Báo cáo Y học: Purification and catalytic properties of a CO-oxidizing:H2-evolving enzyme complex from Carboxydothermus hydrogenoformans doc

Purification and catalytic properties of a CO-oxidizing:H
2
-evolving
enzyme complex from
Carboxydothermus hydrogenoformans
Basem Soboh
1
, Dietmar Linder
2
and Reiner Hedderich
1
1
Max-Planck-Institut f
€
uur terrestrische Mikrobiologie, Karl-von-Frisch-Straße, Marburg, Germany;
2
Biochemisches Institut,
Fachbereich Humanmedizin, Justus-Liebig-Universita
¨
t Giessen, Germany
From the membrane fraction of the Gram-positive bacter-
ium Carboxydothermus hydrogenoformans,anenzyme
complex catalyzing the conversion of CO to CO
2
and H
2
was
purified. The enzyme complex showed maximal CO-oxidi-
zing:H
2
-evolving enzyme activity with 5% CO in the head-

space (450 U per mg protein). Higher CO concentrations
inhibited the hydrogenase present in the enzyme complex.
For maximal activity, the enzyme complex had to be
activated by either CO or strong reductants. The enzyme
complex also catalyzed the CO- or H
2
-dependent reduction
of methylviologen at 5900 and 180 U per mg protein,
respectively. The complex was found to be composed of six
hydrophilic and two hydrophobic polypeptides. The amino-
terminal sequences of the six hydrophilic subunits were
determined allowing the identification of the encoding genes
in the preliminary genome sequence of C. hydrogenofor-
mans. From the sequence analysis it was deduced that the
enzyme complex is formed by a Ni-containing carbon
monoxide dehydrogenase (CooS), an electron transfer pro-
tein containing four [4Fe)4S] clusters (CooF) and a mem-
brane bound [NiFe] hydrogenase composed of four
hydrophilic subunits and two membrane integral subunits.
The hydrogenase part of the complex shows high sequence
similarity to members of a small group of [NiFe] hydro-
genases with sequence similarity to energy conserving
NADH:quinone oxidoreductases. The data support a model
in which the enzyme complex is composed of two catalytic
sites, a CO-oxidizing site and a H
2
-forming site, which are
connected via a different iron–sulfur cluster containing
electron transfer subunits. The exergonic redox reaction
catalyzed by the enzyme complex in vivo has to be coupled to

energy conservation, most likely via the generation of a
proton motive force.
Keywords: membrane-bound hydrogenase; carbon mon-
oxide dehydrogenase; iron–sulfur protein; complex I.
Microorganisms can utilize a variety of exergonic redox-
reactions to gain energy for growth. NADH, H
2
,and
formate play an important role as electron donors, while O
2
,
nitrate, Fe
3+
, fumarate, and sulfate are widespread electron
acceptors [1]. There are also several organisms known that
can oxidize CO using O
2
as electron acceptor. Among these
carboxydotrophic bacteria, Oligotropha carboxidovorans
has been intensively studied [2,3]. Only a few microorgan-
isms have been identified that can grow anaerobically with
CO under chemolithoautotrophic conditions and couple the
oxidation of CO to CO
2
with the reduction of protons to H
2
.
CO þ H
2
O ! CO

2
þ H
2
DG

0
¼À20 kJÁmol
À1
Organisms known to grow at the expense of this reaction
are the gram-negative bacteria Rhodospirillum rubrum and
Rubrivax gelatinosus [4–6] and the Gram-positive bacterium
Carboxydothermus hydrogenoformans [7,8].
The biochemical process underlying the conversion of CO
to CO
2
and H
2
has been most intensively studied in
R. rubrum. The carbon monoxide oxidation system (Coo)
is encoded by the coo regulon, which consists of two gene
clusters regulated by the cooA gene [9]. The cooFSCTJ gene
cluster encodes the catalytic subunit (CooS) of the CO
dehydrogenase, an electron transfer protein (CooF) of the
CO dehydrogenase, and proteins required for the insertion
of Ni into the enzyme (CooC, T, and J) [10,11]. The CO
dehydrogenase is a nickel iron–sulfur protein and has been
purified as a single subunit protein (CooS) [12]. The crystal
structure of this enzyme has been determined [13]. Under
certain purification conditions, CooS copurifies with the
iron–sulfur protein CooF, which mediates the electron

transfer from CooS to a membrane-bound hydrogenase [14].
The second gene cluster cooMKLXUH encodes the
hydrogenase [15,16], which belongs to a small group of
membrane-bound [NiFe] hydrogenases. Members of this
family include Ech hydrogenase from Methanosarcina
barkeri [17,18] and Escherichia coli hydrogenase 3 [19,20].
These membrane-bound hydrogenases characteristically
contain six conserved subunits – four hydrophilic proteins
and two integral membrane proteins. Two of the hydro-
philic subunits (HycE and HycG in E. coli hydrogenase 3)
are related to the hydrogenase large and small subunit
conserved in all [NiFe] hydrogenases. The overall sequence
similarity is, however, very low. Furthermore, the hydro-
genase small subunit is considerably smaller than that of
ÔstandardÕ [NiFe] hydrogenases and contains only the
cysteine ligands for the proximal [4Fe)4S]cluster.The
large and small subunits of the membrane-bound hydro-
genases are more closely related to subunits of the energy-
conserving NADH:quinone oxidoreductase (complex I)
Correspondence to R. Hedderich, Max-Planck-Institut fu
¨
r
terrestrische Mikrobiologie, Karl-von-Frisch-Straße, D-35043
Marburg/Germany. Fax: + 49 6421178299, Tel.: + 49 6421178230,
E-mail:
Abbreviation: Coo, Carbon monoxide oxidation system.
(Received 18 July 2002, revised 17 September 2002,
accepted 30 September 2002)
Eur. J. Biochem. 269, 5712–5721 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03282.x
[21]. The membrane-bound hydrogenases contain at least

four additional subunits not found in standard [NiFe]
hydrogenases, but which have homologues in complex I.
Other members of this class of multisubunit membrane-
bound [NiFe] hydrogenases are Eha and Ehb hydrogenases
from the methanogenic archaeon Methanothermobacter
marburgensis [22] and Mbh hydrogenase from the hyper-
thermophilic archaeon Pyrococcus furiosus [23]. The struc-
ture of these enzymes appears to be more complicated than
that of the related [NiFe] hydrogenases. Ech hydrogenase
from M. barkeri is the only member of this hydrogenase
family that has been purified as an intact membrane
complex; the other members have been found to be too
labile to be purified intact [18]. The characterization of a
mutant strain lacking Ech hydrogenase has led to the
elucidation of the physiological function of this hydrogenase
in M. barkeri [24].
The thermophilic Gram-positive bacterium C. hydrogeno-
formans is also able to utilize CO as sole energy source, and
it was therefore predicted to have a CO oxidation system
similar to that of R. rubrum. The purification of two closely
related CO dehydrogenases from C. hydrogenoformans,
designated as CO dehydrogenase I and CO dehydroge-
nase II, has recently been reported [25]. The two purified
enzymes are homodimers of the catalytic subunit (CooS).
The crystal structure of CO dehydrogenase II has been
solved [26].
Here we report on the purification and catalytic proper-
ties of a membrane-bound enzyme complex from C. hydro-
genoformans composed of a hydrogenase and a CO
dehydrogenases. The complex catalyzes the conversion of

CO to CO
2
and H
2
.
MATERIALS AND METHODS
Materials
Dodecyl-b-
D
-maltoside was from Glycon Biochemicals.
Carbon monoxide (99.997%) was from Messer Griesheim.
All chromatographic materials were from Amersham
Pharmacia Biotech or Bio-Rad. All other chemicals were
from Merck or Sigma.
Growth of the organism
C. hydrogenoformans Z-2901 (DSM 6008) was from the
Deutsche Sammlung fu
¨
r Mikroorganismen und Zellkul-
turen (Braunschweig). C. hydrogenoformans was grown
strictly anaerobically in 10-L fermentors at 70 °Cand
pH 7.0 in the medium described in [7], with slight modifi-
cations. To avoid precipitation, the CaCl
2
and MgCl
2
concentrations were lowered to 1.2 and 1.1 m
M
,respect-
ively; in addition pyruvate was added at a concentration of

10 m
M
. The fermentors were continuously supplied with
350 mL of CO/H
2
S(99.9:0.1,v/v)perminandstirredat
1000 r.p.m. When the culture reached an D
578
of % 2, the
cells were harvested under N
2
andstoredat)20 °C.
Purification of a CO-oxidizing:H
2
-evolving enzyme
complex
All purification steps were carried out under strictly anoxic
conditions under an atmosphere of N
2
/H
2
(95 : 5, v/v). Cell
extracts were routinely prepared from 40 g cells (wet mass)
suspended in 50 mL 50 m
M
Mops/KOH (pH 7.0) contain-
ing 2 m
M
dithiothreitol (buffer A). Lysozyme (5 mg) was
added, and the suspension was incubated for 15 min at

20 °C. Cells were disrupted by sonication at 4 °Cin
intervals of 3 · 6 min using an energy output of 200 W
(Bandelin sonicator) at 18 °C. Undisrupted cells and cell
debris were removed by centrifugation at 10 000 g for
20 min.
Crude membranes were isolated from cell extracts by
ultracentrifugation at 160 000 g for 2 h. Membranes were
resuspended in 60 mL buffer A containing 0.5 m
M
deter-
gent (dodecyl-b-
D
-maltoside) using a Teflon Potter homo-
genizer. After a second ultracentrifugation at 160 000 g for
2 h, washed membranes were homogenized in % 70 mL
buffer A (1.8 mg proteinÆmL
)1
) using a Teflon Potter
homogenizer. Dodecyl-b-
D
-maltoside was added to a con-
centration of 16 m
M
[4.6 mgÆ(mg protein)
)1
]. The suspen-
sion was incubated for 12 h at 4 °C with slight swirling.
After centrifugation at 160 000 g for 40 min, the solubilized
membrane proteins present in the supernatant were loaded
onto a Q-Sepharose HiLoad column (2.6 · 15 cm) equili-

brated with buffer A containing 2 m
M
dodecyl-b-
D
-malto-
side (buffer A + detergent). The column was washed with
60 mL buffer A + detergent. Protein was eluted in a
stepwise NaCl gradient at a flow rate of 5 mLÆmin
)1
(60 mL
each in buffer A + detergent): 0.1, 0.2, 0.3, 0.4, and 0.5
M
.
The CO-oxidizing:H
2
-evolving enzyme complex was recov-
ered in the fractions eluting with 0.3
M
NaCl. Protein
was concentrated and desalted by ultrafiltration (Molecular/
Por cellulose ester ultrafiltration membranes, 100-kDa
cut-off, Spectrum) and further purified by chromato-
graphy on ceramic hydroxyapatite (Bio-Rad). The column
(1.6 · 20 cm) was equilibrated with 0.03
M
potassium
phosphate buffer pH 7.0 containing 2 m
M
dithiothreitol
and 2 m

M
detergent. Protein was loaded and the column
was washed with 50 mL of 0.03
M
potassium phosphate
buffer + detergent. Protein was eluted using a linear
gradient from 0.03
M
to 1
M
potassium phosphate (400 mL).
The CO-oxidizing:H
2
-evolving enzyme complex was recov-
ered in the fractions eluting with 1
M
potassium phosphate
while part of the CO dehydrogenase activity was found in
the 0.03
M
potassium phosphate washing fraction.
Fractions containing CO-oxidizing:H
2
-evolving enzyme
activity were concentrated by ultrafiltration as described
above and applied to a Superdex 200 gel filtration column
(2.6 · 60 cm) equilibrated with buffer A + detergent
+0.1
M
NaCl. Protein was eluted using the same buffer.

The enzyme complex eluted after 187 mL (peak maximum)
corresponding to an apparent molecular mass of 450 kDa.
Thyroglobulin (670 kDa), apoferritin (443 kDa), b-amylase
(200 kDa) and alcohol dehydrogenase (150 kDa) were used
to calibrate the column. Protein was concentrated by
ultrafiltration and stored in buffer A + detergent at a
protein concentration of 3 mgÆmL
)1
at 4 °C.
Purification of CO dehydrogenase
The hydroxyapatite fraction containing CO dehydrogenase
activity but no hydrogenase activity (see above) was further
fractionated by gelfiltration on a Superdex 200 column
(2.6 · 60 cm) equilibrated with buffer A + detergent +
0.1
M
NaCl. CO dehydrogenase eluted after 215 mL
Ó FEBS 2002 CO-oxidizing:H
2
-evolving enzyme complex (Eur. J. Biochem. 269) 5713
corresponding to an apparent molecular mass of 120 kDa.
Protein was concentrated by ultrafiltration and analyzed by
SDS/PAGE.
Determination of enzyme activities
The assays were routinely carried out under anoxic condi-
tions at 70 °C either in 8-mL serum bottles or in 1.5-mL
cuvettes. All assays contained 50 m
M
Mops/KOH (pH 7),
2m

M
dodecyl-b-
D
-maltoside, and 2 m
M
dithiothreitol. One
unit of enzyme activity corresponds to 1 lmol H
2
or CO
formed or consumed per min.
Hydrogen-uptake activity with methylviologen as elec-
tron acceptor was determined by following the reduction of
methylviologen at 578 nm. The 0.8-mL assays contained
20 m
M
methylviologen and 0.1 m
M
sodium dithionite. In
standard assays, cuvettes were allowed to equilibrate with
100% H
2
in the headspace (1.2 · 10
5
Pa). CO-oxidizing
activity was assayed under the same conditions, except that
H
2
was replaced by 100% CO in the headspace. One unit of
H
2

- or CO-oxidation activity is defined as the reduction of
2 lmol of methylviologen per min, which is equivalent to
1 lmol of CO or H
2
oxidized per min. When methylene blue
was used as electron acceptor in the hydrogen-uptake assay
or the CO dehydrogenase assay, the assay mixture con-
tained 1.3 m
M
methylene blue instead of methylviologen.
The H
2
formation activity with reduced methylviologen
as electron donor was measured by following the oxidation
of reduced methylviologen at 578 nm. The standard assay
contained 2 m
M
methylviologen, which was reduced with
sodium dithionite to a DE
578
of 2, and N
2
(1.2 · 10
5
Pa) as
the gas phase. The reaction was started by the addition of
protein.
The CO-dependent formation of H
2
was followed by

determining the H
2
concentration in the gas phase in 1-mL
assays in 8-mL serum bottles. Where indicated, the enzyme
was activated with 1 m
M
Ti(III)citrate prior to the assay and
1m
M
Ti(III)citrate was added to the assay mixture. The gas
phase was 100% CO (1 · 10
5
Pa) or as indicated. The
reaction was started by the addition of enzyme. The solution
was stirred vigorously with a magnetic bar. At 1.5-min
intervals, samples from the gas phase were withdrawn, and
H
2
was quantified after separation by gas chromatography.
OneunitofH
2
formation activity is defined as 1 lmol of H
2
produced min.
The gas chromatograph (model Carlo Erba GC Series
6000) was equipped with a thermal conductivity detector.
Gases were separated by a molecular sieve (5 A
˚
). The oven
and injection port were at 110 °C; the detector was at

150 °C. The carrier gas was N
2
at a flow rate of
30 mLÆmin
)1
.
Analytical methods
Nonheme iron was quantified colorimetrically with neo-
cuproin (2,9-dimethyl-1,10-phenanthroline) and ferrozine[3-
(2-pyridyl)-5,6-bis(4-phenyl sulfonate)-1,2,4-triazine] as
described by Fish [27]. Acid-labile sulfur was analyzed as
methylene blue [28].
Nickel was determined by atomic absorption spectro-
scopy on a 3030 Perkin Elmer atomic absorption spectro-
meter fitted with an HGA-600 graphite furnace assembly
andanAS-60autosampler.
Protein concentration was routinely determined by the
method of Bradford using serum albumin as the standard
[29].
Extraction of hydrophobic proteins from the purified
enzyme complex
For the extraction of hydrophobic proteins a modification
of the recently published procedure was used [30]. 350 lg
purified enzyme complex in 100 lL buffer A + detergent
was added to 900 lL chloroform/methanol (67 : 33, v/v).
The mixture was kept on ice for 15 min before centrifuga-
tion (4 °C) at 10 000 g for 20 min. The organic phase,
which contained proteins soluble in chloroform/methanol,
was dried in a SpeedVac. The dried pellet was solved in SDS
sample buffer and was analysed by SDS/PAGE.

Determination of the stoichiometry of the different
subunits present in the CO-oxidizing:H
2
-evolving
enzyme complex
Two different methods were applied. Both methods rely on
the binding of Coomassie brilliant blue to proteins. First,
Coomassie-stained SDS gels were scanned (Scantouch
210, Nikon) and protein bands were quantified using the
IMAGEQUANT
software (Molecular Dynamics).
Alternatively, Coomassie-stained protein bands were
excised from SDS gels. In general protein bands from four
lanes were combined. Protein-bound Coomassie was
extracted with 200 m
M
NH
4
CO
3
in 50% acetonitrile for
14 h at room temperature under vigorous shaking which
resulted in a complete extraction of protein-bound dye.
Gel-pieces were sedimented by centrifugation at 5000 g.The
amount of dye present in the extract was determined
spectrophotometrically at 590 nm.
In-gel tryptic digestion or CNBr cleavage of proteins
and identification of peptides by MALDI-TOF mass
spectrometry
Protein samples were separated by SDS/PAGE and the gel

bands containing protein were excised after staining with
Coomassie brilliant blue. Gel pieces were completely
destained with 200 m
M
NH
4
CO
3
in 50% acetonitrile (three
times) and dried with a SpeedVac.
Tryptic digestion was started with the addition of a
solution containing 0.2 lg trypsin per lLin40m
M
ammo-
nium hydrogencarbonate buffer pH 8.1 containing 10%
acetonitrile. Gel pieces were covered with this solution. The
protein was digested for 14 h at 35 °C. The CNBr cleavages
were performed for at least 14 h in the dark under N
2
at
37 °C by adding a solution containing 1
M
CNBr in 70%
trifluoroacetic acid to the gel pieces. Peptides were extracted
three times from gel pieces by sonication for 20 min in 60%
acetonitrile, 1% trifluoroacetic acid. Extracts were combined
and concentrated in a SpeedVac. Concentrated solutions
were desalted with a ZipTipC18 (Millipore). Peptides were
eluted from the ZipTipC18 using a solution of 10 mgÆmL
)1

a-cyano-4-hydroxysuccinnamic acid in acetonitrile/H
2
O/
trifluoroacetic acid (70 : 30 : 0.1, v/v/v). Subsequently,
aliquots of 1 lL of the eluate were spotted onto a target
disk and allowed to air-dry. Spectra were obtained using a
Voyager DE RP MALDI time-of-flight mass spectrometer
5714 B. Soboh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Applied Biosystems). The accuracy of external calibration
was, in general, better than 0.02%.
Amino acid sequence analysis
Preliminary sequence data of the C. hydrogenoformans
genome was obtained from The Institute for Genomic
Research website at . For the prediction
of transmembrane helices in proteins, programs at non-
commercial servers were used ( />DAS/ />Multiple sequence alignments were made using the program
at />RESULTS
Purification of a CO-oxidizing:H
2
-evolving enzyme
complex from
Carboxydothermus hydrogenoformans
After cell breakage and separation of the membrane
fraction from the soluble fraction, 80–90% of the hydro-
genase activity and 60–70% of the CO dehydrogenase
activity, both tested with methylviologen as electron accep-
tor, were found in the membrane fraction. The distribution
of the enzyme activities strongly depended on the sonication
conditions; prolonged sonication resulted in higher portions
of the two enzyme activities in the soluble fraction. The

membrane fraction also catalyzed the conversion of CO to
CO
2
and H
2
at high rates (Table 1). Three enzyme assays
were used to follow the purification of hydrogenase, CO
dehydrogenase, and a possible complex of both enzymes
(CO-oxidizing:H
2
-evolving enzyme activity) from the mem-
brane fraction of C. hydrogenoformans (Table 1). The crude
membrane fraction was washed with buffer containing
0.5 m
M
dodecyl-b-
D
-maltoside in order to remove included
soluble proteins and proteins loosely associated with the
membrane. In this washing step normally less than 1% of
the hydrogenase but about 40% of CO dehydrogenase
activity were released into the supernatant. Washed mem-
branes containing tightly bound membrane proteins were
solubilized using dodecyl-b-
D
-maltoside at a concentration
of 16 m
M
. All three activities were almost completely
recovered in the solubilized protein fraction. Proteins were

further fractionated by anion-exchange chromatography on
Q-Sepharose HiLoad, chromatography on hydroxyapatite
and by gel filtration chromatography on Superdex 200. In
these chromatographic steps, the three enzyme activities
coeluted (Table 1). Only after chromatography on hydroxy-
apatite a partial separation was observed. A fraction eluting
with 30 m
M
potassium phosphate from this column only
contained CO dehydrogenase activity while a fraction
eluting at 1
M
potassium phosphate contained CO dehy-
drogenase-, hydrogenase- and CO-oxidizing:H
2
-evolving
activity (Table 1). Proteins in this latter fraction were
further purified by gelfiltration on Superdex 200. Hydro-
genase-, CO dehydrogenase and CO-oxidizing:H
2
-evolving
activity was found in a single peak eluting with an apparent
molecular mass of 450 kDa. A fraction containing only
hydrogenase and no CO dehydrogenase activity has never
been obtained.
The enzyme preparation thus obtained was subjected to
SDS/PAGE. Prior to electrophoresis, the samples were
either boiled in SDS sample buffer or incubated in SDS
sample buffer at room temperature for 1 h. In the nonboiled
samples, nine major polypeptides were observed with

apparent molecular masses of 115, 89, 62, 41, 29, 23, 21,
18, and 16 kDa (Fig. 1A, lane 1). In boiled samples of the
same fraction, the 89-kDa polypeptide was no longer
detectable (Fig. 1A, lane 2). The intensity of the 115-kDa
polypeptide decreased in boiled samples and a smear at the
interface to the stacking gel was observed indicating protein
aggregation. The apparent molecular mass of the 115-kDa
polypeptide varied with the acrylamide concentration in the
gel. This protein band was shifted to higher apparent
molecular masses with increasing acrylamide concentra-
tions. The value given was obtained with a 14% acrylamide
gel. In 12% gels this polypeptide migrated with an apparent
molecular mass of about 90 kDa. To identify hydrophobic
polypeptides the purified protein fraction was extracted with
chloroform/methanol (67 : 33, v/v). The 115-kDa and the
29-kDa proteins were selectively extracted using this solvent
mixture, indicating a hydrophobic nature of these polypep-
tides (Fig. 1A, lane 3).
The hydroxyapatite fraction, which only contained CO
dehydrogenase activity but no hydrogenase activity (see
above), was further purified by gel filtration chromatogra-
phy on Superdex 200. CO dehydrogenase activity eluted
from this column with an apparent molecular mass of
120 kDa. An SDS/PAGE analysis of this fraction showed
one protein band with an apparent molecular mass of
62 kDa in samples boiled prior to SDS/PAGE (Fig. 1B).
This polypeptide was identified as CooSI by peptide mass
Table 1. Purification of a CO-oxidizing:H
2
-evolving enzyme complex from C. hydrogenoformans. Theenzymewaspurifiedfrom40gcells(wet

mass). One unit of enzyme activity is defined as the amount of enzyme that catalyzes the reduction of 2 lmol methylviologen by 1 lmol CO (CO
dehydrogenase activity) or H
2
(hydrogenase activity) or the production of 1 lmol H
2
from 1 lmol CO (complex activity). Activities were
determined with either 100% H
2
or 100% CO in the gas phase. Enzymes were not activated with Ti(III)citrate.
Total protein
Hydrogenase
activity
CO dehydrogenase
activity
Complex
activity
Purification step (mg) (U
total
)(UÆmg
)1
)(U
total
)(UÆmg
)1
)(U
total
)(UÆmg
)1
)
Membrane fraction 821 18 061 21 647 610 789 16 145 20

Washed membrane fraction 493 17 971 36 388 571 788 16 065 33
Solubilized membrane proteins 462 15 085 33 404 081 874 13 420 29
Q-Sepharose HiLoad 162 10 285 63,5 382 040 2358 8604 53
Hydroxyapatite (1
M
potassium phosphate eluate) 88 8080 92 251 428 2857 5720 65
Superdex 200 35 6400 183 207 346 5924 4049 116
Ó FEBS 2002 CO-oxidizing:H
2
-evolving enzyme complex (Eur. J. Biochem. 269) 5715
finger printing. Trypsin digestion of this protein yielded
peptides with the following masses (in Da), which matched a
theoretical digest of CooSI (mass deviations from theore-
tical values are given in brackets): 1069.58 (0.05), 1100.52
(0.02), 1144.57 (0.00), 1144.57 (0.03), 1947.19 ()0.15),
2021.14 ()0.08), 2021.14 ()0.08), 2162.06 ()0.17), 2259.33
()0.13), 2278.31 ()0.17), 2279.35 ()0.17), 2727.59 ()0.17).
Protein analysis by amino-terminal sequencing
and mass fingerprinting, and identification of the
encoding genes
The amino-terminal sequences of seven of the nine
polypeptides detected in nonboiled protein samples were
determined. The results are summarized in Table 2. The
amino-terminal sequences of the 89- and 62-kDa protein
bands were found to be identical. Since the 89-kDa band
was not observed in boiled samples, this may indicate that
this protein band is the dimer of the 62-kDa protein (see
below). This is also supported by the observation that the
intensity of the 62-kDa band increased in boiled samples.
The amino-terminal sequences of the 115-kDa and of the

29-kDa polypeptides could not be determined (see below).
Using the sequence information obtained, the encoding
genes were identified in the genome of C. hydrogenoformans
(Table 2). Preliminary sequence data were obtained from
The Institute for Genomic Research website at http://
www.tigr.org. The genes are organized in one hydrogenase
gene cluster and a CO dehydrogenase gene cluster (Fig. 2).
The amino acid sequences deduced from the nucleotide
sequences of these genes show high similarity to the amino
acid sequence of the subunits of CO-induced hydrogenase
and of subunits of the carbon monoxide dehydrogenase
from R. rubrum [10,16]. Therefore, the same nomenclature
was chosen for the respective genes and proteins from
C. hydrogenoformans (Fig. 2). Four of the polypeptides in
Table 2. Determination of the amino-terminal sequences of the subunits of CO-oxidizing:H
2
-evolving enzyme complex by Edman degradation.
Polypeptides were separated by SDS/PAGE, blotted onto poly(vinylidene difluoride) membranes (Applied Biosystems) as described previously [17].
Sequences were determined using an Applied Biosystems 4774 protein/peptide sequencer and the protocol given by the manufacturer. The names of
the identified gene products are given in parentheses. Protein sequences derived from gene sequences are shown. Amino acids identified by Edman
degradation are highlighted in bold. The 89-kDa protein band was found to be a mixture of two polypeptides. The major sequence corresponded to
CooSI, the minor sequence corresponded to CooSII. For CooSI % 20–40 pmol and for CooSII % 5–10 pmol of each amino acid were found after
each reaction cycle.
89-kDa polypeptides
(CooSI) (main sequence)
NWKNSVDPAVDYLLPIAKK
(CooSII) (minor sequence) AKQNLKSTDRAVQQM
62-kDa polypeptide (CooSI) NWKNSVDPAVDYLLPIAKKAGIE
41-kDa polypeptide (CooH) STYTIPVGPLHVALEEPMYFRVEV
23-kDa polypeptide (CooU) MVRNIQEEFTEKLTRALGAEFSLRW

21-kDa polypeptide (CooFI) MATANENYFIYADPRKCLGCKNCEIA
18-kDa polypeptide (CooX) FLKIALRNLFKSPTTDPYPF
16-kDa polypeptide (CooL) MKKILQKIAKKSPWLYRINAG
Fig. 1. SDS/PAGE of purified CO-oxidizing:H
2
-evolving enzyme complex from C. hydrogenoformans. Protein was separated by electrophoresis in a
14% polyacrylamide slab gel (9 · 7 cm) and subsequently stained with Coomassie brillant blue R250 [38]. The arrows indicate the migration
distance of the protein molecular mass marker from MBI Fermentas (marker 1) or the low molecular mass marker from Amersham Pharmacia
Biotech (marker 2). Samples were carboxymethylated with iodoacetamide prior to SDS/PAGE. (A) Samples of the purified enzyme complex (18 lg
protein) were mixed with SDS-sample buffer and samples were either incubated in SDS-sample buffer for 60 min at 20 °C (lane 1) or were boiled for
5 min prior to SDS/PAGE (lane 2). (Lane 3) Proteins were extracted with chloroform/methanol from 80 lg of the complex. The solvent was
evaporated. Proteins were solved in SDS-sample buffer and incubated for 60 min at 20 °C. (B) Purified CO dehydrogenase (4 lgprotein)was
boiled in SDS-sample buffer prior to analysis by SDS/PAGE in 14% gels.
5716 B. Soboh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the purified enzyme could be clearly assigned as subunits of
the hydrogenase: CooH is the hydrogenase large subunit
that contains the [NiFe] active site, CooL is the hydrogenase
small subunit with one binding motif for a [4Fe)4S] cluster,
CooX is an iron–sulfur protein predicted to ligate two
[4Fe)4S] clusters, and CooU is a small protein with no
known cofactor binding motifs. The hydrogenase gene
cluster contains three additional genes, cooM, cooK,and
hypA. cooM encodes a large integral membrane protein
(136 kDa) predicted to form 34 transmembrane helices. The
cooK gene encodes a second integral membrane protein with
a molecular mass of 34.5 kDa and eight predicted trans-
membrane helices. Possible candidates for these integral
membrane proteins are the 115- and 29-kDa polypeptides of
the enzyme preparation, for which the amino-terminal
sequence could not be determined. Both polypeptides are

hydrophobic as shown by the chloroform/methanol extr-
action experiment. These polypeptides were in-gel trypsin or
CNBr digested and the peptides obtained were analysed by
MALDI-TOF mass spectrometry. The peptide masses were
compared with the masses obtained from theoretical digests
of the cooM and cooK derived polypeptides. Trypsin
digestion of the 29-kDa polypeptide resulted in four major
polypeptides, three of which matched the theoretical digest
of CooK. These peptides with masses of 1557.87 Da
()0.01), 1564.86 Da ()0.01) and 1713.97 Da ()0.005) were
found to be located in a large hydrophilic loop of CooK
between amino acids 28 and 73. Most probably the
hydrophobic regions of the protein were not accessible by
the protease. CNBr cleavage, which is a more appropriate
method to obtain peptides from integral membrane proteins
[31], could not be used for the 29-kDa protein because of the
low methionine content of this protein. CNBr cleavage of
the 115-kDa polypeptide resulted in 12 peptides with
molecular masses between 800 and 3000 Da, which could
be assigned to CooM. Mass deviations of the measured
masses from expected masses are given in brackets (in Da):
1004.3 ()0.07), 1134.54 (0.04), 1440.64 (0.19), 1501.81
(0.09), 1519.77 (0.05), 1568.77 (0.06), 2034.89 (0.08),
2147.07 (0.12), 2307.07 (0.23), 2458.20 (0.18), 2467.25
(0.13), 2584.17 (0.20). A theoretical CNBr cleavage of
CooM resulted in 31 peptides in the mass range between 800
and 3000 Da, which is generally used for MALDI-TOF
analysis. Based on these results the 115- and 29-kDa
subunits can be tentatively assigned as CooM and CooK,
respectively. The apparent molecular masses of these

polypeptides, as determined from SDS gels, differ from
the calculated molecular masses. This is typical for integral
membrane proteins, which often show an anomalous
migration in SDS gels [32]. Both polypeptides shifted to
higher apparent molecular masses at higher acrylamide
concentrations as has been described for other membrane
proteins [32].
A large intergenic region is found upstream of cooM.
Directly downstream of cooH, a gene designated as hypA is
located (Fig. 2). The gene product is closely related to the
E. coli HypA and HybF proteins, which are essential for the
maturation of E. coli hydrogenases [33].
The hypA gene is followed by an intergenic region of 116
base pairs. Downstream of this intergenic region are two
genes designated cooF1 and cooSI. cooFI encodes an
electron transfer protein predicted to ligate four [4Fe)4S]
clusters. cooSI encodes the Ni- and Fe-containing catalytic
subunit of CO dehydrogenase. A catalytically active CooSI
dimer has been previously purified and characterized from
C. hydrogenoformans [25]. The 89- and 62-kDa polypeptides
were assigned as CooSI, and the 21-kDa polypeptide was
assigned as CooF1 via amino-terminal sequencing.
C. hydrogenoformans was previously shown to form a
second CO dehydrogenase, designated CO dehydro-
genase II. Upon inspection of the amino-terminal sequence
of the 89-kDa polypeptide a background sequence was iden-
tified, which corresponds to the amino-terminal sequence of
CooSII (Table 2). The data described in Materials and
methods indicate a CooSI to CooSII ratio of 4 : 1 in the
89-kDa protein band. In contrast, the amino-terminal

sequence of the 62-kDa protein band does not contain a
background sequence corresponding to CooSII. From a
densitometric analysis of Coomassie stained SDS-gels it can
be estimated that % 60–70% of CooS migrate as the
monomer (62 kDa protein band) (data not shown). Hence,
the overall content of CooSII on the total CooS content of
the complex can be estimated to be less than 10%.
The two CooS proteins of C. hydrogenoformans have
been shown to form homodimers [25,26]. This explains the
occurrence of two forms of CooS in the SDS-polyacryl-
amide gels of nonboiled protein samples. The deviation of
the apparent molecular mass of the dimer from the
calculated value of 124 kDa may be related to the fact that
the protein dimer is not completely unfolded and thus shows
an anomalous migration behavior. The CooSII dimer might
be more stable in SDS-containing buffer at room tempera-
ture explaining the observation that the CooSII monomer
was not found under these conditions.
Subunit stoichiometry and cofactor content
of the enzyme complex
In Coomassie-stained SDS gels the CO dehydrogenase
subunits, CooS and CooF, showed a higher intensity
Fig. 2. Genetic organization of the co o genes encoding the subunits of the CO-oxidizing:H
2
-evolving enzyme complex of C. hydrogenoformans. The
cooMKLXUHhypA and the cooFISI gene cluster are located on contig 2340 of the preliminary genome sequence. The genes hypA and cooFI are
separated by a 116-bp noncoding region. The genes within the putative cooMKLXUHhypA and cooFISI transcription units are separated by less
than 28 base pairs or even overlap.
Ó FEBS 2002 CO-oxidizing:H
2

-evolving enzyme complex (Eur. J. Biochem. 269) 5717
compared to the hydrogenase subunits. A densitometric
analysis revealed a molar ratio of CooS to CooH, the
catalytic subunit of the hydrogenase, of 2.1 ± 0.1 : 1 and a
molar ratio of CooF to CooH of 1.9 ± 0.1 : 1. The same
results were obtained when the Coomassie dye, bound to
the different subunits, was quantified after extraction with
200 m
M
ammonium carbonate in 50% acetonitrile. These
data suggest a CO dehydrogenase to hydrogenase stoichi-
ometry of 2 : 1 in the purified complex.
Gel filtration chromatography of the complex on a
calibrated Superdex 200 column revealed an apparent
molecular mass of the complex of 450 kDa. This value is
consistent with the molecular mass of a complex containing
a single copy of each hydrogenase subunit (CooM, K, L, X,
U and H) and two copies of each CO dehydrogenase
subunit (CooF and CooS) with a calculated molecular mass
of 441 kDa.
The purified enzyme had a deep brown color. The air-
oxidized minus sodium-dithionite-reduced difference spec-
trum showed a broad absorbance peak between 350 and
500 nm, indicative for the presence of iron–sulfur centers.
The enzyme complex was found to contain 6.9 nmol Ni per
mg protein, 139.5 nmol nonheme iron and 133 nmol acid-
labile sulfur per mg protein. These values correspond to
3 mol Ni per mol enzyme, 61.5 mol nonheme iron and
59 mol acid labile sulfur per mol enzyme using the
calculated molecular mass of the complex of 441 kDa.

They are consistent with calculated values of 3 mol Ni,
65 mol nonheme iron and 66 mol acid labile sulfur per mol
enzyme complex.
CO dehydrogenase and hydrogenase form a tight
complex
Further experiments were performed to elucidate if CO
dehydrogenase and hydrogenase form a tight complex or
whether the two enzymes coelute on the different chroma-
tography columns by coincidence. The chromatographic
properties of purified CO dehydrogenase I were compared
with the properties of the proposed complex of CO
dehydrogenase I and hydrogenase. CO dehydrogenase I
eluted from the hydroxyapatite column with 0.03
M
potas-
sium phosphate while CO dehydrogenase I associated with
the hydrogenase was strongly bound to this column and
could only be eluted from this column with 1
M
potassium
phosphate. CO dehydrogenase I eluted from a Superdex
200 gel filtration column with an apparent molecular mass
of 120 kDa while the proposed complex eluted with an
apparent molecular mass of 450 kDa. These data together
with kinetic data described below strongly suggest that CO
dehydrogenase is tightly associated with the hydrogenase.
Catalytic properties of the enzyme complex
The purified CO-oxidizing:H
2
-evolving enzyme complex is

composed of a hydrogenase and a CO dehydrogenase. The
activities of the two enzymes were determined individually
with methylviologen as artificial electron donors or accep-
tors. The purified enzyme complex catalyzed the formation
of H
2
with reduced methylviologen as electron donor at a
rate of 170 U per mg protein, the reduction of methyl-
viologen by H
2
at a rate of 180 U per mg protein, and the
reduction of methylviologen by CO at a rate of 5900 U per
mg protein. To determine whether hydrogenase and CO
dehydrogenase are electrically connected, the enzyme pre-
paration was tested for its ability to catalyze the conversion
of CO to CO
2
and H
2
. With 100% CO in the headspace, the
enzyme complex catalyzed this reaction at a rate of 120 U
per mg protein. This value increased to 450 U per mg
protein when the assay was carried out with 5% CO in the
headspace and the enzyme complex was activated with
Ti(III)citrate (see below). A linear increase of activity with
increasing protein concentrations was obtained, even at
protein concentrations as low as 45 lg per mL assay
mixture, corresponding to concentrations of the complex of
0.1 l
M

. This behavior suggests that all components involved
in this reaction form a tight complex with an active site for
CO oxidation, an active site for H
2
formation, and the
required electron transfer components. The presence of two
distinct active sites was also supported by inhibition studies;
CO dehydrogenase activity was specifically blocked by
cyanide, and hydrogenase activity was blocked by CO or
acetylene.
TherateofH
2
formation with CO as electron donor and
the rate with reduced methylviologen as electron donor
showed the same pH dependence, with an optimum at
pH 6.5.
Redox-dependent activation of the
CO-oxidizing:H
2
-evolving enzyme complex
CO dehydrogenase from R. rubrum has recently been
shown to be mostly inactive at redox potentials higher than
)300 mV. The enzyme can be converted to an active form
by the addition of strong reductants or by incubation
with CO. This activation process is dependent on the
concentration of CO and the incubation time [34]. The
CO-oxidizing:H
2
-evolving enzyme activity of the C. hydro-
genoformans enzyme complex was also found to increase

with increasing CO concentrations. Maximal activities were
obtained with approximately 40% CO in the headspace of
the assay mixture, corresponding to 250 l
M
CO in solution
at 70 °C. Since the apparent K
m
value of the purified
C. hydrogenoformans CO dehydrogenase I is 18 l
M
[25], a
maximal H
2
-formation rate from CO was therefore expec-
ted at very low CO concentrations (< 100 l
M
). At higher
CO concentrations, the inhibition of the hydrogenase in the
enzyme complex by CO was expected to predominate (see
below). The finding that maximal activity could only be
observed at rather high CO concentrations indicates that
CO dehydrogenase of the enzyme complex is activated in
the presence of CO. To activate the enzyme independently
of CO, the strong reductant Ti(III)citrate, which has been
successfully used to activate other redox enzymes that
require low redox potentials [35], was used. Incubation of
the enzyme complex in the presence of 1 m
M
Ti(III)citrate
for 1 min at 65 °C resulted in an increase of activity that was

maximally pronounced at low CO concentrations. With 5%
CO in the headspace, corresponding to 31 l
M
CO in
solution, the initial activity of the CO-oxidizing:H
2
-evolving
enzyme complex increased about 45-fold after activation
with Ti(III)citrate. In general, the extent of activation was
dependent on how long the enzyme preparation had been
stored before used. Freshly prepared enzyme showed a
higher specific activity and could only be activated to a
lower extent as compared to older preparations. Figure 3
5718 B. Soboh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
shows the dependence of CO-oxidizing:H
2
-evolving enzyme
activity on the CO concentration with enzyme not activated
by Ti(III)citrate (Fig. 3A) and enzyme activated with
Ti(III)citrate (Fig. 3B).
To elucidate if Ti(III)citrate activates CO dehydrogenase,
hydrogenase or both enzymes the influence of Ti(III)citrate
on CO dehydrogenase- and hydrogenase activity was tested.
In these assays methylene blue was used instead of
methylviologen as electron acceptor. Reduced methyl-
viologen is a strong reductant, which could also activate
the enzyme(s). It was found that preincubation of the
complex with Ti(III)citrate had no influence on the rate of
methylene blue reduction by H
2

which proceeded at a rate
of 30 U per mg protein. However, the initial rate of
methylene blue reduction by CO increased by a factor of 21
to a final activity of 1500 U per mg protein. These findings
indicate that the CO dehydrogenase in the complex is
activated by Ti(III)citrate.
Inhibition of the hydrogenase present in the enzyme
complex by CO and acetylene
The rate of hydrogen production with reduced methylvio-
logen as electron donor was determined at different CO
concentrations. To follow this reaction, it was necessary to
inhibit the CO dehydrogenase present in the enzyme
complex. This was achieved with 3 m
M
potassium cyanide,
which is a potent inhibitor of CO dehydrogenase, but does
not inhibit the hydrogenase [36]. The inhibition curve shows
50% inhibition with 45% CO in the headspace, corres-
ponding to % 300 l
M
CO in solution at 70 °C (Fig. 4). A
similar value (50% inhibition at 300 l
M
CO in solution)
has been determined for CO-induced hydrogenase from
R. rubrum [15]. Hence, both hydrogenases are quite insen-
sitive to inhibition by CO compared to most other [NiFe]
hydrogenases: typically, a 50% inhibition of [NiFe] hydro-
genases at CO concentrations of % 40 l
M

in solution has
been observed [37].
H
2
formation from reduced methylviologen or from CO
was inhibited by acetylene when added to the gas phase. A
50% inhibition was observed with 30% acetylene in the gas
phase. The reduction of methylviologen by CO was not
inhibited by acetylene.
DISCUSSION
C. hydrogenoformans catalyzes the conversion of CO to
CO
2
and H
2
to gain energy for growth. We have obtained
an enzyme preparation composed of eight polypeptides
Fig. 4. Inhibition of the hydrogenase activity of the CO-oxidizing:H
2
-
evolving enzyme complex by CO. The enzyme was assayed for the
production of H
2
with reduced methylviologen as electron donor. The
assay (1 mL) was carried out in 8-mL serum bottles containing buffer A
+ detergent, 2 m
M
methylviologen, 0.8 m
M
sodium dithionite, 3 m

M
potassium cyanide, and 40 lg purified enzyme. CO was added to the
headspace of the serum bottle to yield 0–100% CO in the gas phase in a
mixture with N
2
at 1 · 10
5
Pa, corresponding to 0–640 l
M
CO in
solution. Samples from the gas phase were taken at 1-min intervals and
were analyzed for H
2
.
Fig. 3. Dependence of the CO-oxidizing:H
2
-evolving enzyme activity on
the CO concentration. The production of H
2
with CO as electron donor
was assayed. The assay mixture (1 mL in 8-mL serum bottles) con-
tained buffer A + detergent and 45 lg of the purified enzyme complex.
CO was added to the headspace of the serum bottle to yield 0–100%
CO in the gas phase in a mixture with N
2
at 1 · 10
5
Pa, corresponding
to 0–640 l
M

CO in solution at 70 °C.Samplesfromthegasphasewere
taken at intervals of 1.5 min and were analyzed for H
2
.(A)The
enzyme used was not incubated with Ti(III)citrate. (B) The enzyme was
incubated with 1 m
M
Ti(III) citrate for 1 min at 65 °C prior to the
enzyme assay. In addition, 1 m
M
Ti(III) citrate was added to the assay
mixture. Control experiments carried out in the absence of CO showed
that the enzyme catalyzed the formation of H
2
with Ti(III)citrate as
electron donor only with low background activities.
Ó FEBS 2002 CO-oxidizing:H
2
-evolving enzyme complex (Eur. J. Biochem. 269) 5719
catalyzing this reaction at high specific rates. The enzyme
preparation was found to be composed of a CO dehydro-
genase and a hydrogenase. Amino-terminal sequence
analysis allowed the assignment of four of the polypeptides
present in the enzyme preparation as the hydrophilic
subunits of the hydrogenase. Two hydrophobic proteins
were identified which most likely constitute the membrane
spanning part of the hydrogenase. The genes encoding
these six polypeptides could be identified in the genome
of C. hydrogenoformans. The deduced proteins show
high sequence similarity to proteins encoded by the

cooMKLXUH gene cluster of R. rubrum and to the six
subunits of Ech hydrogenase from M. barkeri [16,18].
Purified Ech hydrogenase is composed of six subunits: four
hydrophilic proteins and two integral membrane proteins.
The two additional polypeptides present in the enzyme
preparation from C. hydrogenoformans could be identified
as subunits CooSI and CooFI of the CO dehydrogenase.
The catalytic subunit CooSI of CO dehydrogenase has
been previously purified from the soluble fraction of
C. hydrogenoformans as a homodimer. In this enzyme
preparation the CooFI protein was lacking [25]. CooFI can
be regarded as a four [4Fe)4S] cluster containing polyfer-
redoxin, which is proposed to mediate the electron transfer
between the catalytic subunit CooS and the hydrogenase.
A homologue of CooF, the HycB protein, is also encoded
by the hyc operon of E. coli, which encodes E. coli
hydrogenase 3. HycB is thought to mediate the electron
transfer between formate dehydrogenase and hydrogenase
3inE. coli [20].
The data obtained for C. hydrogenoformans strongly
suggest that the catalytic subunit of CO dehydrogenase
(CooSI), the electron transfer protein CooF and the
hydrogenase form a tight complex with a fixed stoichiom-
etry. First, a coelution of these proteins on three different
chromatography columns was observed. In particular a very
strong binding of these proteins to hydroxyapatite was
found. Elution of the complex from this column was only
possible at a potassium phosphate concentration of 1
M
.In

contrast, the purified CooSI dimer did not bind to
hydroxyapatite at a concentration of 30 m
M
potassium
phosphate. Second, purified CooSI dimer eluted from a
calibrated gel filtration column with a molecular mass of
120 kDa while the complex eluted with an apparent
molecular mass of 450 kDa under the same conditions.
Third,therateofH
2
formation from CO increased linearly
with protein concentration. For a multicomponent system,
containing CO dehydrogenase, the electron transfer protein
(CooF) and the hydrogenase as individual components, a
nonlinear protein dependence would have been expected.
Fourth, in different enzyme preparations a constant subunit
stoichiometry was always observed, the molar concentra-
tion of CooS and CooF being % twofold higher than the
concentration of CooH, the catalytic subunit of the
hydrogenase. A hydrogenase preparation containing a
lower or zero content of CO dehydrogenase was not
obtained using the purification procedure described. In
contrast, CO dehydrogenase devoid of hydrogenase can
be easily obtained. Svetlitchnyi et al. [25] have recently
purified the CooSI homodimer from the soluble fraction of
C. hydrogenoformans. Also the work presented here indi-
cates that only a part of CooSI present in the cell is tightly
associated with the hydrogenase. A possible explanation
could be that the synthesis of CO dehydrogenase and
hydrogenase are not completely coregulated resulting in an

excess of CooSI in the cell. Svetlitchnyi et al. [25] have
recently purified the CooS dimer of a second CO dehydro-
genase (CO dehydrogenase II) from C. hydrogenoformans.
Electron microscopy studies had shown that in vivo this
enzyme is associated with the cytoplasmic membrane.
Purified CO-oxidizing:H
2
-evolving enzyme complex was
found to contain low amounts of CooSII. CooSII was
estimated to account for approximately 10% of the total
CooS content. Meyer and coworkers [25] have proposed
that CooSI interacts with a membrane-bound hydrogenase
and thus has a function in energy conservation. This
proposal is a based on the in vitro reconstitution of a
CO-oxidizing:H
2
forming system composed of cytoplasmic
membranes, CooSI and a greenish-brown-coloured protein
fraction called factor B. Addition of CooSI resulted in a
fourfold increase of activity while addition of CooSII had
no influence on the activity. The isolation of an enzyme
complex, which in addition to a membrane-bound hydro-
genase and the polyferredoxin CooFI mainly contains
CooSI, supports the data by Meyer and coworkers. It is,
however, not yet clear why small amounts of CooSII are
present in the enzyme complex. The function of CO
dehydrogenase II, which has thus far only been purified as
CooSII dimer, is not yet known. Meyer and coworkers [25]
have suggested that the enzyme provides the cell with
reducing equivalents for anabolic reactions. It is, however,

also possible that CooSI and CooSII are isoenzymes that
both are able to interact with the hydrogenase via their
specific CooF proteins. Directly upstream of the cooSII
gene, a gene encoding a second CooF protein, CooFII, is
located. The CooFII protein, which has a calculated
molecular mass of 20 kDa, has not been detected in the
enzyme complex from C. hydrogenoformans.
Thus far Ech hydrogenase from Methanosarcina barkeri
[18,24] and the enzyme complex described in this work are
the only members of a growing family of energy converting
[NiFe] hydrogenases which can be purified and thus are
accessible to further biochemical studies.
ACKNOWLEDGEMENTS
Preliminary sequence data was obtained from The Institute for
Genomic Research website at . Sequencing of
C. hydrogenoformans is accomplished with support from the United
States Department of Energy. This work was supported by the Max-
Planck-Gesellschaft, by the Deutsche Forschungsgemeinschaft, and by
the Fonds der Chemischen Industrie. We thank Karen Brune for
editing the manuscript.
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