide dismutase 2sod 2.0 Tetrahedral His 3 (3 Nd), Asp


Zinc ®nger 3znf not applicable Tetrahedral Cys 2, His 2 (Ne)
Enolase 6enl 2.2 Trigonal bipyramidal Asp 2, Glu, H2 O, phosphoglycolic acid (O)
Glycerol kinase 1glc 2.65 Tetrahedral (distorted) His 2 (2 Ne), Glu, H2 O
121 asek / J. Inorg. Biochem. 71 (1998) 115±127
122

Table 5
The metalloproteins containing the coordinated cadmium (II) ion

Compound PDB code 


Resolution [A] Coordination geometry Binding residues

Glutaredoxin 1aaz 2.0 (Square planar; tetrahedral (one (H2 O 3, His (Ne); H2 O 2, His (Nd))
empty site))
Azurin (Cd form) 1aiz 1.8 Trigonal His 2 (Nd), Cys
Cd-substituted Ca-binding parvalbumin B 1cdp 1.6 (Octahedral; octahedral) (Asp 2, Glu 2 (1 biden.), Ser, peptide O
(Phe); Asp 3, Glu (biden.), H2 O, peptide O
(Lys))
DNA-binding protein 1cld not applicable Tetrahedral Cys 4
Concanavalin A (Cd substituted for Mg) 1con 2.0 Octahedral Asp 2, H2 O 2, Glu, His (Ne)
L. RulõÂsek, J. Vondr

Cd-6 metallothionein-1 (a-domain) 1dmc not applicable Tetrahedral Cys 4


Cd-6 metallothionein-1 (b-domain) 1dme not applicable Tetrahedral Cys 4
Staphylococcal enterotoxin A 1esf 1.9 Square pyramidal His 2 (Ne, Nd), Ser, terminal N (Ser), Asp
(bidentate)
Ferritin 1ies 2.5 Tetrahedral (one empty site) Glu 3
Thermolysin complexed with Cd 1lne 1.7 (3x octahedral; trigonal bipyramidal) (H2 O 3, Asp 2, peptide O (Gln); H2 O 3, Asp
(biden.), peptide O (His); H2 O 2, Glu 2, Asp,
peptide O (Asn); H2 O 2, His 2 (Ne), Asp)
Phosphotransferase Phosphoglucomutase 1lxt 2.7 Distorted trigonal bipyramidal Asp 3, Ser, SO2ÿ4
Cd-7 metallothionein (a-domain) 1mhu not applicable Tetrahedral Cys 4
Cd-7 metallothionein (b-domain) 2mhu not applicable Tetrahedral Cys 4
Major urinary protein 1mup 2.4 Irregular His 2 (Ne, Nd), Asp (bidentate)
Troponin C 1ncx 1.8 Octahedral Asp 2, Asn (amide O), Glu (bidentate),
peptide O (Arg), H2 O
Metallothionein isoform II 4mt2 2.0 Tetrahedral Cys 4
asek / J. Inorg. Biochem. 71 (1998) 115±127
Table 6
The metalloproteins containing the coordinated mercury (II) ion

Compound PDB code 


Resolution [A] Coordination geometry Binding residues

Carboxypeptidase A (Zn replaced by Hg) 1arm 1.74 (Distorted square pyramid; linear) (Glu (biden.), His 2 (Nd), H2 O, R3 N‡ ; His (Ne),
L. RulõÂsek, J. Vondr

Lys)
Carbonic anhydrase II complexed with nitrate 1can 1.9 Tetrahedral His 3 (2 Ne, Nd), H2 O
Carbonic anhydrase I complexed with HgCl2 1crm 2.0 (Distorted tetrahedral; linear) (His 3 (2 Ne, Nd), Clÿ ; Cys, Clÿ )
Carbonic anhydrase I complexed with inhibitor 1czm 2.0 (Trigonal; (Hg)2 bridged) (peptide O 3 (Cys, Leu, Glu); Cys, H2 O 3 (2
bridging))
Astacin with Zn replaced by Hg 1iac 2.1 Pseudooctahedral (two empty sites) His 3 (Ne),Tyr
Protein R2 of ribonucleotide reductase 1mrr 2.5 (Bent; approximately trigonal) (Cys 2; (Cys-Cys), Tyr, H2 O)
Proliferating cell nuclear antigen 1plq 2.3 (Linear; pseudooctahedral (three empty sites)) (Cys 2; peptide O (Gly, Lys) 2, Cys)
Protease omega 1ppo 1.8 Pseudooctahedral (three empty sites) Cys, His (Nd), H2 O
Proteinase K complexed with Hg 1ptk 2.4 Linear Cys, His (Nd)
Semliki forest virus capsid protein 1vcp 3.0 Linear Cys 2
Cutinase 1xzb 1.75 Linear Cys, acetate (bidentate)
Plastocyanin 3pcy 1.9 Tetrahedral His 2 (Nd), Met, Cys
123 asek / J. Inorg. Biochem. 71 (1998) 115±127
124 L. RulõÂsek, J. Vondr
asek / J. Inorg. Biochem. 71 (1998) 115±127

Table 7
The distribution of coordination geometries and the abundance of the binding residues in metalloproteins (as obtained from PDB)

Metal ion Entries Coordination Binding residues

Co2‡ 9 Octahedral Total 5 His 19 (14 Ne, 5 Nd), H2 O 9,


Distorted 2 Asp 9 (2 bidentate), Glu 7 (3
Tetrahedral 2 bidentate), Tyr, Gln (amide
Trigonal bipyramidal Total 2 oxygen) 1
Distorted 1
(Co)2 bridged 1

Ni2‡ 8 Octahedral Total 5 His 15 (11 Ne, 4 Nd) +1 His


Pseudo 2 bound weakly by Ne, H2 O 9,
Tetrahedral Total 4 Asp 9, Cys 5, Glu, Tyr,
Distorted 3 peptide oxygen (Gly) 1
Pseudo 1
Square pyramidal 1

Cu2‡ 20 Tetrahedral Total 1 His 66 (41 Ne, 25 Nd), Cys


Distorted 1 11, Met, Tyr 5, H2 O 4, Asp 3,
Square planar 3 peptide oxygen (Gly, Met) 2,
Irregular 5 Glu, Gln (amide oxygen),
Trigonal 5 peptide nitrogen (Asp),
Trigonal bipyramidal 4 terminal nitrogen (Asp) 1
Octahedral Total 3
Distorted 1
Linear 1
1

Zn2‡ 49 Tetrahedral Total 4 His 108 (86 Ne, 22 Nd), Cys


Distorted 8 56, Asp 27 (4 bidentate, 1
Pseudo 5 chelated), H2 O 23, Glu 11 (2
Trigonal bipyramidal Total 6 bidentate), Tyr, Lys 2, Ser
Distorted 5 Thr, peptide oxygen,
Pseudo 1 nitrogen (Leu, chelated),
Octahedral Total 1 terminal nitrogen (Thr) 1
Distorted 3
Pseudo 1

Trigonal 1
Square planar 2
Square pyramidal 1
Irregular 1
1
Cd2‡ 16 Tetrahedral Total 8 Cys 25, Asp 19 (3 bidenate),
Pseudo 2 H2 O 19, His 11 (6 Ne, 5 Nd),
Octahedral 7 Glu 10 (3 bidentate), peptide
Trigonal bipyramidal Total 2 oxygen (Phe, Lys, Arg, Asn,
Distorted 1 His, Gln) 6, Ser 3, Asn
Square planar 1 (amide oxygen), terminal
Square pyramidal 1 nitrogen (Ser) 1
Trigonal 1
Irregular 1
Hg2‡ 12 Linear 6 His 16 (8 Ne, 8 Nd), Cys 15,
Octahedral Total 3 H2 O 7, peptide oxygen (Gly,
Pseudo 3 Lys, Cys, Leu, Glu) 5, Tyr 2,
Tetrahedral Total 3 Glu (bidentate), Met, Lys 1
Distorted 1
Trigonal 2
Square pyramidal Total 1
Distorted 1
Bent 1
(Hg)2 bridged 1
L. RulõÂsek, J. Vondr
asek / J. Inorg. Biochem. 71 (1998) 115±127 125

Table 8
The distribution of coordination geometries among the selected transition metal cations (as obtained from CSD)

Coord. number Geometry Co2‡ Ni2‡ Cu2‡ Zn2‡ Cd2‡ Hg2‡

P7 12 7 8 3 25 16
6 210 415 366 108 62 10
Octahedral 131 272 214 64 24 8
5 18 19 499 69 7 4
Trigonal bipyramidal 237 18
Square pyramidal 19 8
4 71 430 523 110 10 28
Tetrahedral 15 2 0 43 4 4
Square planar 26 346 388 22 1 1
3 1 1 4 2 0 9
2 0 1 0 0 0 14
Linear 11

Fig. 1. The preferrred coordination geometries of the selected divalent (2+) metal ions.

Metalloproteins vs. small-molecule crystals. The corre- idues. The complexes of Cu2‡ in octahedral ligand-®eld
lation between the abundance of coordination geome- are subject to Jahn±Teller distortion [19] that results in
tries of selected metal ions in small-molecule crystal the elongation of the molecule along the D4h axis. The
structures and in metal-binding sites of metalloproteins indirect consequence is the preference of square planar
is good. Due to a higher ¯exibility of ligands in smaller coordination in Cu2‡ complexes.
complexes, a metal ion can accommodate its preferred Binding residues. The bonding of metals in metallo-
mode of coordination more easily than in more rigid proteins occurs almost exclusively via side chains of the
metalloprotein structures. The good correlation implies amino acid residues containing sulfur, oxygen or nitro-
that a given metal has greater anity for the metal- gen as donor atoms. Binding through peptide oxygen or
binding site that satis®es its preferred coordination ge- nitrogen or via terminal groups is rare. The following 11
ometry. The explanation for preferred geometries can be residues (binding through their side chains) were found
found in the inorganic chemistry and inorganic bio- in the binding sites of metalloproteins: His (totally 236
chemistry textbooks cited in the introduction. The times), Cys (112), Asp (67), Glu (31), Tyr(11), Met (6),
preference for octahedral coordination by cobalt (II) Ser(4), Lys(3), Gln (2), Asn (1), Thr (1). Histidine is by
and nickel (II) ions stems from the ligand-®eld stabili- far the most common metal-binding residue. It prefers
zation energy gain that is highest in the octahedral li- to bind through Ne rather than Nd. It is rather sur-
gand-®eld. This energy gain cannot be accomplished by prising since in free histidine at neutral pH, the Ne is
closed shell d10 ions of Zn2‡ , Cd2‡ , and Hg2‡ . In their protonated and Nd is deprotonated (pK(Im) ˆ 6.1).
compounds, the arrangement of the ligands is the result However, both N atoms in the imidazole ring are almost
of the equilibrium between the attractive electrostatic equivalent (as the result of its aromaticity) and we
metal-ligand forces and the repulsion of the bound res- suppose that the presence of metal may change the
126 L. RulõÂsek, J. Vondr
asek / J. Inorg. Biochem. 71 (1998) 115±127

Table 9
The mean metal-ligand bond distances in metalloproteins, classi®ed with respect to the binding residues (the numbers in parentheses indicate the
abundance of the given bond, see also Table 7)

Residue\metal Co2‡ Ni2‡ Cu2‡ Zn2‡ Cd2‡ Hg2‡

His (Ne) 2.12 (14) 2.23 (11) 2.11 (41) 2.10 (86) 2.51 (6) 2.36 (8)
His (Nd) 2.09 (5) 2.11 (4) 2.02 (25) 2.14 (22) 2.34 (5) 2.33 (8)
Cys (S) 2.42 (5) 2.15 (11) 2.36 (56) 2.60(25) 2.42 (15)
Asp (Od, Oe) 2.25 (9) 2.11 (9) 2.12 (3) 2.17 (27) 2.30 (19)
Glu (Od, Oe) 2.20 (7) 2.21 (1) 2.21 (1) 2.14 (11) 2.42 (10) 2.54 (1)
Tyr (O) 2.34 (1) 2.27 (1) 2.26 (5) 2.30 (2) 2.89 (2)
Met (S) 2.55 (5) 3.02 (1)
Ser (O) 2.24 (1) 2.26 (3)
Lys (N) 2.26 (2) 2.27 (1)
Gln (O) 2.20 (1) 2.21 (1)
Asn (O) 2.37 (1)
Thr (O) 2.66 (1)
Peptide O 2.36 (1) 2.59 (2) 2.20 (1) 2.33 (6) 2.63 (5)
Peptide N 2.06 (1) 2.23 (1)
Terminal N 2.05 (1) 2.27 (1) 2.41 (1)

above protonation equilibrium. Ne is sterically more order of magnitude less accurate than of small-molecule
accessible and upon approaching of the metal, the hy- crystals (with R-factor less than 5%).
drogen moves from Ne to Nd. Cysteine is second in the However, there are at least three noticeable trends.
abundance row. It binds mainly to softer metals, Cd2‡ First, the distance between a metal and imidazole ni-
and Hg2‡ . Glutamic and aspartic acids are also very trogen of histidine is approximately by 0.07 A  smaller
abundant and they prefer to bind in the monodentate when histidine is bound by Nd (with the exception of
(through one carboxyl oxygen) rather than bidentate zinc (II) cation) rather than Ne. In spite of a higher
mode. It has been already mentioned that coordination abundance of metal±Ne bonding, we presume it implies
through oxygen or nitrogen of the peptide bond is rare that metal-nitrogen bond is stronger for Nd. There is a
in metalloproteins, though carbonyl oxygen and de- small energy loss when deprotonating Ne (and pro-
protonated amide nitrogen are good ligands in smaller tonating Nd) that probably destabilizes the metal±Ne
coordination compounds. A detailed analysis of the bond.
coordination properties of the amide bond can be found Second, the metal±sulfur distance in metal±cysteine
in a review of Sigel and Martin [20]. We presume that it bond is shorter for copper (II) than for cobalt (II) and
is worse steric accessibility of the peptide bond in me- nickel (II) cations, which implicates a stronger bond. As
talloproteins that prevents the peptide oxygen or nitro- has been discussed above, all the ®rst row transition
gen to be among the more abundant ligands. If they metal cations studied here belong to the same class of
participate in the binding, it seems that it is irrelevant borderline metal ions. The fact that anity of copper
what residue they come from. It implies important (II) ions for sulfur is substantially higher (corroborated
consequences for the binding of metals in proteins. The by the high abundance of copper±methionine bonding)
peptide bond retains its chemical behavior even in the seems surprising to us.
complex environment in proteins. Apart from steric re- Third, the bond between a metal and the oxygen in
straints, it is not in¯uenced by the neighboring residues. phenolic ring of tyrosine is weaker as can be inferred
The selectivity of a site for a particular metal should be from the higher interatomic distances (when compared
therefore achieved (besides the discussed preference for to metal±H2 O distance). We presume it is the result of
coordination geometry) primarily by the participation of aromaticity and conjugation of oxygen p-electrons with
di€erent side chains in the binding of that metal. the benzene ring, which weakens the metal±oxygen
The binding residues can be also analyzed with re- bond.
spect to the HSAB concept. The Co2‡ , Ni2‡ , Cu2‡ , and
Zn2‡ belong to the borderline metal ions. They have a
high anity for nitrogen and oxygen, mainly from 4. Conclusions
neutral ligands. On the other hand, Cd2‡ and Hg2‡ be-
long to softer metal ions and prefer binding to softer This contribution is intended to summarize the
donors, such as sulfur. These trends can be seen in Ta- structural information about the metalloproteins and
ble 7. small-molecule crystals that has been collected mainly in
Interatomic metal±ligand distances. The mean inter- the last two decades and is available in PDB and CSD.
atomic metal±ligand distances are summarized in Ta- Single coordination geometry has been assigned to each
ble 9. When examining the results, it must be kept in of the metals investigated here except for cadmium
mind that structure determinations of proteins are by an where the octahedral and tetrahedral coordinations are
L. RulõÂsek, J. Vondr
asek / J. Inorg. Biochem. 71 (1998) 115±127 127

of approximately the same abundance. Good correla- [2] C. Sousa, A. Cebolla, V. de Lorenzo, Nature Biotechnology 14
tion has been found between the coordination arrange- (1996) 1017.
ments in small-molecule crystals and the metal-binding [3] R.G. Pearson, J. Am. Chem. Soc. 85 (1963) 3533.
[4] R.G. Parr, R.G. Pearson, J. Am. Chem. Soc. 105 (1983) 7512.
sites in metalloproteins. It implies that the principles
[5] H. Sigel, D.B. McCormick, Acc. Chem. Res. 3 (1970) 201.
governing the binding of metals in smaller complexes [6] R.B. Martin, J. Chem. Educ. 64 (1987) 402.
and metalloproteins are similar and that pre-designed [7] Metal Ions in Biological Systems, H. Sigel (Ed.), Marcel Dekker,
binding sites with certain coordination geometry will New York, 1974.
exhibit greater anity towards metals that prefer this [8] Metal Ions in Biology, T.G. Spiro (Ed.), Wiley Interscience, New
geometry. York, 1980.
The observed abundance of the binding residues is in [9] Handbook of Metal±Ligand Interactions in Biological Fluids, G.
agreement with the semiquantitative concepts of coor- Berthon (Ed.), vol. 1, Marcel Dekker, New York, 1995.
dination chemistry. The participation of di€erent amino [10] J.J.R. Frausto da Silva, R.J.P. Williams, The Biological Chem-
acid side chains in the binding site is another factor istry of the Elements, Clarendon Press, Oxford, 1993.
[11] A. Vedani, D.W. Huhta, J. Am. Chem. Soc. 112 (1990) 4759.
determining its speci®city.
[12] J.P. Glusker, Adv. Protein Chem. 42 (1991) 1.
[13] F.A. Cotton, G. Wilkinson, Advanced Inorganic Chemistry,
Wiley, New York, 1980.
Acknowledgements [14] Protein Data Bank: http://pdb.pdb.bnl.gov.
[15] A.G. Orpen, L. Brammer, F.H. Allen, O. Kennard, D.G.
This work was supported by the Grant Agency of the Watson, R. Taylor, International Tables for Crystallography,
Czech Republic (grants 203/98/0650 and 203/96/0111) vol. C, Chs. 9.5 and 9.6, Kluwer Academic Publishers, Dordrecht,
and by the Grant Agency of the Academy of Sciences of 1992.
[16] F.H. Allen, J.E. Davies, J.J. Galloy, O. Johnson, O. Kennard,
the Czech Republic (grant A4055801/1998). We would
C.F. Macrae, E.M. Mitchell, G.F. Mitchell, J.M. Smith, D.G.
like to thank to Dr. Ivana Cõsarov
a for several hints Watson, J. Chemical Information and Computer Science 31
about e€ective searches in CSD. (1991) 187.
[17] F.H. Allen, O. Kennard, Chemical Design Automation News 8
(1993) 31.
References [18] H.B. Burgi, J.D. Dunitz, Acc. Chem. Res. 16 (1983) 153.
[19] R. Engelman, The Jahn±Teller E€ect in Molecules and Crystals,
[1] Handbook of Metal-Ligand Interactions in Biological Fluids, G. Wiley Interscience, New York, 1972.
Berthon (Ed.), vol. 2, Marcel Dekker, New York, 1995. [20] H. Sigel, R.B. Martin, Chem. Rev. 82 (1982) 385.

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