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Characterization of the carbonylation and methylation sites in carbon monoxide dehydrogenase from clostridium thermoaceticum.
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KINETICS
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CHARACTERIZATION OF THE CARBONYLATION AND METHYLATION SITES IN CARBON MONOXIDE DEI-IYDROGENASE FROM CLDSllUDIUiU THERMOACElXXM. . Kumar and Stephen W. Ragsdale+ Department of Biochemistry,East Campus, Universityof Nebrask-aLincoln Lincoln, h?E-68583-0718, USA. Clostridiwnthermoaceticumconverts Hz + CO* and CO to acetyl-CoA by a mechanism called the reductive acetyl-CoA (Wood) pathway’. Three one-carbon carriers in this pathway have been established: a NiFeS complex on the enzyme, carbon monoxide dehydrogenase (CODH), a cobalt center in a corrinoid/iron sulfur protein (C/Fe-SP) and tetrahydrofolate (H,folate). CODH contains 2 Ni, 11-14 Fe, 1 Zn, and - 14 acid-labile S’ per a,p unit. The redox-active metals are arranged into at least three separate complexes. CO is the precursor of the carbonyl group of acetyl-CoA and bind to CODH at a site which has been studied by EPR, Miissbauer, ENDOR, and EXAFS spectroscopies and by spectroelectrochemistry’. Based on these combined studies, the site at which CO binds has been called a [NiF&&j cluster. We published* the results of an infrared spectroscopic study of the binding site for CO in CODH. CO was found to be terminally coordinated to one metal in the active-site mixed metal cluster best described as [Ni-X-FhS,]-C = 0. This site was shown to act as the precursor of the carbonyl group of acetyl-CoA. More recently, we have used IR spectroscopy to evaluate an unresolved question of whether a separate site exists for binding of CO for CO oxidation to CO,. We also have studied the binding of CO to CODH by f&exe quench EPR and determined the rate of reduction of each of the metal centers in CODH. Using stopped flow kinetic studies, we have found that methylation of CODH by the methylated C/Fe-SP (Equation 1) appears to be the rate limiting step CH,-[Co”]-C/Fe-SP + CODH # [Co’+]-C/Fe-SP + CH,-CODH (Eq. 1) in the synthesis of acetyl-CoA from CH,-H,folate, CO and CoA. Two possible mechanisms of the methyl transfer, involving heterolytic or homolytic cleavage of the methyl-Co bond, can be envisioned. The two mechanisms can be distinguished by observing whether Co’+ or Co*+ is the product of the transmethylation reaction. The rates of decay of methylcobahunin and of formation of Co’+ are identical and spectral analysis of the time course shows clean isosbestic points. Thus, that the mechanism involves a heterolytic cleavage in which the reductively activated metal center on CODH performs a nucleophilic attack on the methyl group of methylcob(III)amide, generating Co’+ and methylated CODH (Eq. 1). The reaction is dependent on the concentration of CODH and on the ionic strength of the buffer indicating the interaction of opposite charges. 1. S.W. Ragsdale, CRC Crit. Rev. Biochem. Mol. Biol. 26, 261 (1991). 2. M. Kumar and S.W. Ragsdale, J. Am. C7zem.Sot. 114, 8713 (1992).
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CHARACTERIZATION OF THE CARBONYLATION AND METHYLATION SITES IN CARBON MONOXIDE DEI-IYDROGENASE FROM CLDSllUDIUiU THERMOACElXXM. . Kumar and Stephen W. Ragsdale+ Department of Biochemistry,East Campus, Universityof Nebrask-aLincoln Lincoln, h?E-68583-0718, USA. Clostridiwnthermoaceticumconverts Hz + CO* and CO to acetyl-CoA by a mechanism called the reductive acetyl-CoA (Wood) pathway’. Three one-carbon carriers in this pathway have been established: a NiFeS complex on the enzyme, carbon monoxide dehydrogenase (CODH), a cobalt center in a corrinoid/iron sulfur protein (C/Fe-SP) and tetrahydrofolate (H,folate). CODH contains 2 Ni, 11-14 Fe, 1 Zn, and - 14 acid-labile S’ per a,p unit. The redox-active metals are arranged into at least three separate complexes. CO is the precursor of the carbonyl group of acetyl-CoA and bind to CODH at a site which has been studied by EPR, Miissbauer, ENDOR, and EXAFS spectroscopies and by spectroelectrochemistry’. Based on these combined studies, the site at which CO binds has been called a [NiF&&j cluster. We published* the results of an infrared spectroscopic study of the binding site for CO in CODH. CO was found to be terminally coordinated to one metal in the active-site mixed metal cluster best described as [Ni-X-FhS,]-C = 0. This site was shown to act as the precursor of the carbonyl group of acetyl-CoA. More recently, we have used IR spectroscopy to evaluate an unresolved question of whether a separate site exists for binding of CO for CO oxidation to CO,. We also have studied the binding of CO to CODH by f&exe quench EPR and determined the rate of reduction of each of the metal centers in CODH. Using stopped flow kinetic studies, we have found that methylation of CODH by the methylated C/Fe-SP (Equation 1) appears to be the rate limiting step CH,-[Co”]-C/Fe-SP + CODH # [Co’+]-C/Fe-SP + CH,-CODH (Eq. 1) in the synthesis of acetyl-CoA from CH,-H,folate, CO and CoA. Two possible mechanisms of the methyl transfer, involving heterolytic or homolytic cleavage of the methyl-Co bond, can be envisioned. The two mechanisms can be distinguished by observing whether Co’+ or Co*+ is the product of the transmethylation reaction. The rates of decay of methylcobahunin and of formation of Co’+ are identical and spectral analysis of the time course shows clean isosbestic points. Thus, that the mechanism involves a heterolytic cleavage in which the reductively activated metal center on CODH performs a nucleophilic attack on the methyl group of methylcob(III)amide, generating Co’+ and methylated CODH (Eq. 1). The reaction is dependent on the concentration of CODH and on the ionic strength of the buffer indicating the interaction of opposite charges. 1. S.W. Ragsdale, CRC Crit. Rev. Biochem. Mol. Biol. 26, 261 (1991). 2. M. Kumar and S.W. Ragsdale, J. Am. C7zem.Sot. 114, 8713 (1992).