Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii

Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum. Acetobacterium woodii, like C. thermoaceticum contains high levels of CODH. In this work we show that crude extracts of A. woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA). The purified CODH from A. woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C. thermoaceticum enzyme, indicating the CODH of A. woodii, like that of C. thermoaceticum is an acetyl-CoA synthetase. Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue. The UV absorption spectra and the amino acid compositions of A. woodii and C. thermoaceticum CODHs are very similar. Evidence is presented using purified enzymes from A. woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C. thermoaceticum.     

Purification and characterization of membrane-associated CooC protein and its functional role in the insertion of nickel into carbon monoxide dehydrogenase from Rhodospirillum rubrum

The accessory protein CooC, which contains a nucleotide-binding domain (P-loop) near the N terminus, participates in the maturation of the nickel center of carbon monoxide dehydrogenase (CODH). In this study, CooC was purified from the chromatophore membranes of Rhodospirillum rubrum with a 3,464-fold purification and a 0.8% recovery, and its biochemical properties were characterized. CooC is a homodimer with a molecular mass of 61-63 kDa, contains less than 0.1 atom of Ni(2+) or Fe(2+) per dimer, and has a lambda(max) at 277.5 nm (epsilon(277.5) 32.1 mm(-1) cm(-1)) with no absorption peaks at the visible region. CooC catalyzes the hydrolysis of ATP and GTP with K(m) values of 24.4 and 26.0 microm and V(max) values of 58.7 and 3.7 nmol/min/mg protein for ATP and GTP hydrolysis, respectively. The P-loop mutated form of K13Q CooC was generated by site-specific replacement of lysine by glutamine and was purified according to the protocol for wild-type CooC purification. The K13Q CooC was inactive both in ATP hydrolysis and in vivo nickel insertion. In vitro nickel activation of apoCODH in the cell extracts from UR2 (wild type) and UR871 (K13Q CooC) showed that activation of nickel-deficient CODH was enhanced by CooC and dependent upon ATP hydrolysis. The overall results suggest that CooC couples ATP hydrolysis with nickel insertion into apoCODH. On the basis of our results and models for analogous systems, the functional roles of CooC in nickel processing into the active site of CODH are presented.     

Chemical modification of the functional arginine residues of carbon monoxide dehydrogenase from Clostridium thermoaceticum

Carbon monoxide dehydrogenase (CODH) is the key enzyme of autotrophic growth with CO or CO2 and H2 by the acetyl-CoA pathway. The enzyme from Clostridium thermoaceticum catalyzes the formation of acetyl-CoA from the methyl, carbonyl, and CoA groups and has separate binding sites for these moieties. In this study, we have determined the role of arginine residues in binding of CoA by CODH. Phenylglyoxal, an arginine-specific reagent, inactivated CODH, and CoA afforded about 80-85% protection against this inactivation. The other ligands, such as the carbonyl and the methyl groups, gave no protection. By circular dichroism, it was shown that the loss of activity is not due to extensive structural changes in CODH. Earlier, we showed that tryptophan residues are located at the CoA binding site of CODH [Shanmugasundaram, T., Kumar, G. K., & Wood, H. G. (1988) Biochemistry 27, 6499-6503]. A comparison of the fluorescence spectra of the native and phenylglyoxal-modified enzymes indicates that the reactive arginine residues appear to be located close to fluorescing tryptophans. Fluorescence spectral studies with CoA analogues or its components showed that CoA interacts with the tryptophan(s) of CODH through its adenine moiety. In addition, evidence is presented that the arginines interact with the pyrophosphate moiety of CoA.     

Channeling of carbon monoxide during anaerobic carbon dioxide fixation

Carbon monoxide is an intermediate in carbon dioxide fixation by diverse microbes that inhabit anaerobic environments including the human colon. These organisms fix CO(2) by the Wood-Ljungdahl pathway of acetyl-CoA biosynthesis. The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) catalyzes several key steps in this pathway. CO(2) is reduced to CO at a nickel iron-sulfur cluster called cluster C located in the CODH subunit. Then, CO is condensed with a methyl group and coenzyme A at cluster A, another nickel iron-sulfur cluster in the ACS subunit. Spectroscopic studies indicate that clusters A and C are at least 10-15 A apart. To gain a better understanding of how CO production and utilization are coordinated, we have studied an isotopic exchange reaction between labeled CO(2) and the carbonyl group of acetyl-CoA with the CODH/ACS from Clostridium thermoaceticum. When solution CO is provided at saturating levels, only CO(2)-derived CO is incorporated into the carbonyl group of acetyl-CoA. Furthermore, when high levels of hemoglobin or myoglobin are added to remove CO from solution, there is only partial inhibition of the incorporation of CO(2)-derived CO into acetyl-CoA. These results provide strong evidence for the existence of a CO channel between cluster C in the CODH subunit and cluster A in the ACS subunit. The existence of such a channel would tightly couple CO production and utilization and help explain why high levels of this toxic gas do not escape into the environment. Instead, microbes sequester this energy-rich carbon source for metabolic reactions.     

Involvement of tryptophan residues at the coenzyme A binding site of carbon monoxide dehydrogenase from Clostridium thermoaceticum

Carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum plays a central role in the newly discovered acetyl-CoA pathway [Wood, H.G., Ragsdale, S.W., & Pezacka, E. (1986) FEMS Microbiol. Rev. 39, 345-362]. The enzyme catalyzes the formation of acetyl-CoA from methyl, carbonyl, and CoA groups, and it has specific binding sites for these moieties. In this study, we have determined the role of tryptophans at these subsites. N-Bromosuccinimide (NBS) oxidation of the exposed and reactive tryptophans (5 out of a total of approximately 20) of CODH at pH 5.5 results in the partial inactivation of the exchange reaction (approximately 50%) involving carbon monoxide and the carbonyl group of the acetyl-CoA. Also, about 70% of the acetyl-CoA synthesis was abolished as a result of NBS modification. The presence of CoA (10 microM) produced complete protection against the partial inhibition of the exchange activity and the overall synthesis of acetyl-CoA caused by NBS. Additionally, none of the exposed tryptophans of CODH was modified in the presence of CoA. Ligands such as the methyl or the carbonyl groups did not afford protection against these inactivations or the modification of the exposed tryptophans. A significant fraction of the accessible fluorescence of CODH was shielded in the presence of CoA against acrylamide quenching. On the basis of these observations, it appears that certain tryptophans are involved at or near the CoA binding site of CODH.     

Rubrivivax gelatinosus acsF (Previously orf358) Codes for a Conserved, Putative Binuclear-Iron-Cluster-Containing Protein Involved in Aerobic Oxidative Cyclization of Mg-Protoporphyrin IX Monomethylester

This study describes the characterization of orf358, an open reading frame of previously unidentified function, in the purple bacterium Rubrivivax gelatinosus. A strain in which orf358 was disrupted exhibited a phenotype similar to the wild type under photosynthesis or low-aeration respiratory growth conditions. In contrast, under highly aerated respiratory growth conditions, the wild type still produced bacteriochlorophyll a (Bchl a), while the disrupted strain accumulated a compound that had the same absorption and fluorescence emission spectra as Mg-protoporphyrin but was less polar, suggesting that it was Mg-protoporphyrin monomethylester (MgPMe). These data indicated a blockage in Bchl a synthesis at the oxidative cyclization stage and implied the coexistence of two different mechanisms for MgPMe cyclization in R. gelatinosus, an anaerobic mechanism active under photosynthesis or low oxygenation and an aerobic mechanism active under high-oxygenation growth conditions. Based on these results as well as on sequence analysis indicating the presence of conserved putative binuclear-iron-cluster binding motifs, the designation of orf358 as acsF (for aerobic cyclization system Fe-containing subunit) is proposed. Several homologs of AcsF were found in a wide range of photosynthetic organisms, including Chlamydonomas reinhardtii Crd1 and Pharbitis nil PNZIP, suggesting that this aerobic oxidative cyclization mechanism is conserved from bacteria to plants.     

The acetyl-CoA pathway of autotrophic growth

Abstract The most direct conceivable route for synthesis of multicarbon compounds from CO₂ is to join two molecules of CO₂ together to make a 2-carbon compound and then polymerize the 2-carbon compound or add CO₂ successively to the 2-carbon compound to make multicarbon compounds. Recently, it has been demonstrated that the bacterium, Clostridium thermoaceticum , grows autotrophically by such a process. The mechanism involves the reduction of one molecule of CO₂ to a methyl group and then its combination with a second molecule of CO₂ and CoA to form acetyl-CoA. We have designated this autotrophic pathway the acetyl-CoA pathway [1]. Evidence is accumulating that this pathway is utilized by other bacteria that grow with CO₂ and H₂ as the source of carbon and energy. This group includes bacteria which, like C. thermoaceticum , produce acetate as a major end product and are called acetogens or acetogenic bacteria. It also includes the methane-producing bacteria and sulfate-reducing bacteria.
The purpose of this review is to examine critically the evidence that the acetyl-CoA pathway occurs in other bacteria by a mechanism that is the same or similar to that found in C. thermoaceticum . For this purpose, the mechanism of the acetyl-CoA pathway, as found in C. thermoaceticum , is described and hypothetical mechanisms for other organisms are presented based on the acetyl-CoA pathway of C. thermoaceticum . The available data have been reviewed to determine if the hypothetical schemes are in accord with presently known facts. We conclude that the formation of acetyl-CoA by other acetogens, the methanogens and sulphate-reducing bacteria occurs by a mechanism very similar to that of C. thermoaceticum .     

Interaction of ferredoxin with carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Acetogenic bacteria, as determined with Clostridium thermoaceticum, synthesize acetate by the acetyl-CoA pathway which involves the reduction of CO2 to a methyl group and then combination of the methyl with CoA and a carbonyl group formed from CO or CO2 (Wood, H.G., Ragsdale, S.W., and Pezacka, E. (1986) Trends Biochem. Sci. 11, 14-18). Carbon monoxide dehydrogenase (CODH), the key enzyme in this pathway not only catalyzes the oxidation of CO to CO2 but also the final step, the synthesis of acetyl-CoA from a methyl group, CO, and CoA. Previously, it has been shown that ferredoxin can stimulate exchange of CO with CH3 14COSCoA (Ragsdale, S.W., and Wood, H.G. (1985) J. Biol. Chem. 260, 3970-3977). In the present study, it has been observed that ferredoxin and CODH can form an electrostatically stabilized complex. In order to identify the ferredoxin binding region on CODH, the ferredoxin and CODH were cross-linked by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked CODH-ferredoxin adduct was enzymatically as active as the uncross-linked complex. The native CODH and cross-linked CODH-ferredoxin complex were subjected to cyanogen bromide cleavage. By comparison of the high-performance liquid chromatography peptide profiles, it was observed that the mobility of at least one peptide is altered in the CODH-ferredoxin cross-linked complex. The peptide was identified with residues 229-239 of the alpha-subunit of CODH.     

Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed.     

Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed.     

Acetyl-coenzyme A synthesis from methyltetrahydrofolate, CO, and coenzyme A by enzymes purified from Clostridium thermoaceticum: attainment of in vivo rates and identification of rate-limiting steps.

Many anaerobic bacteria fix CO2 via the acetyl-coenzyme A (CoA) (Wood) pathway. Carbon monoxide dehydrogenase (CODH), a corrinoid/iron-sulfur protein (C/Fe-SP), methyltransferase (MeTr), and an electron transfer protein such as ferredoxin II play pivotal roles in the conversion of methyltetrahydrofolate (CH3-H4folate), CO, and CoA to acetyl-CoA. In the study reported here, our goals were (i) to optimize the method for determining the activity of the synthesis of acetyl-CoA, (ii) to evaluate how closely the rate of synthesis of acetyl-CoA by purified enzymes approaches the rate at which whole cells synthesize acetate, and (iii) to determine which steps limit the rate of acetyl-CoA synthesis. In this study, CODH, MeTr, C/Fe-SP, and ferredoxin were purified from Clostridium thermoaceticum to apparent homogeneity. We optimized conditions for studying the synthesis of acetyl-CoA and found that when the reaction is dependent upon MeTr, the rate is 5.3 mumol min-1 mg-1 of MeTr. This rate is approximately 10-fold higher than that reported previously and is as fast as that predicted on the basis of the rate of in vivo acetate synthesis. When the reaction is dependent upon CODH, the rate of acetyl-CoA synthesis is approximately 0.82 mumol min-1 mg-1, approximately 10-fold higher than that observed previously; however, it is still lower than the rate of in vivo acetate synthesis. It appears that at least two steps in the overall synthesis of acetyl-CoA from CH3-H4folate, CO, and CoA can be partially rate limiting. At optimal conditions of low pH (approximately 5.8) and low ionic strength, the rate-limiting step involves methylation of CODH by the methylated C/Fe-SP. At higher pH values and/or higher ionic strength, transfer of the methyl group of CH3-H4folate to the C/Fe-SP becomes rate limiting.     

The two CO-dehydrogenases of Thermococcus sp. AM4

Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO₂ as a source of carbon. The recent detailed characterizations of some of them have evidenced a great diversity in terms of catalytic properties and resistance to O₂. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp. AM4 (Tc). We have also crystallized CooS2, which is coupled in vivo to a hydrogenase. CooS1 and CooS2 are homodimers, and harbour five metalloclusters: two [Ni4Fe-4S] C clusters, two [4Fe-4S] B clusters and one interfacial [4Fe-4S] D cluster. We show that both are dependent on a maturase, CooC1 or CooC2, which is interchangeable. The homologous protein CooC3 does not allow Ni insertion in either CooS. The two CODHs from Tc have similar properties: they can both oxidize and produce CO. The Michaelis constants (K_m) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO₂. Product inhibition is observed only for CO₂ reduction, consistent with CO₂ binding being much weaker than CO binding. The two enzymes are rather O₂ sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O₂ than any other CODH for which these data are available.     

Redox, haem and CO in enzymatic catalysis and regulation

The present paper describes general principles of redox catalysis and redox regulation in two diverse systems. The first is microbial metabolism of CO by the Wood-Ljungdahl pathway, which involves the conversion of CO or H2/CO2 into acetyl-CoA, which then serves as a source of ATP and cell carbon. The focus is on two enzymes that make and utilize CO, CODH (carbon monoxide dehydrogenase) and ACS (acetyl-CoA synthase). In this pathway, CODH converts CO2 into CO and ACS generates acetyl-CoA in a reaction involving Ni·CO, methyl-Ni and acetyl-Ni as catalytic intermediates. A 70 ? (1 ?=0.1?nm) channel guides CO, generated at the active site of CODH, to a CO 'cage' near the ACS active site to sequester this reactive species and assure its rapid availability to participate in a kinetically coupled reaction with an unstable Ni(I) state that was recently trapped by photolytic, rapid kinetic and spectroscopic studies. The present paper also describes studies of two haem-regulated systems that involve a principle of metabolic regulation interlinking redox, haem and CO. Recent studies with HO2 (haem oxygenase-2), a K+ ion channel (the BK channel) and a nuclear receptor (Rev-Erb) demonstrate that this mode of regulation involves a thiol-disulfide redox switch that regulates haem binding and that gas signalling molecules (CO and NO) modulate the effect of haem.     

Effect of nitrate on the autotrophic metabolism of the acetogens Clostridium thermoautotrophicum and Clostridium thermoaceticum.

Although nitrate stimulated the capacity of Clostridium thermoautotrophicum and Clostridium thermoaceticum to oxidize (utilize) substrates under heterotrophic conditions, it inhibited autotrophic H2-CO2-dependent growth. Under basal medium conditions, nitrate was also inhibitory to the use of one-carbon substrates (i.e., CO, formate, methanol, or the O-methyl groups of vanillate or syringate) as sole carbon energy sources. This inhibitory effect of nitrate was bypassed when both O-methyl groups and CO were provided concomitantly; H2-CO2 did not replace CO. These results indicated that nitrate blocked the reduction of CO2 to the methyl and carbonyl levels. On the basis of the inability of acetogenic cells (i.e., cells cultivated without nitrate) to consume or reduce nitrate in resting-cell assays, the capacity to dissimilate nitrate was not constitutive. Nitrate had no appreciable effect on the specific activities of enzymes central to the acetyl-coenzyme A (CoA) pathway. However, membranes obtained from cells cultivated under nitrate-dissimilating conditions were deficient in the b-type cytochrome that was typical of membranes from acetogenic cells, i.e., cells dependent upon the synthesis of acetate for the conservation of energy. Collectively, these findings indicated that (i) C. thermoautotrophicum and C. thermoaceticum cannot engage the carbon-fixing capacities of the acetyl-CoA pathway in the presence of nitrate and (ii) the nitrate block on the acetyl-CoA pathway occurs via an alteration in electron transport.     

Controlled potential enzymology of methyl transfer reactions involved in acetyl-CoA synthesis by CO dehydrogenase and the corrinoid/iron-sulfur protein from Clostridium thermoaceticum

Many anaerobic bacteria fix CO2 via the Wood pathway of acetyl-CoA synthesis. Carbon monoxide dehydrogenase (CODH), also called acetyl-CoA synthase, accepts the methyl group from the methylated corrinoid/iron-sulfur protein (C/Fe-SP), binds a carbonyl group from CO, CO2, or the carboxyl of pyruvate, and binds coenzyme A. Then CODH catalyzes the synthesis of acetyl-CoA from these enzyme-bound groups. Here, we have characterized the methyl transfer steps involved in acetyl-CoA synthesis. We have studied the reactions leading to methylation of CODH by methyl iodide and shown an absolute requirement of the C/Fe-SP in this reaction. In addition, we have discovered and partly characterized two previously unknown exchange reactions catalyzed by CODH: between the methylated C/Fe-SP and methylated CODH and between methylated CODH and the methyl moiety of acetyl-CoA. We have performed these two exchange reactions, methylation of the C/Fe-SP, and methylation of CODH at controlled potentials. The rates of all these reactions except the exchange between methylated C/Fe-SP and methylated CODH are accelerated (from 1 to 2 orders of magnitude) when run at low potentials. Our results provide strong evidence for a nucleophilic redox-active metal center on CODH as the initial acceptor of the methyl group from the methylated C/Fe-SP. This metal center also is proposed to be involved in the cleavage of acetyl-CoA in the reverse reaction.     

Spectroscopic studies of nickel-deficient carbon monoxide dehydrogenase from Rhodospirillum rubrum: nature of the iron-sulfur clusters

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum utilizes three types of Fe-S clusters to catalyze the reversible oxidation of CO to CO(2): a novel [Ni4Fe5S] active site (C cluster) and two distinct [4Fe4S] electron-transfer sites (B and D clusters). While recent X-ray data show the geometric arrangement of the five metal centers at the C cluster, electronic structures of the various [Ni4Fe5S] oxidation states remain ambiguous. These studies report magnetic circular dichroism (MCD), variable temperature, variable field MCD (VTVH MCD), and resonance Raman (rR) spectroscopic properties of the Fe-S clusters contained in Ni-deficient CODH. Essentially homogeneous sample preparations aided in the resolution of the reduced [4Fe4S](1+) (S = (1)/(2)) B cluster and the reduced Ni-deficient C cluster (denoted C, S > (1)/(2)) by MCD. The three Fe atoms derived from the [Ni3Fe4S] cubane component appear to dominate the reduced C cluster MCD spectrum, while the presence of a fourth Fe center can be inferred from the ground state spin. The same underlying MCD features present in Ni-deficient CODH spectra are also observed for Ni-containing CODH, suggesting that both proteins contain the same C cluster Fe-S component. Overlooked in all spectroscopic studies to date, the D cluster was confirmed by rR to be a typical [4Fe4S] site with cysteinyl coordination. Together, MCD and rR data show that the D cluster remains in the oxidized [4Fe4S](2+) (S = 0) state at potentials > or = -530 mV (versus SHE), thus exhibiting an unusually low redox potential for a standard [4Fe4S](2+/1+) electron-transfer site.     

CO-dependent H2 evolution by Rhodospirillum rubrum: role of CODH:CooF complex

Upon exposure to CO during anaerobic growth, the purple phototrophic bacterium Rhodospirillum rubrum expresses a CO-oxidizing H(2) evolving enzymatic system. The CO-oxidizing enzyme, carbon monoxide dehydrogenase (CODH), has been purified and extensively characterized. However the electron transfer pathway from CODH to the CO-induced hydrogenase that evolves H(2) is not well understood. CooF is an Fe-S protein that is the proposed mediator of electron transfer between CODH and the CO-induced hydrogenase. Here we present the spectroscopic and biochemical properties of the CODH:CooF complex. The characteristic EPR signals observed for CODH are largely insensitive to CooF complexation. Metal analysis and EPR spectroscopy show that CooF contains 2 Fe(4)S(4) clusters. The observation of 2 Fe(4)S(4) clusters for CooF contradicts the prediction of 4 Fe(4)S(4) clusters based on analysis of the amino acid sequence of CooF and structural studies of CooF homologs. Comparison of in vivo and in vitro CO-dependent H(2) evolution indicates that approximately 90% of the activity is lost upon cell lysis. We propose that the loss of two labile Fe-S clusters from CooF during cell lysis may be responsible for the low in vitro CO-dependent H(2) evolution activity. During the course of these studies, a new assay for CODH:CooF was developed using membranes from an R. rubrum mutant that did not express CODH:CooF, but expressed high levels of the CO-induced hydrogenase. The assay revealed that the CO-induced hydrogenase requires the presence of CODH:CooF for optimal H(2) evolution activity.     

In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ.

The products of cooCTJ are involved in normal in vivo Ni insertion into the carbon monoxide dehydrogenase (CODH) of Rhodospirillum rubrum. Located on a 1.5-kb DNA segment immediately downstream of the CODH structural gene (cooS), two of the genes encode proteins that bear motifs reminiscent of other (urease and hydrogenase) Ni-insertion systems: a nucleoside triphosphate-binding motif near the N terminus of CooC and a run of 15 histidine residues regularly spaced over the last 30 amino acids of the C terminus of CooJ. A Gm(r)omega-linker cassette was developed to create both polar and nonpolar (60 bp) insertions in the cooCTJ region, and these, along with several deletions, were introduced into R. rubrum by homologous recombination. Analysis of the exogenous Ni levels required to sustain CO-dependent growth of the R. rubrum mutants demonstrated different phenotypes: whereas the wild-type strain and a mutant bearing a partial cooJ deletion (of the region encoding the histidine-rich segment) grew at 0.5 microM Ni supplementation, strains bearing Gm(r)omega-linker cassettes in cooT and cooJ required approximately 50-fold-higher Ni levels and all cooC insertion strains, bearing polar or nonpolar insertions, grew optimally at 550 microM Ni.