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-

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

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

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

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-

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.

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

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

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

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

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

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

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

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

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

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

. 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

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

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

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

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

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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