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Carbon monoxide dehydrogenase (CODH) is a key enzyme in the pathway of carbon monoxide and carbon dioxide fixation by anaerobic bacteria. It performs the oxidation of CO to CO2, the reduction of CO2 to CO, and the synthesis of acetyl-CoA from a methylated corrinoid/iron-sulfur protein, CO, and CoA. These reactions occur at metal centers on CODH and involve metal-carbon bond formation and transformation. There are three iron-containing centers that play distinct roles in CODH: Centers A, B, and C. Center A is the site of synthesis of acetyl-CoA and catalyzes an exchange reaction between CO and acetyl-CoA. Center C is the site of CO oxidation and CO2 reduction. In the work described here, inhibition of CODH by carbon disulfide was studied. CS2 was found to serve as a probe of the interaction of CODH with CO at Center A. EPR spectroscopic and steady-state kinetic studies demonstrated that CS2 mimics the binding of CO to the nickel/iron-sulfur cluster at Center A; however, CS2 itself does not undergo oxidation-reduction and does not appear to bind to Center C as does CO. In the isotope exchange reaction between acetyl-CoA and CO, CS2 was found to be a competitive inhibitor with respect to CO (Ki = 0.47 mM) and a mixed inhibitor with respect to acetyl-CoA (Ki1 = 0.30 and Ki2 = 1.1 mM). The reaction of dithionite-reduced CODH with CS2 resulted in an EPR spectrum with g values of 2,200, 2,087, and 2,017.(ABSTRACT TRUNCATED AT 250 WORDS)

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 final steps in the synthesis of acetyl-CoA by CO dehydrogenase (CODH) have been studied by following the exchange reaction between CoA and the CoA moiety of acetyl-CoA. This reaction had been studied earlier (Pezacka, E., and Wood, H. G. (1986) J. Biol. Chem. 261, 1609-1615 and Ramer, W. E., Raybuck, S. A., Orme-Johnson, W. H., and Walsh, C. T. (1989) Biochemistry 28, 4675-4680). The CoA/acetyl-CoA exchange activity was determined at various controlled redox potentials and was found to be activated by a one-electron reduction with half-maximum activity occurring at -486 mV. There is approximately 2000-fold stimulation of the exchange by performing the reaction at -575 mV relative to the rate at -80 mV. Binding of CoA to CODH is not sensitive to the redox potential; therefore, the reductive activation affects some step other than association/dissociation of CoA. We propose that a metal center on CODH with a midpoint reduction potential of less than or equal to -486 mV is activated by a one-electron reduction to cleave the carbonyl-sulfur bond and/or bind the acetyl group of acetyl-CoA. Based on a comparison of the redox dependence of this reaction with that for methylation of CODH (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) and CO2 reduction and formation of the Ni-Fe-C EPR signal (Lindahl, P. A., Münck, E., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3873-3879), we propose that the assembly of the acetyl group of acetyl-CoA, i.e. binding the methyl group of the methylated corrinoid/iron-sulfur protein, binding CO, and methyl migration to form the acetyl-CODH intermediate, occur at the novel Ni-Fe3-4-containing site in CODH. CO has two effects on the CoA/acetyl-CoA exchange: it activates the reaction due to its reductive capacity and its acts as a noncompetitive inhibitor. We also discovered that the CoA/acetyl-CoA exchange was inhibited by nitrous oxide via an oxidative mechanism. In the presence of a low-potential electron donor, CODH becomes a nitrous oxide reductase which catalytically converts N2O to N2. This study combined with earlier results (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) establishes that the two-subunit form of CODH is completely active in all reactions known to be catalyzed by CODH.

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.

An 88-kDa corrinoid/iron-sulfur protein (C/Fe-SP) is the methyl carrier protein in the acetyl-CoA pathway of Clostridium thermoaceticum. In previous studies, it was found that this C/Fe-SP contains (5-methoxybenzimidazolyl)cobamide and a [4Fe-4S]2+/1+ center, both of which undergo redox cycling during catalysis, and that the benzimidazole base is uncoordinated to the cobalt (base off) in all three redox states, 3+, 2+, and 1+ [Ragsdale, S.W., Lindahl, P.A., & Münck, E. (1987) J. Biol. Chem. 262, 14289-14297]. In this paper, we have determined the midpoint reduction potentials for the metal centers in this C/Fe-SP by electron paramagnetic resonance and UV-visible spectroelectrochemical methods. The midpoint reduction potentials for the Co3+/2+ and the Co2+/1 couples of the corrinoid were found to be 300-350 and -504 mV (+/- 3 mV) in Tris-HCl at pH 7.6, respectively. We also removed the (5-methoxybenzimidazolyl)cobamide cofactor from the C/Fe-SP and determined that its Co3+/2+ reduction potential is 207 mV at pH 7.6. The midpoint potential for the [4Fe-4S]2+/1+ couple in the C/Fe-SP was determined to be -523 mV (+/- 5 mV). Removal of this cluster totally inactivates the protein; however, there is little effect of cluster removal on the midpoint potential of the Co2+/1+ couple. In addition, removal of the cobamide has an insignificant effect on the midpoint reduction potential of the [4Fe-4S] cluster. A 27-kDa corrinoid protein (CP) also was studied since it contains (5-methoxybenzimidazolyl)cobamide in the base-on form.(ABSTRACT TRUNCATED AT 250 WORDS)