THE ! J O U R N A L OF BIOLOGICAL CHEMISTRY c) 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 27, Issue of September 25, pp . 18395-18403,1391 Printed in U.S.A.

Characterization of the CO Oxidation/H2 Evolution System of Rhodospirillum rubrum ROLE OF A 22-kDa IRON-SULFUR PROTEIN IN MEDIATING ELECTRON TRANSFER BETWEEN CARBON MONOXIDE DEHYDROGENASE AND HYDROGENASE*

(Received for publication, April 12, 1991)

Scott A. Ensign$§ and Paul W. LuddenSlI From the $Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison,

~~~

Madison, Wisconsin 53706

The response of the membrane-associated carbon monoxide dehydrogenase (CODH) from Rhodospiril- lum rubrum to solubilization by detergents and organic solvents, the properties of solubilized CODH, and the mechanism for coupling CO oxidation to hydrogen ev- olution via a CO-induced hydrogenase activity have been investigated. The release of CODH by a variety of ionic and nonionic detergents occurs in a redox- dependent fashion: CODH is solubilized in the presence of low-potential reductants (dithionite, CO, and Hz) but is resistant to solubilization from membranes prepared in the absence of reductant or membranes prepared in the presence of reductant and subsequently dye-oxi- dized. This redox-dependent response to detergent sol- ubilization has been exploited to release CODH from the membranes in a purified state. CODH can also be solubilized from deoxycholate-washed membranes in a redox-independent manner with 20% ethanol. CODH solubilized by deoxycholate or ethanol, when purified to homogeneity by the protocol previously described for heat-solubilized CODH (Bonam, D., and Ludden, P. W. (1987) J. Biol. Chem. 262, 2980-2987), i s associ- ated with a previously unobserved 22-kDa protein. The 22-kDa protein can be dissociated from CODH with acetonitrile and can be reconstituted with CODH, after removal of acetonitrile, in a stoichiometric (1:l) fashion. The isolated 22-kDa protein contained 4.0 iron atoms, a reducible Fe-S center, and was Oz- and heat-labile. The 22-kDa protein did not alter the cat- alytic properties of CODH as assayed in vitro with methyl viologen as the electron acceptor for CO oxi- dation, but was required for reconstituting CO oxida- tion to hydrogen evolution via the CO-induced mem- brane-bound hydrogenase. Other electron carrier pro- teins (ferredoxins and flavodoxin) were ineffective a t coupling CO oxidation and hydrogen evolution. We conclude that the 22-kDa protein is a reversibly dis- sociable subunit of CODH that mediates electron trans- fer to hydrogenase.

* This research was supported in part by the College of Agricultural and Life Sciences at the University of Wisconsin-Madison and by grant DE-FG02-87ER13691 from the United States Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by Training Grant 5T32GM07215-14 from the Na- tional Institute of Health and by a fellowship from the College of Agricultural and Life Sciences during the course of these studies.

1 To whom correspondence should he addressed.

A diverse set of bacteria is capable of oxidizing CO through the action of carbon monoxide dehydrogenases (CODH).’ These include the aerobic carboxydobacteria (1, 2 ) and the anaerobic sulfate reducers (3, 4), acetogens (5, 6), methano- gens (7,8), and phototrophs (9,lO). The properties of CODHs purified from the different classes of bacteria and the meta- bolic roles of these enzymes vary considerably and have been reviewed elsewhere (6, 11, 12).

CODH from the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized as a monomeric, O,-labile, nickel- and iron-sulfur-containing enzyme of mo- lecular weight 62,000 (13). The enzyme is induced to high levels (2-5% of cell protein) in cultures of R. rubrum growing photoheterotrophically with malate as a carbon source and ammonium as a nitrogen source by exposing the cells to CO (13, 14). A CO-insensitive hydrogenase is induced along with CODH (14), and both proteins are associated with the chro- matophore membranes, suggesting that the in vivo oxidation of CO to COS and subsequent reduction of protons to H, may be coupled to the generation of a proton motive force and the synthesis of ATP. In noninduced cultures of R. rubrum, the CO-inducible hydrogenase is absent, and very low levels of CODH are found in the supernatant fraction (13, 15).

The basis for the association of CODH with the chromat- ophore membranes of CO-induced R. rubrum cells, and the mechanism for coupling electron transfer between the CODH and hydrogenase proteins, have not previously been investi- gated in detail. CODH is remarkably stable to heat, with no loss of activity upon 80 ”C heat treatment for 5 min, and this stability to heat was exploited to release CODH from the membranes during purification (13). In this report we inves- tigate alternative methods for solubilizing CODH from chro- matophore membranes, identify and characterize a previously unidentified 22-kDa iron-sulfur-containing subunit of CODH, and show that this subunit is required for reconstituting a CO-oxidizing/H,-evolving system with solubilized CODH and hydrogenase-containing membranes.

MATERIALS AND METHODS

Cell Growth and Chromatophore Membrane Preparation-R. rub- rum (ATCC 11170) was grown photoheterotrophically on the medium of Ormerod et al. (16) as described previously (13, 17). The cells were harvested by tangential flow filtration (15) and stored in liquid

The abbreviations used are: CODH, carbon monoxide dehydro- genase; CHAPS, 3-[(3-~holamidopropyl)dimethylammonio]-l-pro- panesulfonate; SB-12, 3-(N,N-dimethyllaurylammonio)-propane- sulfonate; Tween 80, polyoxyethylenesorbitan monooleate; MOPS, 3- (N-morpho1ino)propanesulfonic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography.

18395

18396 22-kDa Fe-S-containing Subunit of CO Dehydrogenase nitrogen until used. Fifty-g portions of cell paste were thawed in 100 ml of 200 mM MOPS buffer, pH 8.0, containing 2 mM dithiothreitol, 4 mM sodium dithionite, 2 mg ofDNase, 2 mg of RNase, and 30 mg of lysozyme. Cells were disrupted using a bead-beater (Biospec prod- ucts, Barlesville, OK) in an anaerobic glovebox (Vacuum/Atmos- pheres Dri-Lab glovebox model HE-493) with an NP atmosphere containing less than 1 ppm of oxygen. The extract was centrifuged a t 53,000 X g for 16 h, and the chromatophore membranes were resus- pended in 100 mM MOPS, pH 8.0, using a 55-ml plastic-coated tissue grinder (Wheaton). The manipulation of membrane suspensions and loading of centrifuge tubes was carried out in the anaerobic box.

Detergent Solubilization of CODH-Chromatophore membrane pel- lets were resuspended in 100 mM MOPS, pH 8.0, containing 3 mM dithionite or 1 mM indigo carmine to the concentrations indicated in the table legends. Stock detergent solutions in 100 mM MOPS, pH 8.0, were added to give 1% final concentrations, and the suspensions were incubated at 25 "C for 60 min prior to centrifugation at 203,000 X g for 2.5 h. The membrane pellets were resuspended in dithionite- free or dithionite-containing MOPS buffer, and both the supernatant and membrane suspensions were diluted into dithionite-containing buffer and assayed for CODH activity as described below.

Ethanol Solubilization of CODH-Deoxycholate-washed mem- branes were resuspended in 100 mM MOPS, pH 7.5, in the anaerobic box. The suspension (5.02 mg protein/ml) was stirred a t room tem- perature while adding ethanol dropwise to give a final concentration of 20% (v/v). After a 10-min incubation the suspension was centri- fuged for 10 min a t 39,000 X g in 40-ml screw-capped Teflon Oak Ridge tubes (Nalgene). The supernatant and membrane pellet (after resuspension in 5 volumes of MOPS buffer) were assayed for CODH activity.

Purification of CODH from Deoxycholate- and Ethanol-solubilized Membranes-Indigo carmine-oxidized membranes (a typical prepa- ration consisted of 100 ml containing 28 mg of protein/ml and 410,000 units of CODH activity) were washed twice with 1% deoxycholate as described above. CODH was released by deoxycholate or ethanol as described above after reduction of the twice-washed membrane sus- pension with buffer containing 3 mM dithionite. After centrifugation, the supernatant solutions were subjected to the same purification protocol described previously for CODH solubilized from chromato- phore membranes by heat treatment (ion-exchange and hydroxylapa- tite chromatography and preparative gel electrophoresis) (13) with minor modifications as noted below. For ethanol-solubilized CODH, the hydroxylapatite chromatography step was eliminated, and for deoxycholate-solubilized CODH the order of columns (ion-exchange, then hydroxylapatite) was reversed to remove chromatophore pig- ments more efficiently. Purification of CODH after heat --solubiliza- tion was carried out as described previously (13). Homogenous CODH preparations from heat-solubilized, deoxycholate-solubilized, and ethanol-solubilized membranes consistently exhibited specific activi- ties for CO oxidation of 5000-6000 units/mg (1 unit = 1 pmol of CO oxidized per rnin).

Separation of the 62- and 22-kDa Subunits of Ethanol-solubilized CODH-Ethanol-solubilized purified CODH (10-80 mg) was applied to an anaerobic 1.5 X 9-cm column of DE52-cellulose (Whatman) equilibrated in 100 mM MOPS, pH 7.5, containing 50 mM NaC1. The 22-kDa protein was eluted by passing 250 ml of buffer containing 100 mM MOPS, pH 7.5, 50 mM NaCI, and 30% acetonitrile through the column a t a flow rate of 5 ml/min. Under these conditions the 22- kDa protein eluted as a brown band, and the 62-kDa protein remained tightly bound to the column. The column was then reequilibrated in acetonitrile-free MOPS buffer and CODH was eluted with 400 mM NaCl in MOPS buffer. The 22-kDa protein was diluted 3-fold into 25 mM Tris buffer, pH 8.0, and was applied to a second anaerobic DE52 column (1.0 X 2 cm) that had been equilibrated in 25 mM Tris, pH 8.0. The column-bound protein was reequilibrated in 25 mM Tris, pH 8.0, washed with several column volumes 100 mM MOPS, pH 7.5, and then eluted in 400 mM NaCl in MOPS buffer. All column buffers contained 2 mM dithionite to remove traces of 0,.

Preparation of CODH-depleted Hydrogenase-containing Mem- branes-Ten g of CO-induced R. rubrum cells were thawed anaero- bically in 25 ml of 200 mM MOPS, pH 8.0, containing 3 mM dithionite and lysozyme, DNase, and RNase as described above. The thawing buffer, and all subsequent buffers used in handling of hydrogenase membranes, contained dithionite, 15% glycerol, 0.2 mM phenylmeth- ylsulfonyl fluoride, 0.5 pg/ml leupeptin, and 1 mM EDTA. The inclusion of these stabilizing agents was necessary to prevent loss of hydrogenase activity in subsequent steps. Thawed cells were disrupted with a French pressure cell a t 16,000 pounds/inch2, and the chromat-

ophore membranes were sedimented by centrifugation at 203,000 X g for 2.5 h. The membranes were resuspended in 100 mM MOPS, pH 8.0, and CHAPS detergent was added to 1.6%. The membrane/ CHAPS suspension (volume = 50 ml) was incubated on ice for 30 min and the membranes then sedimented by centrifugation a t 203,000 X g for 2.5 h. The membranes were subsequently extracted as de- scribed above three times: once with 0.8% CHAPS and twice with MOPS buffer containing no detergent. The final membrane pellet was resuspended to 49 ml in 100 mM MOPS, pH 7.5, and beaded into liquid N, for storage. All membrane manipulations were performed in the anaerobic box.

Preparation of Other Proteins-Purified Azotobacter uinelnndii fer- redoxin (18) and flavodoxin (18) were gifts from Dr. Vinod Shah in our laboratory, and purified R. rubrum ferredoxins I and I1 (19) were gifts from Mr. Scott Murrell, previously of our laboratory.

Oxidation of Protein Samples and UVIVisible Spectroscopy-Pro- tein samples (in 0.2-1.5 ml volume) were stripped of dithionite in the anaerobic box on 1.5 X 10-cm columns of Sephadex G-25. The samples were then oxidized by the addition of indigo carmine to 1 mM final

on a 0.5 X 5-cm column of Bio-Rad AG1-X8, followed by chromatog- concentration. Indigo carmine was removed from oxidized samples

eluted from the DEAE column with 400 mM NaCl and diluted with raphy on a 0.5 X 5-cm column of DEAE-cellulose. Samples were

100 mM MOPS buffer, pH 7.5, to give a final NaCl concentration of 50 mM. UV/visible spectra of samples were obtained on a Shimadzu UV-160 UV/visible recording spectrophotometer.

Assay Procedures-CO-oxidizing activity was measured spectro- photometrically at 25 "C and pH 7.5 using the CO-dependent methyl viologen reduction assay described previously (17). Hydrogenase ac- tivity in chromatophore membranes was measured by following the production of hydrogen gas with a Shimadzu GC-8A gas chromato- graph equipped with a thermal conductivity detector and a molecular sieve 5A column. Either reduced methyl viologen or CO were used as the source of reducing equivalents for the reduction of protons to hydrogen gas. Assays were performed on 20-50-pl samples of mem- brane suspensions in 13-ml serum-stoppered vials containing a 2-ml total volume of buffer (100 mM MOPS, pH 7.5) under NP in a shaking water bath at 30 "C. Hydrogenase assays with reduced methyl violo- gen as reductant contained 2 mM methyl viologen and 40 mM dithi- onite in the assay vial. Hydrogenase assays with CO as reductant contained a gas phase of 13% CO and 1 mM dithionite in the assay vial. Protein concentration was determined by the bicinchoninic acid (BCA) assay (20) using Sigma grade A bovine serum albumin as standard. A variety of protein assays were compared versus quanti- tative amino acid analysis, and the BCA assay gave the most accurate results for quantitation of samples of the 22-kDa protein. Quantita- tion of protein in samples of 62-kDa CODH gave identical concentra- tions when assayed with either the BCA method or the modified Lowry assay (21) which we had used to quantitate CODH in previous studies. SDS-polyacrylamide gel electrophoresis (10% total, 2.7% cross-linker running gel) was performed as described by Laemmli (22) with modifications described previously (23). Electrophoresed proteins were fixed in formaldehyde (24) and visualized by silver staining (25, 26) or Coomassie Blue staining (27). Metal analyses were performed on an Applied Research Laboratories model 34000

University of Wisconsin Soil and Plant Analysis Laboratory. inductively coupled plasma atomic emission spectrophotometer at the

Data Analysis-Kinetic constants ( K , and V,,,) for CO oxidation catalyzed by CODH were calculated by fitting initial rates to the equation for a rectangular hyperbola using the computer program described by Cleland (28).

Chemicals-CHAPS, sodium cholate, sodium deoxycholate, Tween 80, indigo carmine, and methyl viologen were purchased from Sigma. MOPS was purchased from United States Biochemical Cop. Triton X-100 and dithiothreitol were purchased from Boehringer Mannheim. SB-12 was purchased from Fluka. Ethanol was purchased from Aaper. Acetonitrile was purchased from Baker.

RESULTS

Solubilization Properties of CODH-CODH from R. rubrum has been shown previously to be associated with the chro- matophore membrane fraction of broken cells after centrifu- gation (13). As shown in Table I, the release of CODH from the chromatophore membranes in the presence of the solubi- lizing detergent deoxycholate is dramatically influenced by the redox state of the membrane suspension. When mem-

22-kDa Fe-S-containing Subunit of CO Dehydrogenase 18397

TARLE I Effect of oxidant and reductant on dao.u\cholate-.solubilization of carbon monoxide deh.vdrogenase from anaerobic chromatophores

Membrane preparation" Percent of CODH soluhilizedh

Dithionite-free 4.2 Dithionite-reduced, in buffer con- 74.6

Dit hionite-reduced, resuspended in 60.9

0 3 mM indigo carmine-oxidized 6.85 1 .0 mM indigo carmine-oxidized 4.4

"The dithionite-free detergent/membrane suspension (10.9 mg protein/ml) was prepared from cells broken in the absence of dithio- nite; the other membrane preparations (21.05 mg protein/ml) were prepared from cells broken in the presence of 4 mM dithionite.

" The percent of CODH solubilized was determined from the ratio o f units of CODH activity recovered in the supernatant fraction to the total units recovered in both the supernatant and membrane pellet fractions. Total recovery of activity was a t least 8 0 1 in every case.

taining 3 mM dithionite

dithionite-free buffer

TABLE I1 Effect of oxidant and reductant on the solubilization of membrane-

bound carbon monoxide dehydrogenase Membrane suspensions were prepared and incubations performed

a s described under "Materials and Methods." The protein concentra- tion in the detergent/chromatophore suspensions was 4.11 mg/ml. Total recovery of activity was 124 ? 16%.

Solubilizing agent solubilized solubility in Oxidized,, Reducedh reduced suspensions

1% SB-12 22.8 88.9 3.67 1% sodium cholate 14.4 81.6 5.67 1% CHAPS 7.07 73.5 10.4 1% Tween 80 3.95 64.9 16.4 1% Triton X-100 4.49 51.6 11.5

I' Membrane suspensions were oxidized by the addition of indigo- carmine to 1.0 mM from a stock solution of 20 mM prepared in 100 mM MOPS, pH 8.0.

Membrane suspensions were reduced by the addition of sodium dithionite to 3.0 mM from a stock solution of 100 mM prepared in 100 mM MOPS 8.0.

branes were prepared and treated with 1% deoxycholate in the absence of dithionite, CODH remained associated with the membrane fraction. In contrast, CODH from membranes prepared and treated with deoxycholate in the presence of dithionite was largely solubilized. When dithionite-reduced membranes were resuspended in dithionite-free buffer and then treated with deoxycholate, a greater percentage of CODH activity remained associated with the membrane fraction. When the reduced dithionite-free membranes were oxidized with indigo carmine ( E " = -125 mV) prior to treatment with deoxycholate, CODH remained quantitatively associated with the membrane. Dithiothreitol a t 10 mM concentration was unable to stimulate deoxycholate-solubilization of CODH from dithionite-free membranes.

Several other detergents were tested for their ability to release CODH from indigo carmine-oxidized and dithionite- reduced membranes. As shown in Table 11, each of the deter- gents behaved similarly to deoxycholate, releasing CODH from reduced membranes much more efficiently than from oxidized membranes. The zwitterionic detergents SB-12 and CHAPS and the bile salts cholate and deoxycholate were found to be more effective solubilizing agents than the non- ionic detergents triton X-100 and Tween 80.

The resistance of oxidized membranes to detergent solubi- lization was used as a basis for selectively releasing CODH from reduced membranes after releasing the majority of the

detergent-soluble proteins from oxidized membranes. The purification obtained by this approach is shown in Table I11 and Fig. 1. After two extractions of oxidized membranes with deoxycholate, CODH was released by reduction of the mem- branes in the presence of deoxycholate. Dithionite, CO, and Hr were each suitable reductants for stimulating release of

TABLE I11 Deoxycholate and ethanol solubilization of carbon monoxide dehydrogenase from oxidized and reduced chromatophores

Detergent treatment of membrane suspensions and assay of CODH activity was performed as described under "Materials and Methods."

Percent of CODH in each ~~~~l recovery fraction from previous Treatment''

Supernatant Memhranes step

First deoxycholate elu- 5.8 94.2 (233)h tion, oxidized mem- branes

tion, oxidized mem- branes

tion, dithionite-re- duced membranes

tion, CO-reduced membranes

tion, H1-reduced mem- branes

membranes

Second deoxycholate elu- 5.5 94.5 (240.2)

Third deoxycholate elu- 76.5 (2617) 23.5

Third deoxycholate elu- 81.1 (3167) 18.9

Third deoxycholate elu- 62.2 (1919) 37.8

Ethanol elution, oxidized 78.8 (3465) 21.2

Ethanol elution, reduced 90.6 (4236) 9.4

P. /o

111

99.8

92.2

103

98.6

79.8

8.75 membranes " The protein concentrations in the seven detergent/membrane

suspensions were, from top to bottom, 12.0,6.22,8.90,9.88,8.89,4.01, and 4.01 mg/ml.

"Specific activities of CODH in the predominant fractions are shown in parentheses.

67 -

43- - . " ,

. - -CODH

30-

17.3 - . A ~.

L a n e 1 2 3 4 5 6 7 8 9

FIG. 1. Gel electrophoretic profiles of proteins released by deoxycholate and ethanol extraction of oxidized and reduced chromatophores. Suspensions of R. rubrum chromatophores which had been oxidized by 1 mM indigo carmine were extracted t.wice with 1% deoxycholate. The chromatophore suspension was then reduced with dithionite, CO, or Hz, and CODH was selectively extracted by 1% deoxycholate or 20% ethanol. The gel was st.ained with silver as described under "Material and Methods." Lane loadings: Lone I , molecular weight standards; lane 2, first deoxycholate-extracted su- pernatant (6 pg); lane 3, 2nd deoxycholate-extracted supernatant (6 pg); lane 4, second deoxycholate-extracted chromatophores (6 pg); lane 5, dithionite/deoxycholate extracted supernatant (2 pg); lane 6, CO/deoxycholate-extracted supernatant (2 pg); lane 7, HJdeoxycho- late-extracted supernatant (2 pg); lane 8, ethanol-extracted superna- tant (2 pg); lane 9, heat-released purified CODH standard (1 pg).

18398 22-kDa Fe-S-containing Subunit of CO Dehydrogenase

CODH from the membranes. Identical patterns of protein bands on a silver-stained SDS gel were observed for the supernatants of membranes extracted in the presence of the three reductants (Fig. 1, lanes 5-7), with CODH the predom- inant protein released. Densitometry scans of the gel indicated CODH was greater than 35% pure for each of the extractions, with specific activities ranging from 1919 units/mg (He-re- leased) to 3167 units/mg (CO-released).

Ethanol was found to be an even more selective agent for the solubilization of CODH from deoxycholate-washed mem- branes. No loss of CODH activity was seen when membrane preparations were exposed to 5-25% ethanol for incubation periods of 1 h at 25 “C (data not shown). After addition of 20% ethanol the membranes could be removed by low speed centrifugation (39,000 X g for 10 min) with CODH the pre- dominant protein released into the supernatant solution (see Fig. 1, lane 8). A densitometry scan of the gel indicated that CODH was greater than 55% pure. In contrast to the results with detergents (Tables I1 and 111), ethanol was effective at releasing CODH from both oxidized and reduced membranes (see Table IV). Dimethyl sulfoxide also selectively released CODH from oxidized and reduced deoxycholate-washed mem- branes (data not shown).

Identification, Reversible Dissociation, and Characterization of a 22-kDa CODH Subunit-Deoxycholate- and ethanol- solubilized CODH were purified to homogeneity by our pub- lished purification protocol for heat-solubilized CODH (13). As shown in Fig. 2 (lanes 2-4), ethanol-and deoxycholate- solubilized CODH appeared identical to heat-solubilized CODH on SDS-PAGE, except for the presence of an addi- tional peptide with an apparent molecular mass of -22 kDa which was absent in heat-solubilized CODH preparations. This peptide copurified with CODH through hydroxylapatite chromatography, DEAE and Mono Q FPLC ion-exhange chromatography, alkyl Superose FPLC chromatography, Su- perose 12 FPLC and G-75 gel filtration chromatography, and native gel electrophoresis, suggesting that it is specifically and stoichiometrically associated with the catalytic 62-kDa pep- tide.

In order to determine the role of this peptide as a possible CODH subunit, a protocol was devised for reversibly disso- ciating it from the 62-kDa peptide under nondenaturing con-

ditions. This was accomplished by applying a CODH prepa- ration containing both the 62- and 22-kDa proteins to a column of DEAE-cellulose anion-exchange resin and washing the column with 50 mM NaCl in 30% acetonitrile. Under these conditions the 22-kDa peptide dissociated from CODH and eluted from the column as a brown band, leaving the 62-kDa peptide tightly bound to the column. Separation of the two peptides was strictly dependent upon a proper ionic strength of the acetonitrile/buffer solution: at lower NaCl concentra- tions, the 22-kDa peptide remained bound to the DEAE column with the 62-kDa peptide, and at higher NaCl concen- trations (i.e. 100 mM) the two peptides coeluted from the column. SDS-PAGE of the separated peptides (Fig. 2, lanes 5-7) demonstrates that they have been quantitatively sepa- rated by the NaCl/acetonitrile treatment. Full recovery of CODH activity (as measured with methyl viologen as electron acceptor) was associated with the 62-kDa protein.

There are slight differences in the mobility of the 22-kDa peptide on SDS-PAGE, depending on the conditions under which it was prepared. The peptide complexed with ethanol- solubilized CODH consistently electrophoresed at a slightly higher apparent molecular weight than the peptide complexed with deoxycholate-solubilized CODH (Fig. 2, lane 2 versus lane 3 ) , and the isolated peptide electrophoresed at a slightly lower molecular weight after its separation from the 62-kDa peptide with acetonitrile (Fig. 2, lanes 6 and 7). The isolated peptide electrophoresed with an apparent molecular mass of 21,600 daltons, and it is this molecular mass that we have used to quantitate molar amounts of the peptide.

Based on the observation that the 22-kDa protein eluted from the DEAE column as a brown band, we suspected that it contained an Fe-S center. This was confirmed by the UV/ visible spectra of the isolated protein under oxidized and reduced conditions, which clearly show that it contains a reducible center (Fig. 3). The difference spectrum for oxidized minus dithionite-reduced 22-kDa protein (inset to Fig. 3) has an absorption maximum at 413 nm, a characteristic feature of Fe-S centers. The UV/visible spectra for acetonitrile-sep- arated 62-kDa CODH in the oxidized, CO-reduced, and dithi- onite-reduced states are identical to the spectra which we have reported previously for heat-solubilized CODH (29), whereas the spectra of ethanol-solubilized CODH are similar

TABLE IV Separation of CODH from the CO-induced membrane-bound hydrogenase by selective solubilization with CHAPS

detergent Hydrogenase” CODHb Hpase/CODH

Step Total activity, Total activity, Percent Yield, Total activity, Total activity, Percent Yield, m membranes supernatant solubilized membranes membranes supernatant Solubilized membranes membranes‘

units % units % ratio Broken cell extract 2,000 NAd NA 100 176,400 NA NA 100 1.00 Ultracentrifugation 1,803 20.5 1.1 90.2 160,930 16,600 9.4 91.2 1.01

1.6% CHAPS wash 1,800 164 8.3 90.0 41,700 113,700 73.2 23.6 3.81 of broken extract

of membranes 1.0% CHAPS wash 1.725 65.9 3.7 86.2 9,960 40,900 80.4 5.65 15.4

of membranes

membranes 2 X buffer wash of 1,436 0.0 0.0 71.8 2,818 6,975 71.2 1.6 45.0

a Hydrogenase activity was determined by following hydrogen evolution with methyl viologen as reductant as described under “Materials and Methods.” One unit of hydrogenase activity is defined as 1 Mmol of HZ evolved/ min.

CODH activity was determined spectrophotometrically by following CO-dependent methyl viologen reduction as described under “Materials and Methods.”

The ratio of hydrogenase to CODH after each step was based on the ratio of activity units of hydrogenase and CODH and was normalized to 1.00 for the broken cell extract.

NA, not applicable in the broken cell extract.

22-kDa Fe-S-containing 5 ’ 1

30- \

- 4 0 17..7- \

i.allc i 2 j 3 5 6 7

FIG. 2. SDS-PAGE of deoxycholate-, ethanol-, and heat- released carbon monoxide dehydrogenase. The gel was run in a mini-protean I1 system (Bio-Rad) and was stained with Coomassie Blue as described under “Materials and Methods.” Lane 1, molecular weight standards (1.5 pg each); lane 2, ethanol-solubilized CODH (5 pg); lane 3, deoxycholate-solubilized CODH (5 pg); lane 4 , heat- solubilized CODH (4 pg); lane 5, acetonitrile-separated 62-kDa sub- unit (6 pg); lanes 6 and 7, acetonitrile-separated 22-kDa subunit (2.5 and 6 p a ) .

I I I I I I I 250 300 350 400 450 500 550 600

Wavelength (nm)

FIG. 3. UV/visible absorption spectra of the acetonitrile- separated 22-kDa CODH subunit. Spectra are of an indigo car- mine-oxidized protein sample (0.178 mg/ml) under N2 before (-) and after (- - - -) reduction with 1 mM dithionite. The inset shows the difference spectrum for oxidized minus reduced protein.

except for a greater magnitude of Fe-S center absorption in the 400-nm region due to the additional absorption of the 22- kDa subunit . The extinction coefficients for the Fe-S centers of the various protein preparations in their oxidized, CO-treated, and dithionite-reduced forms are tabulated in Table V. The 62-kDa single subunit CODH forms have nearly identical coefficients for their oxidized and reduced states, whereas the coefficients for ethanol-solubilized CODH are the sum of the individual contributions of the 62- and 22-kDa subunits. The Fe-S center of the 22-kDa subunit was reduced by CO when complexed with the 62-kDa subunit but was not reduced by CO in its isolated state.

Metal analysis of the isolated 22-kDa protein by plasma emission spectroscopy shows 4.0 Fe/mol, suggesting that i t may contain a single reducible low-potential 4Fe-4S cluster. No other transition metals, including nickel, which is an integral component of the active site of the 62-kDa subunit (29), were observed in the peptide. Metal analysis of the acetonitrile-separated 62-kDa subunit showed 8.0 Fe and 0.93 Ni uersus 8.1 Fe and 1.2 Ni for heat-solubilized CODH (Table

xbunit of CO Dehydrogenase 18399

V). Metal analysis of ethanol-solubilized CODH showed 14 Fe and 1 Ni.

In order to determine whether the acetonitrile-separated 22-kDa and 62-kDa subunits could reassociate, the various CODH forms were characterized by gel filtration chromatog- raphy. As shown in Fig. 4A, ethanol-solubilized CODH, which contains the 62- and 22-kDa subunits, elutes from a Superose- 12 gel filtration column as a single peak and at a lower K,, value (0.29) than heat-solubilized CODH (0.32), which lacks the 22-kDa subunit. After separation of the two subunits of ethanol-solubilized CODH by acetonitrile, they were individ- ually chromatographed (Fig. 4B) . The 62-kDa subunit eluted with the same retention time as heat-solubilized CODH, and the 22-kDa subunit eluted a t a K., value of 0.46. Adding the 22-kDa subunit back to the acetonitrile-separated 62-kDa subunit resulted in the reassociation of the two subunits, as evidenced by their migration as a single peak at the position of ethanol-solubilized CODH (Fig. 4C). Adding excess (more than 1 mol/mol) 22-kDa subunit to 62-kDa subunit did not change the elution profile of the reconstituted protein, and the excess 22-kDa subunit eluted at the same position as isolated 22-kDa subunit (Fig. 4C), demonstrating that there is a single saturable binding site for the 22-kDa subunit on the 62-kDa subunit. Heat-solubilized CODH could also be reconstituted with isolated 22-kDa subunit, yielding a com- plex which eluted with a K., of 0.29 (Fig. 40) .

The CODH forms with and without the 22-kDa subunit could also be resolved by anaerobic native gel electrophoresis. As shown in Fig. 5 (lanes 1-4) deoxycholate- and ethanol- solubilized CODH electrophoresed more slowly than did heat- solubilized CODH or acetonitrile-separated 62-kDa CODH. Brief heat treatment of ethanol-solubilized CODH (80 “C, 0.5 min) led to its partial conversion from the slower to faster migrating form (lane 5), indicating that the 22-kDa subunit had been partially denatured and dissociated. The reconsti- tution of the separated 62- and 22-kDa subunits was readily followed on the gel and could be quantitated by densitometry scan. In lane 7, the 22- and 62-kDa subunits, when reconsti- tuted in a ratio of 0.32 to 1, were converted partially to the slower migrating form. A densitometry scan indicated that 35% had been converted to the slower migrating form. In lane 8, reconstitution of 22- to 62-kDa subunit in a ratio of 0.63 to 1 resulted in 66% migrating as the slower form. Addition of an excess of 22-kDa subunit to either the acetonitrile-sepa- rated 62-kDa subunit (lane 9 ) or heat-solubilized CODH (lane IO) resulted in full conversion to the slower migrating forms.

Heat-solubilized CODH and the acetonitrile-extracted 62- kDa subunit are competent to catalyze the in uitro oxidation of CO with methyl viologen as the electron acceptor a t rates comparable with ethanol- or deoxycholate-solubilized CODH complexed with the 22-kDa subunit (Table V), demonstrating that the 22-kDa subunit is not an integral component of the CO oxidation site of CODH. As shown in Table V, the three enzyme forms have similar turnover numbers ( b ) for CO oxidation and virtually identical K, values for methyl violo- gen. The isolated 22-kDa subunit has no detectable CO oxi- dation activity.

Requirements for Reconstituting CO-dependent Hydrogen- ase Activity with Hydrogenase-containing Membranes and Pu- rified CODH-Since there is no obvious in uitro role for the 22-kDa protein in catalyzing CO oxidation, we considered the possibility that it functions to couple the oxidation of CO to the reduction of protons by the R. rubrum CO-induced hydro- genase. In order to determine the requirements for reconsti- tuting CO oxidation and hydrogen evolution, it was necessary to prepare a membrane suspension depleted in CODH but

18400 22-kDa Fe-S-containing Subunit of CO Dehydrogenase TABLE V

Properties of purified CODH f o r m

Characteristic CODH form

Ethanol-solubilized Heat-solubilized 62-kDa subunit AN-separated

22-kDa subunit AN-separated

Subunit composition Metal analysis f l p , . s (mM")'

Oxidized CO-treated Dithionite-reduced

Kinetics of CO oxidation Vmax (units/mg)

K m , ~ v (mM) k w (s-')

K,,, on Superose-12 CO-linked hydrogenase activityd

(nmol/min/mg)

CY@ (62 and 22 kDa) 1.0 Ni, 14.2 Fe

74.8 35.8 35.5

6,600 9,100 3.4 0.29 95.6

CY (62 kDa) 1.2 Ni, 8.1 Fe

34.4 18.7 18.3

7,700 8,000 3.1 0.32 (0.29)' 2.5 (120)'

(Y (62 kDa) 0.93 Ni, 8.0 Fe

36.0 19.6 19.2

9,900 10,200 3.2 0.32 (0.29)' 2.1 (103)'

@ (22 kDa) 4.0 Fe

37.3 37.3 16.6

ND

0.46 ND'

"The extinction coefficients of 62-kDa subunit-containing proteins were measured a t 418 nm; the extinction coefficients of isolated 22-kDa subunit were measured at 413 nm.

No detectable CO oxidation activity. The values in parentheses for heat-solubilized and acetonitrile (AN)-separated 62-kDa CODH are those

The values reported were obtained by subtracting the background hydrogenase activity supported by 2 mM obtained after reconstitution with the 22 kDa subunit.

dithionite (7.7) and CO (2.1) alone. e No detectable stimulation of CO-dependent hydrogenase activity.

containing active hydrogenase. This was made possible by the extreme resistance of hydrogenase to solubilization with de- tergents. As shown in Table IV, repeated washing of a dithi- onite-reduced membrane suspension with CHAPS detergent resulted in the selective release of CODH. More than 98% of CODH was solubilized, whereas 72% of hydrogenase activity was recovered in the membrane fraction, resulting in a 45- fold increase in the relative ratio of hydrogenase to CODH.

The CO-dependent evolution of Hz by the hydrogenase membranes, in the absence and presence of added CODH, is shown in Fig. 6A. As expected, very low levels of CO-depend- ent HP evolution were observed in the absence of added CODH. Addition of ethanol-solubilized CODH, which con- tains both the 62- and 22-kDa subunits, to the membranes led to a 50-fold stimulation in the rate of CO-dependent Hz evolution, demonstrating that the CO-oxidizing/Hz-evolving system can be reconstituted upon the addition of CODH. The addition of the same number of units of heat-solubilized or acetonitrile-separated 62-kDa CODH to the membranes re- sulted in virtually no stimulation of Hz evolution activity. The 62-kDa single subunit CODH forms were, however, com- petent in catalyzing CO-dependent Hz evolution upon recon- stitution with the isolated 22-kDa subunit. As shown in Fig. 6B, there is a linear relationship between the rates of CO- dependent hydrogenase activity and the equivalents of 22- kDa subunit added to 62-kDa CODH. Maximal hydrogenase rates were obtained a t a 1 to 1 (mol/mol) ratio of subunits. The 22-kDa subunit alone, in the presence of CO, had no stimulatory effect on the rate of CO-dependent hydrogen evolution.

Other electron carriers which might serve to couple electron transfer between CODH and hydrogenase, and the specificity of CO as a reductant for hydrogenase, were examined. A comparison of the abilities of R. rubrum ferredoxins I and 11, A. vinelandii ferredoxin, A. vinelandii flavodoxin, and the 22- kDa protein to catalyze electron transfer to the hydrogenase complex are shown in Table VI. Dithionite was found to be a poor reductant for hydrogenase, either in the presence or absence of an added electron carrier protein. In the presence of the electron carrier proteins, the addition of 62-kDa CODH

led to a -3-fold increase in the low rates of dithionite- dependent hydrogen evolution. The addition of CO resulted in a significant stimuiation of hydrogenase only in the pres- ence of the 22-kDa subunit, demonstrating that the subunit is highly specific in mediating electron transfer from CODH to hydrogenase and that CO is a much preferred reductant for the system relative to dithionite.

A sample of 22-kDa protein was exchanged into dithionite- free buffer on a G-25 gel filtration column in the anaerobic box to determine whether it was labile to Oz. The ability of the protein to restore CO-dependent hydrogenase activity after its addition to 62-kDa CODH was used as a basis for assessing its sensitivity to Oz. The protein was found to be extremely 02-labile: incubation under an atmosphere of 1% oxygen at 30 "C led to its inactivation with a t l p of less than 1 min. No restoration of activity occurred upon removal of Oz and reduction of samples with dithionite. By way of compar- ison, a sample of the 22-kDa protein which was stripped of dithionite and oxidized anaerobically with indigo carmine remained fully catalytically competent after reduction with dithionite and addition to 62-kDa CODH. Heat treatment of the isolated 22-kDa subunit at 80 "C, the temperature used in the heat-solubilization of CODH during purification, also led to its irreversible inactivation and denaturation (data not shown).

DISCUSSION

In this paper we demonstrate that the release of R. rubrum CODH from chromatophore membranes is dependent on re- dox state, describe protocols for releasing CODH from the membrane in a highly purified state, and identify and char- acterize a 22-kDa Fe-S protein required for electron transfer from CODH to hydrogenase. The observation that the binding affinity of CODH to the membrane is redox-dependent has interesting implications regarding its mode of binding. The in vivo and in vitro (in intact membranes) oxidation of CO to COz by CODH is coupled to the reduction of protons catalyzed by a coinduced CO-insensitive hydrogenase (14). CODH and hydrogenase may be closely associated within the membrane, with the oxidation of CO being directly coupled to the reduc-

22-kDa Fe-S-containing Subunit of CO Dehydrogenase 18401

i n I A = 0.04

0 20 Kav 0

FIG. 4. Superose-12 gel filtration elution profiles of CODH in the presence, absence, and after reconstitution with the 22- kDa subunit. Protein samples (0.5 ml) were anaerobically chromat- ographed a t a flow rate of 0.7 ml/min on a 10 X 300-mm Superose- 1 2 FPLC column (Pharmacia LKB Biotechnology Inc.) which was equilibrated with 100 mM MOPS, pH 7.5, containing 0.5% CHAPS, 100 mM NaCI, 0.05% NaN:%, and 2 mM dithionite. The absorption of protein samples eluting from the column was monitored a t 420 nm using a Gilson UV/visible detector. The absorption of protein samples at 420 nm is plotted uersus Knv. A, elution profiles of 3.0 (-) nmol nf ethanol- , and 6.1 (- - - -) nmol of heat-solubilized CODH. R, elution profiles of the 62-kDa (3.9 nmol (-)) and 22-kDa (2.6 nmol ( - - - -)) subunits of ethanol-solubilized CODH after separation by acetonitrile. C, elution profiles of acetonitrile-separated 62-kDa CODH subunit (3.9 nmol) in the absence of added 22-kDa subunit (-); 2.5 nmol of 62-kDa CODH + 2.3 nmol of 22-kDa subunit (- - - -); 3.5 nmol of 62-kDa CODH + 5.1 nmol of 22-kDa subunit (- - - -). D, 6.1 nmol of heat-solubilized CODH (-) and 2.4 nmol of heat-solubilized CODH + 2.1 nmol of 22-kDa subunit (- - - -). A N . acetonitrile.

tion of protons. Alternatively, one or more electron carrier proteins may mediate the transfer of electrons between the two proteins, in which case CODH would interact with a distinct membrane anchor protein. The redox effect on CODH solubilization may result from an increase in the strength of the protein-protein interactions between CODH and an in- tegral membrane anchor protein when one or both of the proteins are in their oxidized form, either due to conforma- tional changes induced upon oxidation of the protein(s) or due to direct electronic effects. A second possibility is that the affinity of CODH for membranous binding sites is affected by redox-induced conformational changes in the enzyme. The ability of CO, a direct reductant for CODH, and Hz, an indirect reductant for CODH through the mediating action of hydrogenase, to stimulate the solubilization of CODH sup- ports our hypothesis that the redox state of CODH and/or a redox protein which is an electron acceptor for CODH is responsible for the differential binding affinity.

The effect of redox on CODH binding affinity has also provided an excellent tool for releasing the enzyme from the membranes in a purified state. The stability of CODH to detergents, both in its oxidized and reduced states, allows for the removal of the majority of detergent-soluble proteins by

1 . a w I 2 7 4 5 6 7 x 0 I O

FIG. 6. Native anaerobic PAGE of CODH samples. Lane 1, deoxycholate-solubilized CODH (23.5 pg); lane 2, et.hanol-solubilized CODH (20.4 pa) ; lane 3 , heat-solubilized CODH (30.9 pg); lane 4 , acetonitrile-separated 62-kDa CODH (17.9 p g ) ; lane 5 , heat-treated deoxycholate-solubilized CODH (23.5 pg); lane 6, acetonitrile-sepa- rated 22-kDa subunit. (20.1 pg); lane 7, acetonitrile-separated 62-kDa CODH (23.3 pg; 0.38 nmol) + 22-kDa subunit (1.98 pg; 0.092 nmol); lane 8, acetonitrile-separated 62-kDa CODH (26.6 pg; 0.43 nmol) + 62-kDa CODH (17.3 pg; 0.28 nmol) + 22-kDa subunit (8.24 pg; 0.38 22-kDa subunit (5.75 pg; 0.27 nmol); lane 9, acetonitrile-separated

nmol); lane 10, heat-solubilized 62-kDa CODH (31.1 pg; 0.50 nmol) + 22-kDa subunit (12.7 pg; 0.59 nmol).

repeated washings under oxidized conditions, followed by subsequent reduction and release of CODH with detergent or ethanol in a highly purified state. An additional advantage of this protocol is that it avoids the harsh heat treatment method of releasing CODH from the membranes. Although heat treat- ment of CODH has no obvious effect on the competence of CODH to catalyze CO oxidation with methyl viologen as electron acceptor, it does lead to the denaturation and release of the 22-kDa CODH subunit. In the original purification of CODH from heat-solubilized membranes (13) a small amount of CODH (typically 5-10%) was observed to migrate as a second slower band during preparative scale native gel elec- trophoresis, the final stage of purification. This second CODH form, termed peak 2 CODH, was partially characterized and found to contain additional iron and sulfur (-2 additional mol/mol CODH) and an additional reducible Fe-S center as judged by EPR spectroscopy (13). In light of the data pre- sented in this paper it is now apparent that peak 2 CODH arose from an incomplete release of the 22-kDa subunit from 62-kDa CODH during the heat-treatment of the membranes. The 22-kDa subunit was not identified on silver-stained SDS slab gels of purified peak 2 fractions (13), which is not surprising, based on the observation that the subunit stains very lightly with silver (Fig. 1; data not shown). In more recent preparations of heat-solubilized CODH a 30% increase in the time of heat-treatment of the chromatophore mem- branes has resulted in CODH electrophoresing as a homoge- nous peak 1 preparation (data not shown).

As shown in Fig. 6, the 22-kDa subunit is required for coupling CO oxidation to H2 evolution via the CO-induced membrane-bound hydrogenase. Three lines of evidence sug- gest that the 22-kDa subunit participates directly in transfer- ring electrons from the 62-kDa subunit to hydrogenase: (i) the subunit contains a reducible Fe-S center; (ii) this Fe-S center is reduced by CO when the 22-kDa subunit is com- plexed with the 62-kDa subunit but is not reduced by CO in the isolated 22-kDa protein (Table V); and (iii) the 22-kDa

18402 22-kDa Fe-S-containing Subunit of CO Dehydrogenase

2000

- 1500

- E

2 1000

9

0 W

w

I 500 Tu

0 0 10 20 30 40 50 60

TIME (min)

I40 LB

0.0 0.5 1.0 1.5 2.0 2.5 3.0

EQUIVALENTS 22 kDa SUBUNIT ADDED

FIG. 6. Coupling of CO oxidation to hydrogen evolution re- quires both the 62- and 22-kDa CODH subunits. The prepara- tion of CODH-depleted hydrogenase-containing chromatophore membranes and assay of CO-dependent hydrogenase activity is de- scribed under "Materials and Methods." A, CO-dependent Hz accu- mulation uersus time for hydrogenase membranes (40 pl) under 13% CO after the addition of 750 units of ethanol-solubilized purified CODH containing the 22-kDa subunit (0); 750 units of acetonitrile- separated 62-kDa CODH (0); 750 units of heat-solubilized 62-kDa CODH (A); no added CODH (0). B, hydrogenase specific activity uersus equivalents (mole/mol) of 22-kDa subunit added to 1.1 nmol of acetonitrile-separated 62-kDa CODH (0); 2.1 nmol of heat-solu- bilized 62-kDa CODH (0); 0.91 nmol of ethanol-solubilized purified CODH containing the 22-kDa subunit (A). The rate of Hz evolution in the membrane suspensions due to dithionite alone (7.7 nmol/min/ mg) has been subtracted.

TABLE VI Specificity of the 22-kDa CODH subunit as a mediator in coupling

CO oxidation and hydrogen evolution Relative rate of hydrogenase

in the presence of". Electron carrier'

Dithionite 62-kDa CODH, 62-kDa CODH, dithionite CO. dithionite

~

None 3.8 5.4 7.8 22-kDa subunit 6.5 16.2 100.0 R. rubrum ferredoxin 1 3.1 9.2 9.1 R. rubrum ferredoxin 2 3.3 9.2 11.8 A. uinelandii ferredoxin 3.3 9.2 10.3 A. uinelandii flavodoxin 3.3 9.7 13.0

a The rates of HP evolution are expressed as percentages relative to the rate of H, evolution with CO, 62-kDa CODH, and 22-kDa subunit.

* 2.0 nmol of 62-kDa CODH was reconstituted with 2.3 nmol of 22- kDa subunit prior to initiating the assay. 10 nmol of ferredoxin or flavodoxin was added to 2.0 nmol of CODH prior to initiating the assay.

protein is extremely labile to 02. Previous mechanistic studies with 62-kDa CODH have provided strong evidence that nickel is the site of CO binding, that nickel mediates the transfer of electrons from CO oxidation to the Fe-S centers of CODH, and that the Fe-S centers in turn interact with and reduce the added cosubstrate methyl viologen (17, 29, 30, 31). As an extension of these observations it seems likely that the Fe-S center of the 22-kDa subunit accepts electrons from the Fe-S centers of the 62-kDa subunit and donates them to hydrogen- ase, either directly or through an intermediate electron car- rier. An alternative possibility is that the 22-kDa subunit is solely a structural component of CODH, being required so that the 62-kDa subunit is able to directly reduce the mem- brane-bound electron acceptor. This possibility seems un- likely based on the arguments presented above. An additional point of interest is whether or not the 22-kDa subunit partic- ipates directly in electron transfer when the enzyme is assayed spectrophotometrically with methyl viologen as the electron acceptor for CO oxidation. As shown in Table V, both forms of CODH (with and without the 22-kDa subunit present) have quite similar turnover numbers and identical K, values for methyl viologen. During catalysis, it is likely that the rate determining step for CO oxidation involves either the binding of CO at the Ni center, the oxidation of CO to COz, or the release of COz from the enzyme. If this is the case, an additional and rapid electron transfer from the Fe-S center(s) of the 62-kDa subunit to the Fe-S center of the 22-kDa subunit would not noticeably affect the rate of catalysis. If the Fe-S center of the 22-kDa subunit in turn reduces methyl viologen with the same relative affinity as do the Fe-S center(s) of the 62-kDa subunit, no differences in kcat or K,,, would be observed.

The nature of the immediate physiological electron acceptor for CODH is not currently known. If hydrogenase is not the direct elecron acceptor, the acceptor must be a component integral to the chromatophore membrane, because coupling of CO oxidation and H, evolution is dependent only on adding purified dimeric CODH to membranes containing hydrogen- ase which have been extensively washed with CHAPS deter- gent (Table IV). The CHAPS detergent washes would be expected to remove most peripherally bound proteins from the membrane.

In summary, this paper describes the identification and characterization of a 22-kDa subunit of CODH, the develop- ment of a protocol for reversibly dissociating it from the 62- kDa catalytic subunit without loss of activity, and the discov- ery of a role for the subunit in coupling CO oxidation and H, evolution with purified CODH and hydrogenase membranes. In an analogous report Terlesky and Ferry (32,33) identified and purified a ferredoxin from the methanogenic bacterium Methanosarcina thermophila which was required for coupling CO oxidation and Hz evolution using purified CODH and a membrane-bound hydrogenase. Like the 22-kDa subunit of R. rubrum CODH, the M. thermophila ferredoxin was found to be the direct electron acceptor for the M. thermophila CODH complex (32). I t was not determined whether addi- tional electron carrier proteins were involved in mediating the transfer of electrons from the ferredoxin to the membrane- bound hydrogenase. One notable difference between the R. rubrum and M. thermophila sytems is that CODH from M. thermophila is found in the supernatant fraction of cells lysed by french press, suggesting that the enzyme is a soluble protein (34), whereas CODH from R. rubrum is tightly bound to the chromatophore membrane in the absence of solubilizing detergents. I t is, however, possible that a loose association of the M. thermophila CODH complex with the membranes exists and that this interaction is disrupted by cell lysis (32).

22-kDa Fe-S-containing Subunit of CO Dehydrogenase 18403

I t should also be noted that the properties, structures, and physiological roles of the R. rubrum versus M. thermophila CODHs are quite different: M. thermophila CODH is a mul- timeric enzyme complex consisting of five subunits which functions physiologically as a key enzyme in methanogesis from acetate, catalyzing both the oxidation of CO to C02 and the degradation of acetyl-coA (32,34). In contrast, R. rubrum CODH functions solely to catalyze the oxidation of CO to co,.

The physiological role of the R. rubrum CO oxidation/Hz evolution system is not yet well defined. Uffen (10) has shown that the phototrophic bacterium Rhodocyclus gelatinosa is capable of growth in the dark with CO as a sole carbon and energy source. This bacterium does so by oxidizing CO to CO, and H2, in the process generating a proton motive force for the synthesis of ATP. Carbon from CO, is then assimilated into cell material using H, as an indirect reductant by the enzymes of the ribulose bisphosphate carboxylase cycle (35). I t is likely that the CO oxidation/H2 evolution system of R. rubrum plays a similar role under autotrophic growth condi- tions. In support of this hypothesis Uffen and co-workers (36) have also reported that two strains of R. rubrum will grow slowly in the dark with CO as their sole carbon and energy source. The R. rubrum cultures induced for CODH and hy- drogenase in our current and previous studies were grown under photoheterotrophic conditions and hence do not require CODH and hydrogenase for growth. In order to better char- acterize the actual physiological roles of hydrogenase and the 62- and 22-kDa CODH subunits, it will be necessary to grow R. rubrum under autotrophic conditions and to study the effects of mutations in the gene products encoding the pro- teins. Experiments directed toward these ends are currently in progress.

Acknowledgments-We are indebted to Dr. V. K. Shah for many insightful discussions and his critical reading of the manuscript. We also thank M. Madden and Dr. R. Kerby for helpful discussions and for their critical reading of the manuscript and thank K. Dick for providing information regarding the properties of the CO-induced R. rubrum hydrogenase.

1. 2. 3. 4.

5.

6.

REFERENCES Meyer, O., and Schlegel, H. G. (1980) J. Bacteriol. 141, 74-80 Meyer, 0. (1982) J. Biol. Chem. 257, 1333-1341 Yagi, T. (1958) Biochim. Biophys. Acta 30, 194-195 Jansen, K., Thauer, R. K., Widdel, F., and Fuchs, G. (1984) Arch.

Diekert, G. B., and Thauer, R. K. (1978) J. Bacteriol. 136, 597-

Wood, H. G., Ragsdale, S. W., and Pezacka, E. (1986) Biochem.

Microbiol. 138, 257-262

606

Int. 12, 421-440

7.

8.

9. 10. 11.

12.

13.

14.

15.

16.

17.

18.

19. 20.

21. 22. 23.

24.

25.

26. 27.

28. 29.

30.

31.

32.

33.

34.

35. 36.

Daniels, L., Fuchs, G., Thauer, R. K., and Zeikus, J . G. (1977) J.

Krzycki, J. A., and Zeikus, J. G. (1984) J. Bacteriol. 158, 231-

Uffen, R. L. (1981) Enzyme Microb. Technol. 3, 197-206 Uffen, R. L. (1976) Proc. Natl. Acad. Sei. U. S. A. 73, 3298-3302 Meyer, 0. (1988) in Biotechnology Focus 1 (Finn, R. K., Prave,

P., Schlingmann, M., Crueger, W., Esser, K., Thauer, R., and Wagner, F., eds) pp. 3-31, Hanser, Munich, Federal Republic of Germany

Ragsdale, S. W., Wood, H. G., Morton, T. A., Ljungdahl, L. G., and DerVartanian, D. V. (1988) in T h e Bioinorganic Chemistry of Nickel (Lancaster, J. R., Jr., eds) pp. 311-332, VCH Publish- ers, Inc., New York

Bonam, D., and Ludden, P. W. (1987) J. Biol. Chem. 262,2980- 2987

Bonam, D., Lehman, L., Roberts, G. P., and Ludden, P. W. (1989) J . Bacteriol. 171, 3102-3107

Bonam, D., Murrell, S. A., and Ludden, P. W. (1984) J. Bacterid.

Ormerod, J. G., Ormerod, K. S., and Gest, H. (1961) Arch.

Ensign, S. A,, Hyman, M. R., and Ludden, P. W. (1989) Biochern-

Yoch, D. C. (1979) in A Treatise on Dinitrogen Fixation (Hardy, R. W. F., Bottomley, F., and Burns, R. C., eds) pp. 605-652, John Wiley & Sons, New York

Bacteriol. 132, 118-126

237

159,693-699

Biochem. Biophys. 94,449-463

i s t v 28, 4973-4979

Yoch, D. C., and Arnon, D. I. (1975) J . Bacteriol. 121,743-745 Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,

Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85

Peterson, G. L. (1977) Anal. Biochem. 83, 346-356 Laemmli, U. K. (1970) Nature 227, 680-685 Saari, L. L., Triplett, E. W., and Ludden, P. W. (1984) J. Biol.

Steck, G., Leuthard, D., and Burk, R. R. (1980) Anal. Biochem.

Eschenbruch, M., and Burk, R. R. (1982) Anal. Biochem. 125,

O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 Kanemoto, R. H., and Ludden, P. W. (1984) J. Bacteriol. 158,

Cleland, W. W. (1979) Methods Enzymol. 63, 103-138 Ensign, S. A., Bonam, D., and Ludden, P. W. (1989) Biochemistry

Stephens, P. J., McKenna, M.-C., Ensign, S. A,, Bonam, D., and

Ensign, S. A,, Campbell, M. J., and Ludden, P. W. (1990) Bio-

Terlesky, K. C., and Ferry, J. G. (1988) J. Biol. Chem. 263,4075-

Terlesky, K. C., and Ferry, J. G. (1988) J. Biol. Chem. 263,4080-

Terlesky, K. C., Nelson, M. J. K., and Ferry, J. G. (1986) J .

Uffen, R. L. (1983) J. Bacteriol. 155, 956-965 Dashekvicz, M. P., and Uffen, R. L. (1979) Int. J . Syst. Bacteriol.

Chem. 259,15502-15508

118, 197-203

96-99

713-720

28,4968-4973

Ludden, P. W. (1989) J. Biol. Chem. 264, 16347-16350

chemistry 29, 2162-2168

4079

4082

Bacteriol. 168, 1053-1058

29,145-148