Eur. J. Biochem.

261, 1±9 (1999) q FEBS 1999

R E V IE W A RT I C L E

Cobalt proteins
Michihiko Kobayashi1,2 and Sakayu Shimizu1
1
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan; 2Department of Plant Biology,
Carnegie Institution of Washington, Stanford, USA

In the form of vitamin B12, cobalt plays a number of crucial roles in many biological functions. However, recent
studies have provided information on the biochemistry and bioinorganic chemistry of several proteins containing
cobalt in a form other than that in the corrin ring of vitamin B12. To date, eight noncorrin-cobalt-containing enzymes
(methionine aminopeptidase, prolidase, nitrile hydratase, glucose isomerase, methylmalonyl-CoA carboxytransfer-
ase, aldehyde decarbonylase, lysine-2,3-aminomutase, and bromoperoxidase) have been isolated and characterized.
A cobalt transporter is involved in the metallocenter biosynthesis of the host cobalt-containing enzyme, nitrile
hydratase. Understanding the differences between cobalt and nickel transporters might lead to drug development for
gastritis and peptic ulceration.
Keywords: cobalt; noncorrin; transporter; metal; inorganic chemistry; methionine aminopeptidase; prolidase; nitrile
hydratase; glucose isomerase.

Cobalt is a transition metal that occupies a position in the
periodic table between iron and nickel. It has two naturally
occurring oxidation states (Co2+ and Co3+), but can exhibit
oxidation states from ±I to +IV. This metal is rare in comparison
with all known essential trace elements except cadmium and
tungsten: 0.0025% (w/w) in the Earth's crust and 4 £ 10 ±8 %
(w/v) in sea water. Although cobalt is less frequently
encountered in metalloenzymes than the other first-row transi-
tion metals (e.g. manganese, iron, copper and zinc), it is
nevertheless an important cofactor in vitamin-B12-dependent
enzymes. Vitamin B12 [1] contains cobalt in a substituted corrin
macrocycle (a porphyrin relative). The B12 coenzyme possesses
an axial Co(III)-alkyl (5 0 -deoxyadenosine or methyl) group.
Much effort has been concentrated on biochemical studies of
this corrin cobalt. In contrast, only a few proteins containing
noncorrin cobalt have been characterized.
Radioactive cobalt (60Co), which is produced by thermal
neutron bombardment of the natural isotope 59Co, has a half-life
of 5.3 years and decays by b- and g-emission to nonradioactive
nickel (60Ni). It is used as a concentrated source of g radiation in
cancer therapy and food sterilization, and as a radioactive tracer
in biological and industrial applications [2]. Because cobalt has
characteristic spectra correlated with structural properties (such
as observed co-ordination numbers for Co2+ and Co3+), it has
also served as a spectroscopic probe in metalloenzymes.
Substituting cobalt for zinc has often been a useful tool to
investigate the structural basis of catalytic properties in zinc
enzymes and the co-ordination environment of active zinc sites
in other proteins [3].
Correspondence to M. Kobayashi, Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University, Kitashirakawa-
Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. Tel: +81 75 753 6114,
Fax: +81 75 753 6128.
Enzymes: methionine aminopeptidase (EC 3.4.11.18); proline aminopepti-
dase (EC 3.4.11.9); prolidase (EC 3.4.3.7); creatinase (EC 3.5.3.3); nitrile
hydratase (EC 4.2.1.84); glucose isomerase (EC 5.3.1.5) methylmalonyl-
CoA carboxytransferase (EC 2.1.3.1); lysine-2,3-aminomutase (EC 5.4.3.2)
(Received 3 September 1998, revised 13 November 1998, accepted 8
December 1998)
Non-corrin cobalt is receiving increased interest not only in
bioinorganic chemistry but also in biotechnology, and its
availability and remarkable chemical versatility makes cobalt
an invaluable catalyst in the chemical industry (e.g. hydro-
formylation as seen in the oxo process [4]). To date, eight
noncorrin-cobalt-containing enzymes have been isolated
(Table 1). Very recently, a novel protein has been discovered
with a cobalt transporter function [5]. This review summarizes
the structural and functional features of such novel types of
cobalt proteins.
Methionine aminopeptidase
Methionine aminopeptidase is the only protein containing
noncorrin cobalt that has been crystallized and studied by X-
ray crystallography [6]. It is a ubiquitous enzyme that cleaves
the N-terminal methionine from many newly translated poly-
peptide chains in both prokaryotes and eukaryotes. Methionine
aminopeptidase is an important catalyst for the N-terminal
modification to be involved in functional regulation, intracel-
lular targeting and protein turnover [7].
All forms of methionine aminopeptidase examined to date
appear to be metalloproteins and are activated by, or are
sensitive to, metals such as Mn2+, Co2+ and Zn2+. The enzyme
from Salmonella typhimurium is stimulated only by Co2+, not
by Mg2+, Mn2+ or Zn2+, and is inhibited by EDTA. However,
when purified, the native S. typhimurium enzyme does not
contain significant amounts of any metals; enzymatically
important cobalt is loosely associated [8] and so the identity
of the metal ion bound in vivo has not been firmly established.
Determination of the three-dimensional structure of methionine
aminopeptidase from Escherichia coli has, for the first time,
revealed the presence of cobalt ions [6].
The E. coli methionine aminopeptidase is a monomeric
protein of 29 kDa consisting of 263 residues that binds two
Co2+ ions in its active site. The enzyme has several weak
absorption bands between 550 and 700 nm, which arise from
d!d transitions of the d7 Co(II) ions [9]. The crystal structure
displays pseudosymmetry about a pair of cobalt ions [6]. The
2 M. Kobayashi et al. (Eur. J. Biochem. 261) q FEBS 1999

Table 1. Known cobalt-containing proteins.

Enzyme or protein Source Cofactor content Postulated role for cobalt References
Methionine aminopeptidase Animals, yeast, bacteria 2 Co per subunit Hydrolysis [6±9,11±13]
Prolidase Archae 1±2 Co per subunit Hydrolysis [14]
Nitrile hydratase Actinomycetes and bacteria 1 Co in each a-subunit H2O activation, CN-triple-bond [15±18,21,22,24±26]
hydration and protein folding
Glucose isomerase Actinomycetes 1 Co per 4 subunits Isomerization [29,30]
Cobalt transporter Actinomycetes and yeast Cobalt uptake [5,31±33]
Methylmalonyl-CoA Bacteria 1 Co, 1 Zn per subunit Carboxytransferation [39±41]
carboxytransferase
Aldehyde decarbonylase Algae 1 Co-porphyrin per Decarbonylation for aldehyde [43]
ab subunit
Lysine-2,3-aminomutasea Bacteria 0Š.5±1 Co per subunit Mutation [44±46]
Bromoperoxidase Bacteria <0Š.35 Co per 2 subunits Bromination [47]
Cobalt-porphyrin- Bacteria 1 Co-porphyrin per protein Electron carrier [48±50]
containing protein
a
Lysine-2,3-aminomutase also contains iron-sulfur cluster, zinc and PLP as cofactors.

two Co2+ ions are situated between two double b-antiparallel
sheets that constitute part of the active site of the enzyme. In the
enzyme, the cobalt ions are co-ordinated by the residues Asp97,
Asp108, His171, Glu204 and Glu235 (Fig. 1). These five amino
acid residues are conserved in the corresponding sequence of
four other methionine aminopeptidases, with the exception of
Glu235, which is substituted by Gln in the Bacillus subtilis
enzyme. The distance between the cobalt ions is somewhat
larger than twice the covalent radius for cobalt (2.32 A Ê ) and is
similar to that between the two zinc atoms of leucine
aminopeptidase (Zn2+ ± Zn2+ : 2.9 A Ê ).
The three-dimensional structure of a protein often provides
information about its biological role, and in this case has helped
us understand the principles by which bimetallic centers
function. The presence of two Co2+ ions at the active site of
the E. coli enzyme demonstrates the special ability of such two-
metal centers to catalyze hydrolytic reactions. The protein±
cobalt and cobalt±cobalt interactions for each cobalt atom in
methionine aminopeptidase are organized in an approximately
octahedral geometry, with the sixth position unoccupied in the
crystal structure. The two cobalt ions and six of the ligand atoms
(i.e. ligands other than the two oxygen atoms of Glu235 that
Fig. 1. Structure of the catalytic bimetallic core of the Escherichia coli
methionine aminopeptidase in which two cobalt ions are co-ordinated by
the side chain atoms of the five amino acid residues. Co-ordinates are
from the Brookhaven Databank.
directly bind to two cobalts) are very nearly coplanar [6].
Solvent molecules may be bound to both cobalt ions in the
direction of the `absent' octahedral ligands (Fig. 1).
The catalytic mechanism of methionine aminopeptidase is not
known in detail but it appears to share common features with
other metalloproteases, particularly, the bovine leucine amino-
peptidase containing two zinc ions [6]. Recently, Wilce et al.
[10] reported the crystal structure and proposed reaction
mechanism of proline aminopeptidase from E. coli, which
contains two manganese ions per subunit and shows similarity
to the E. coli methionine aminopeptidase in sequence and
structure. Based on these findings, a possible reaction mechan-
ism of the methionine aminopeptidase, which does not require
direct co-ordination of the substrate to the cobalt ions, can be
proposed (Fig. 2). If glutamate and aspartate, which provide
effective binding sites for the cationic cobalt ions, have
carboxylate anions as their side chains, this would lower the
effective charge on the complex, making the formation of a
hydroxide nucleophile less likely. However, the existence of
proximate substrate and metal-bound water (maybe through the
activation by hydrogen-bonding interactions by protein side
chain residues) would generate the catalytic power. In the form
Co2+ ±(OH ± )±Co2+ , an H2O molecule or OH ± ion bridging two
cobalts should have a considerably lowered pKa. This OH ±
group might act as the attacking nucleophile while the substrate
is bound to the binuclear metal core. The next step could be
either of the following: the proton of HB (where B represents a
base) is transferred to the leaving polypeptide (represented by
RII), and the resultant B ± , as a nucleophile, activates a water
molecule to generate OH ± in the binuclear site (scheme I in
Fig. 2) where a hydrogen bond may exist between HB and the
bridging OH ± ; or the proton of a water molecule is transferred to
the leaving polypeptide (scheme II in Fig. 2).
There are two subfamilies of cobalt-containing methionine
aminopeptidases: a prokaryotic class (designated Type I) and a
human class (designated Type II) [11]. Type I includes the
Saccharomyces cerevisiae (type I) enzyme as well as the E. coli,
S. typhimurium, and B. subtilis enzymes, while Type II includes
the S. cerevisiae (type II) enzyme as well as the human and
porcine enzymes. The yeast type-II enzyme is 80% identical to
the human enzyme in its catalytic domain, including complete
conservation of the putative Co2+ ligands. However, in
comparison with the prokaryotic methionine aminopeptidases,
the human and yeast type-I enzymes have N-terminal extensions
in which no obvious sequence similarity is observed. In the
portion of the protein common to the human enzyme and the
yeast type-I and bacterial enzymes, there is 20±40% sequence
q FEBS 1999
Fig. 2. Postulated catalytic mechanisms of methionine aminopeptidase. B, a basic group of the enzyme; RI, N-terminal methionine residue (except its
carboxyl group) of a polypeptide substrate; RII, the leaving polypeptide.
identity, while the yeast type-I enzyme is 40% identical to the
prokaryotic enzymes in this region. However, model building
studies have established that the human methionine aminopep-
tidase could be readily accommodated into the E. coli enzyme
structure and that the Co2+ ligands are fully preserved [11].
Moreover, three-dimensional structure of methionine amino-
peptidase from a hyperthermophilic archaeon Pyrococcus
furiosus [12] which belongs to the Type II group very recently
confirmed the similarity of the catalytic domains between Type I
and II [13].
The E. coli methionine aminopeptidase also displays a fold
that is strikingly similar to the structure of Pseudomonas putida
creatine amidinohydrolase (also known as creatinase) [7], which
hydrolyses creatine to urea and sarcosine, even though the latter
is not a metal-dependent enzyme. This suggests the enzyme
is a member of a diverse superfamily that consists of members
with amidohydrolytic, imidohydrolytic and amidinohydrolytic
functions.
Prolidase
Prolidase, also known as, proline dipeptidase is widespread in
nature, and has been isolated from bacteria [14]. This enzyme
specifically cleaves Xaa-Pro dipeptides, while proline amino-
peptidase (discussed above) [10] specifically releases the N-
terminal residue from a polypeptide, where the penultimate
residue is proline. In concert with other endopeptidases and
exopeptidases, prolidase is thought to be involved in the
terminal degradation of intracellular proteins and may also
function in the recycling of proline [14]. Very recently, prolidase
from P. furiosus has been found to be another cobalt-containing
member (together with methionine aminopeptidase) of the
binuclear N-terminal exopeptidase family [14]. This enzyme is a
homodimer (39.4 kDa per subunit) and contains one cobalt atom
per subunit. Its catalytic activity requires the addition of Co2+
ions, indicating that the enzyme has a second metal ion binding
site. The second Co2+ can be replaced by Mn2+ (resulting in a
25% decrease in activity), but not by other heavy metals such as
Mg2+ , Fe2+ , Zn2+ , Cu2+ , and Ni2+. To summarize, the
prolidase has at least two binding sites per subunit for Co2+ ions:
one is an integral part of the protein (and not removed through
purification or dialysis); and the other has an association
constant of approximately 0.3 mm and is essential for catalysis.
In this regard, the P. furiosus prolidase is similar to members of
Cobalt proteins (Eur. J. Biochem. 261) 3
binuclear metallohydrolases represented by the N-terminal
exopeptidases, the active site of which contain two metal ions.
Interestingly, although the amino acid sequence of the
P. furiosus prolidase shows no significant similarity to those
of methionine aminopeptidases, all five of the cobalt-co-
ordinating residues are conserved in the former enzyme
(Asp209, Asp220, His280, Glu313 and Glu327) [14]. It will
be interesting to investigate whether the reaction mechanism
of this enzyme is similar to that of the methionine
aminopeptidases.
Nitrile hydratase
Nitrile hydratase, which catalyzes hydration of nitriles to amides
as shown below, is a key enzyme involved in the metabolism of
the toxic compounds [15].
R±C;N ‡ H2 O ! R±…CˆO†±NH2
This enzyme is also industrially used in the kiloton-scale
production of acrylamide and nicotinamide from the corre-
sponding nitriles [15±17]. An actinomycete, Rhodococcus
rhodochrous J1 produces two kinds of nitrile hydratases, a
high-molecular mass enzyme (520 kDa) and a low-molecular
mass form (130 kDa) depending on the inducer added to the
culture. Both enzymes are composed of a and b subunits: the
larger enzyme has nine or 10 of each subunit, and the smaller
enzyme has two of each. When this organism is cultured in a
medium containing urea or cyclohexanecarboxamide in the
presence of cobalt ions, the high-molecular mass and low-
molecular mass enzymes, respectively, are selectively induced.
This strain has an obligate requirement for cobalt ions for the
nitrile hydratase activity: both enzymes contain one cobalt ion
per ab pair of subunits as a prosthetic group, but no other
transition metals. The cobalt ions are tightly bound to the
enzymes and are not liberated by dialysis. The enzymes have a
maximum absorption at 410 nm and do not show corrin
absorbance in the visible spectrum [16]. Sequencing of the
genes encoding these enzymes [18] revealed that they share a
characteristic amino acid sequence in each a-subunit: CXXCSC
(Fig. 3). This motif has also been observed in the iron-
containing nitrile hydratases from microorganisms such as
Pseudomonas chlororaphis B23, Rhodococcus sp. N-774 and
Rhodococcus sp. R312 (formerly reported as Brevibacterium
R312) [19]. The side chains of three cysteine residues and the
4 M. Kobayashi et al. (Eur. J. Biochem. 261)
Fig. 3. Alignment of amino acid sequences for the metal-binding site of
the a-subunit of each nitrile hydratase and the g-subunit of T. thioparus
thiocyanate hydrolase. J1-H, R. rhodochrous J1 high-molecular mass
nitrile hydratase [18]; J1-L, R. rhodochrous J1 low-molecular mass nitrile
hydratase [18]; P.put, P. putida [22]; N774, Rhodococcus sp. N-774 [57]; R.
ery, R. erythropolis; B23 [58], P. chlororaphis B23 [59]; Rho, Rhodococcus
sp. [60]; Thio, T. thioparus THI115 thiocyanate hydrolase [23]. The first
three nitrile hydratases contain cobalt ions, whereas the last four NHases
contain ferric ions. Numbers on the right indicate the position of the last
amino acid residue in the motif. The g-subunit sequence of thiocyanate
hydrolase shows sequence similarity to the a-subunit of the nitrile
hydratases. The metal-binding cysteine residues are shown by asterisks.
main-chain amide nitrogens of both the serine and the third
cysteine residue in the motif are ligands in the iron center of the
latter enzyme, which has recently been studied by X-ray
analysis [20].
The high sequence similarity between the cobalt and iron
nitrile hydratases suggests that the cobalt ion is co-ordinated by
the two backbone amide nitrogens and the three cysteine
thiolates in the a-subunit (Fig. 4), which correspond to those of
the iron-containing Rhodococcus sp. R312 nitrile hydratase.
This is supported by the similarities between the cobalt K-edge
X-ray absorbance spectrum of high-molecular mass nitrile
hydratase and the Fe3+ K-edge spectrum of the Rhodococcus
sp. R312 nitrile hydratase, indicating that the ligand environments
Fig. 4. Proposed cobalt center of a high-molecular mass nitrile
hydratase. The cobalt center of high-molecular mass nitrile hydratase is
formed with three cysteine thiolates and the two main-chain amide nitrogens
as ligands in a square-pyramidal geometry represented by the dotted lines.
This drawing is based on the stereoview of the iron center of the
Rhodococcus sp. R312 nitrile hydratase [20].
q FEBS 1999
of the metal ions in the cobalt-and iron-containing nitrile
hydratases are very similar [21]. The cobalt K-edge spectrum of
the high-molecular mass nitrile hydratase also suggests six
co-ordinate cobalt and a distorted octahedral symmetry of the
cobalt center in the enzyme. The native protein is EPR-silent, as
is expected for low-spin Co3+. Chemical reduction of the native
enzyme by dithionite and the artificial dye methylviologen
results in a species with the EPR signature of a low-spin Co2+
complex [21]. Consequently, the cobalt-containing nitrile
hydratase is the first example of a native protein that contains
a noncorrin Co3+ ion with a mixed S and N ligand field of
constituent amino acid of the enzyme.
Very recently, another cobalt-containing nitrile hydratase,
which exhibits enantioselectivity for nitriles such as 2(S)-(4 0 -
chlorophenyl)-3-methylbutyronitrile as a substrate, was isolated
from P. putida [22]. In this enzyme, cobalt exists as a noncorrin
low-spin Co3+ ion with a distortion from the octahedral
symmetry. This enzyme is similar to the R. rhodochrous J1
nitrile hydratases and contains the CXXCSC sequence con-
served in all sequenced nitrile hydratases (Fig. 3), suggesting
that these three cysteines and serine were co-ordinated to the
Co3+ ion. This sequence motif was also found in thiocyanate
hydrolase from Thiobacillus thioparus THI115 [23], which
catalyzes the conversion of thiocyanate ( ± SCN) to carbonyl
sulfide (S = C = O) and ammonia, although the involvement of
metals in the latter enzyme remains unknown.
Whether the cobalt center of the cobalt-containing nitrile
hydratases is the site for nitrile-binding and hydrolytic cleavage
of a CN triple bond in the substrate is unknown, but the EPR
spectra described above strongly suggest a role for the cobalt ion
as a Lewis acid in the catalytic reaction. From these studies on
the structure and function of the cobalt-containing and iron-
containing nitrile hydratases, it is possible to propose the
following scheme for the biocatalysis of nitrile hydration by the
cobalt nitrile hydratases [17]. Firstly, binding of a nitrile occurs
close to a OH ± ion (or a metal-bound water molecule). Then
either OH ± (scheme I in Fig. 5) or a water molecule (activated
by OH ± as a general base, scheme II in Fig. 5) attacks the nitrile
carbon atom to form an imidate [R±C(± OH) = NH]. Finally,
the imidate is converted to an amide.
Recent molecular analyses have furthered our understanding
of the regulation of nitrile hydratase synthesis [24±26]. The
addition of cobalt ions to the culture medium is necessary for
both catalytic activity and synthesis of nitrile hydratases in
R. rhodochrous M8 [26] and R. rhodochrous J1 [15±17], and no
other metals can substitute for cobalt ions. R. rhodochrous M8
shows cobalt-dependent transcription of the gene encoding
nitrile hydratase, a rare example of the specific regulation of
intracellular enzymes at the transcriptional level by the metal
ions that are the prosthetic groups of the enzymes. Metallor-
egulatory proteins, which exert metal-responsive control of
genes involved in metabolism (e.g. iron uptake and storage,
copper efflux, mercury detoxification) have been well charac-
terized [27], in contrast to the absence of such information about
cobalt-dependent enzymes.
In R. rhodochrous J1, by contrast, the expression of both the
high [24] and low [25] molecular mass enzymes nitrile
hydratases are regulated at the transcriptional level by amide
(the reaction product), not by cobalt ions. NhlD, a negative
regulator of the expression of the low-molecular mass enzyme,
has a notable sequence similarity to MerR, CadC and ArsR,
which regulate the transcription of heavy-metal resistance
systems (i.e. detoxification or transport of heavy metals) [25].
These three regulatory proteins repress the expression of the
respective structural genes, which confer heavy-metal resistance;
q FEBS 1999
Fig. 5. Proposed catalytic mechanisms of
nitrile hydratase. B represents a basic group of
the enzyme.
the repression may be relieved by the presence of the heavy-metal
ions, Hg2+ , Cd2+ and AsO33-, respectively. These findings raise
the possibility that the NhlD repressor might have lost the function
of a heavy-metal sensor during the course of evolution [25].
Why do R. rhodochrous J1 NHases use cobalt as an essential
cofactor? There may be two main reasons: firstly, cobalt is a
very good catalyst for CN-triple-bond hydration; and, secondly,
cobalt is required for the folding of the host enzyme. In addition
to the function of the active center, the cobalt ions may play a
role in enhancing the folding or the stabilization of the subunit
polypeptides of the enzyme. However, the addition of nickel or
iron to the growth medium results in the formation of nitrile
hydratase protein that possesses no catalytic activity (M.
Kobayashi et al. unpublished results). This indicates that both
nickel and iron may be able to play a role similar to that
proposed for cobalt in enzyme folding.
Glucose isomerase
Glucose isomerase is one of the most highly used (in terms of
weight) enzymes in industry [28]. It catalyses the reversible
isomerization of d-glucose to d-fructose. Isomerization of
glucose to fructose is of commercial importance in the
production of high-fructose corn syrup. Although this enzyme
requires divalent cations for the activity, its specific requirement
depends on the source of enzyme. Co2+ is essential for glucose
isomerase production by Streptomyces strain YT-5 [29] and
Streptomyces albus [30]. The enzyme from S. albus has been
characterized in detail, and was the first noncorrin-cobalt-
containing enzyme reported. It is tetrameric (16.5 kDa) and
contains one noncorrin cobalt, which shows a low-spin Co2+
complex in EPR measurements. The cobalt ion can be removed
by EDTA treatment, resulting in a loss of activity. Extended X-
ray-absorption fine-structure analyses showed a distortion of the
octahedral symmetry of the cobalt center, a binding to N-ligands
or O-ligands of protein donor atoms (constituent amino acids of
the enzyme), and an absence of S-ligands, while electron nuclear
double resonance spectra suggested only one type of N-ligand,
which appeared to be imidazoles nitrogens from histidine
residues. The visible spectrum of the enzyme exhibits only one
shoulder peak at 410 nm and a weak maximum at 735 nm, in
agreement with a distorted octahedral co-ordination of Co2+.
When treated with cyanide in the presence of dithionite, the
shoulder shifts from 410 nm to 385 nm. These spectra were also
observed for the cyanide derivative of Co2+-substituted carbonic
Cobalt proteins (Eur. J. Biochem. 261) 5
anhydrase which showed a low-spin complex, indicating that the
absent octahedral ligand of the glucose isomerase is replaced by
cyanide.
It is important to reduce the amount of cobalt added to the
culture medium during high-fructose corn syrup production
because of the health hazards from consumption of Co2+ and the
environmental pollution arising from the disposal of spent
media. The substitution of other metals for cobalt during
production is therefore preferable [28].
Cobalt transporters
Several transition metals that play an essential role as cofactors
in many biochemical processes, must be actively incorporated
into cells against concentration gradients, because of the trace
concentrations outside cells and the substantial amounts present
within cells [31]. Although these metals are normally found at
relatively low concentrations in the environment, concentrations
of trace metals in excess of normal physiological levels can be
toxic.
COT1 [32] has been isolated as a suppressor of cobalt toxicity
in S. cerevisiae by sequestration or compartmentalization within
the mitochondria of cobalt ions that cross the plasma membrane.
COT1 is involved in cobalt uptake, but is not solely responsible
for it. This protein contains staggered histidine residues in the
hydrophilic loop between the transmembrane domains 4 and 5.
Together with the above sequence, these sequences
[HGHGHGHSH (positions 140±148) and HTHTHAH (posi-
tions 163±169)] might be responsible for the co-ordination of
cobalt ions [32]. COT1 also participates in zinc uptake,
decreasing the free cytoplasmic concentration of zinc ions,
either by transportation into the mitochondrial matrix or by
direct co-ordination [33]. GRR1 (formerly known as COT2) is
also involved in both cobalt and zinc uptake in S. cerevisiae
cells [33]. However, it has recently been reported that this might
not be the case but that GRR1 may play a more general role in
yeast physiology and indirectly control the permeability of the
membrane to cobalt and zinc ions, or the driving force for the
uptake of cobalt and zinc [33].
In R. rhodochrous J1, on the other hand, two distinct systems
are involved in the accumulation of cobalt ions: a nonspecific,
low-affinity magnesium transporter (M. Kobayashi et al.
unpublished data) and a high-affinity system specific for cobalt
[5]. In R. rhodochrous J1, there is an open reading frame (nhlF),
located between the genes encoding the low-molecular mass
6 M. Kobayashi et al. (Eur. J. Biochem. 261) q FEBS 1999

Fig. 6. Secondary structure model of the cobalt transporter (NhlF) based on its hydropathy profile [5] and on the model of the nickel transporter
(HoxN) [34]. Segments shown in boxes are potential transmembrane a-helical domains, connected by hydrophilic loops. His68, Asp73 and His74 in NhlF
(indicated by the bold open circles) correspond to His62, Asp67 and His68 in HoxN, respectively. Amino acids (other than the above three residues) that are
identical in NhlF and all the nickel transporters, are indicated by solid circles, and the residues conserved in both NhlF and HoxN are noted by solid boxes. The
motif VXLHVLGXAL, which is also found in COT1, is in the first transmembrane region.

nitrile hydratase and amidase, which has sequence similarity
with genes encoding nickel-transporters [34,35]. Expression of
nhlF significantly enhances nitrile hydratase activity in
R. rhodochrous ATCC12674 transformant cultured in cobalt-
limited conditions and confers the ability to take up 57Co on
both R. rhodochrous and E. coli hosts. The nhlF-dependent
cobalt uptake activity is markedly inhibited by the addition of
excess Ni2+ to the cell, although other metals such as Mn2+,
Fe2+ and Cu2+ have no effect. The inhibitory effect by
uncouplers (i.e. CCCP and SF6847) [5] on cobalt uptake
suggests that NhlF mediates cobalt transport into the cell in an
energy-dependent manner [5]. The sequence of this transporter
shows eight putative hydrophobic membrane-spanning domains,
similar to those of nickel transporters [34,35]. The topological
model of the cobalt transporter (Fig. 6) is based upon that of the
nickel transporter (HoxN) [34] from Alcaligenes eutrophus.
NhlF contains nine histidine residues and two cysteine residues;
one histidine (His306) and one cysteine (Cys301) are located on
the fourth outside loop and may comprise a cobalt-binding site
for the initial fixation of this metal. The transmembrane
segments could form a cobalt channel in which the four
histidines might function as transient cobalt-binding ligands.
Recently, Eitinger et al. [34] reported that His62, Asp67 and
His68 in the second transmembrane segment of HoxN are
crucial for nickel uptake. This HXXXXXDH-motif is present in
NhlF as well. This putative cobalt-binding motif is likely to play
a similar role in the cobalt transporter, and could explain the
inhibition of nhlF-dependent cobalt uptake by nickel and that of
HoxN-dependent nickel uptake by cobalt.
There are a few transporters that act on a broad-range of
divalent cations (Zn2+, Ni2+, Cd2+ and Co2+) [36]. NhlF does
not show sequence similarity to either the CorA Mg2+ transport
system [37], which mediates influx of Co2+ as well as
Mg2+ and Ni2+, or to the proteins involved in the active efflux
system of broad specificity for metals, which have been
characterized in detail [38]. NhlF shows structural similarity
only to the nickel transporters suggesting a novel transporter
superfamily. NhlF shows little sequence similarity to COT1
(only the motif VXLHVLGXAL) [5,32]. This 10-amino acid
motif is found in a region of helix 1 of NhlF that corresponds to
helix 5 of COT1. In this homologous region, both NhlF and
COT1 contain a histidine residue (His34 in NhlF) that is a
potential metal-binding amino acid. None of the nickel
transporters contain histidine at the corresponding site,
suggesting it has a functional role in cobalt-specific recogni-
tion [5].
Considering the distribution of the cobalt-containing methio-
nine aminopeptidases in both prokaryotes and eukaryotes, cobalt
q FEBS 1999
transporters may also exist in humans. Further analyses of cobalt
transport systems will be invaluable for increasing our under-
standing of cobalt homeostasis in all living organisms.
Other proteins involving cobalt
Methylmalonyl-CoA carboxytransferase. The biotin-containing
enzyme methylmalonyl-CoA carboxytransferase (transcarbox-
ylase) as found in Propionibacterium shermanii [39] is a
complex multisubunit enzyme (26S) composed of three types of
subunits (1.3S, 5S, and 12S) that catalyzes the transfer of a
carboxyl group from methylmalonyl-CoA to pyruvate to form
propionyl-CoA and oxaloacetate. The 5S (120 kDa) subunit is a
homodimer containing Co2+ and Zn2+ (1 mol of each per
monomer), and assembles with two 1.3S (12 kDa) biotin-
containing subunits to form a stable complex termed the 6S
subunit. The 12S subunit is a hexamer composed of six identical
monomers and combines with 6S subunits to form active
methylmalonyl-CoA carboxytransferase.
There are three types of cobalt in methylmalonyl-CoA
carboxytransferase [39]. The first type is catalytic cobalt.
Detection of the tightly-bound cobalt in the enzyme at
approximately 12K by EPR measurements is consistent with
high-spin Co2+ in a distorted octahedral environment. Two
rapidly exchanging water ligands are proposed to be co-
ordinated to the enzyme-bound Co2+ [40]. The second type
consists of six cobalt ions (per 26S enzyme) that are detected
when the methylmalonyl-CoA carboxytransferase is incubated
with exogeneous Co2+ at pH 8, during which some Co2+
becomes tightly bound. The third type of cobalt in the enzyme
is more weakly bound and is in equilibrium with the free Co2+ ,
which must be maintained at about 2 mm in order to stabilize the
enzyme at alkaline pH. This weakly bound Co2+ is involved in
the equilibrium of the association and dissociation of the
enzyme [39].
Although the three-dimensional structure of the 1.3S subunit
of the P. shermanii methylmalonyl-CoA carboxytransferase [41]
is known, that of Co2+-containing 5S subunit is not. The
structure of the corresponding biotin carboxylase of E. coli [42]
has been determined. High-resolution analysis of this structure
should clarify the detailed catalytic mechanism in which the
biotin carboxyl carrier protein provides a `swinging arm' [42]
that moves between the biotin carboxylase and carboxytrans-
ferase subunits of the complex.
Aldehyde decarbonylase. The cobalt-containing enzyme alde-
hyde decarbonylase converts a fatty aldehyde to a hydrocarbon
and CO [43]. This enzyme is responsible for a key step in the
biosynthesis of hydrocarbon compounds, which is ubiquitous in
living organisms. This compound is an important energetic
source and plays a role in waterproofing, which prevents
desiccation. The aldehyde decarbonylase in the green colonial
alga Botryococcus braunii consists of a and b subunits (66 and
55 kDa, respectively) and consists of one cobalt±porphyrin per
ab pair of subunits [43]. This is the first report of enzymatic
reactions involving noncorrin-cobalt in plants.
Lysine 2,3-aminomutase. Lysine 2,3-aminomutase from Clos-
tridium SB4 catalyzes the reversible isomerization of l-lysine
into l-b-lysine [44], a reaction in which the hydrogen in the 3-
pro-R position of lysine is transferred to the 2-pro-R position of
b-lysine and the 2-amino group of lysine migrates to carbon-3 of
b-lysine. The enzyme contains three cofactors ± pyridoxal
phosphate, Fe±S centers and cobalt or zinc. S-Adenosylmethio-
nine is required to activate the enzyme and this activation is
Cobalt proteins (Eur. J. Biochem. 261) 7
thought to take place by a reaction with a reduced metal center,
leading to the formation of a reactive adenosyl-metal cofactor.
The enzyme purified from cells cultured in media supplemented
with CoCl2 contains higher levels of cobalt than that from cells
grown in media without added cobalt. The enzyme seems to
contain either one or two Co2+ per dimer of subunits and for
Clostridium SB4, the level of cobalt in the enzyme depends on
the concentration of CoCl2 in the growth medium [45]. Electron
paramagnetic resonance measurements suggest a high-spin
Co2+ in the enzyme and therefore an octahedral co-ordination.
The enzyme does not contain, and is not activated by, vitamin
B12. The absence of 59Co-hyperfine splitting in the EPR
spectrum of the Fe±S cluster in lysine 2,3-aminomutase rules
out the possibility that cobalt and iron coexist within a mixed
metal±sulfur cluster [45].
The following mechanism is proposed as one possibility [46].
S-Adenosylmethionine may adenosylate the reduced [Fe±S]
cluster, and the resulting adenosyl-(Fe±S) complex may
transiently and reversibly generate the 5 0 -deoxyadenosyl radical,
which is captured and stabilized by reaction with Co2+ in a
nearby binding site. The identification of the putative adenosyl-
cofactor and the role of Fe±S clusters and cobalt in the
activation of lysine 2,3-aminomutase is under continuing
investigation [46].
Bromoperoxidase. Bromoperoxidase from Pseudomonas putida
[47], which catalyzes the formation of a carbon±bromine bond
in the presence of peroxides, is activated by incubation with
cobalt ions but not with other transition metals such as iron,
nickel, zinc and vanadate. The enzyme is dimeric, 68 kDa in
size and contains cobalt (0.035 nm ^ 0.1 mol´mol enzyme ±1),
while bromoperoxidase from the marine red alga Corallina
pilulifera contains non-heme iron, and bromoperoxidase from
the fungus Curvularia inaequalis contains zinc and iron. The
low concentration of cobalt in the P. putida enzyme can be
explained by the partial dissociation of cobalt from the enzyme
during the purification [47]. Although the enzyme is likely to
contain cobalt as an essential metal for bromination [47], further
studies are required to elucidate its function.
Cobalt±porphyrin-containing proteins. In addition to the
existence of Co±porphyrin-containing proteins in plants as
described above, such proteins have also been found in sulfate-
reducing bacteria. Desulfovibrio gigas and Desulfovibrio
desulfuricans produce cobalt-containing proteins (0.8±0.9 mol´-
mol protein ±1) [48±50]. These proteins (16.7 kDa and 13 kDa,
respectively) are violet in solution with absorption bands
characteristic of a metal-porphyrin center exhibiting maxima
at around 420 nm and 580 nm, with a shoulder at 550 nm [49].
The cobalt±porphyrins are not covalently bound to the proteins.
Although the electron transfer reaction requires catalytic
amounts of cytochrome c3 as reported for the other non-heme-
containing electron carriers of Desulfovibrio, the Co±porphyrin-
containing protein from D. desulfuricans is reduced by hydro-
genase under hydrogen, suggesting this cobalt protein may
function as an electron carrier [50]. Prosthetic groups in these
cobalt-containing proteins are found to be cobalt isobacterio-
chlorins. The oxidized form of these prosthetic groups appears
to be Co3+-sirohydrochlorin or a derivative of the macrocycle.
C ON C L U S I O N
Although metal ions play a variety of roles in natural proteins,
including nucleophilic catalysis, electron transfer, and the
stabilization of protein structure, the functions of cobalt have
8 M. Kobayashi et al. (Eur. J. Biochem. 261)
rarely been studied. Cobalt is used in a limited number of
enzymes, unlike iron and zinc. However, there are several
enzymes that are activated by cobalt ions: E. coli acetylornithi-
nase [51], Methanobacterium thermoautotrophicum cyclic 2,3-
diphosphoglycerate hydrolase [52], and rat liver a-d-mannosi-
dase [53]. However, it is not clear whether cobalt ions are
involved in these enzymes.
It is important to bear in mind the adjacent arrangement of
iron, cobalt and nickel in the periodic table. A nickel
tetrapyrrole coenzyme (factor F430) that is a cofactor of
methyl-CoM reductase has a hybrid macrocyclic structure
between that of corrin and porphyrin, and seems to be an
missing evolutionary link between cobalt-containing corrin and
iron-containing porphyrin systems [54,55].
Although the structures, spectral signatures and catalytic
properties for metallocenters have been characterized, the
mechanisms by which appropriate metal ions are incorporated
into the corresponding proteins are less well understood.
Consequently, the discovery of cobalt transporters could provide
invaluable information on metallocenter biosynthesis and may
stimulate new research in the field of biological metal
incorporation. Understanding the structural and functional
differences between cobalt and nickel transporters may even
allow the development of new therapeutic agents for peptic
ulceration caused by the infection of the human gastric pathogen
Helicobacter pylori, in which the nickel transporter (NixA) [35]
provides nickel for the enzyme urease, which is essential for
H. pylori colonization in the very acid pH of the stomach.
Spectroscopic and structural analyses of a novel type of nitrile
hydratase (containing cobalt and iron in Agrobacterium
tumefaciens) [56], which is involved in the biosynthesis of the
plant hormone indole-3-acetic acid from indole-3-acetonitrile,
will contribute to major advances in our understanding of the
metal functions.
The differences in the physiological functions of each cobalt
protein determine the nature of the ligands and the structures of
the cobalt-binding sites. Further research on cobalt proteins will
allow major advances in our understanding of their biological
function as well as their structure.
A C K N O W L E D G E ME NT S
We wish to thank Dr Akihiko Fujie (Stanford University) for his valuable
discussion and encouragement. We would like to thank H. Ueno for
preparing Fig. 1. This work was supported in part by a Grant-in-Aid for
Scientific Research on Priority Areas from the Ministry of Education,
Science and Culture of Japan.
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