Freeze-Quench Resonance Raman Spectroscopic Evidence for an Fe-CO Adduct during Acetyl-CoA Synthesis and Ni Involvement in CO Oxidation by Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum

摘要

Carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum is a Ni—Fe metalloenzyme that has two separate but linked activities of CO metabolism. One molecule of CO is oxidized to CO_2 at an EPR-detectable site called center C, while another molecule of CO is combined with a methyl group and coenzyme A to form acetyl-CoA at a separate EPR-detectable site called center A. In the work reported here, we have adapted the freeze-quench technique for resonance Raman (RR) spectroscopy. We report that Ni is involved in CO oxidation at center C. We also confirm our previous inference tot CO binds to center A at an Fe atom, and not at Ni. The CODH from acetogenic bacteria contains two Ni atoms, as well as 11 —14 Fe atoms. One Ni is associated with an Fe—S duster at center A. The location of the second Ni atom has been uncertain. Center C has been suspected of harboring the second Ni, by analogy with the CO oxidation site of the R. rubrum CODH. This enzyme lacks acetyl-CoA synthesis activity and has a single Ni atom. Its EPR spectrum shows a sight increase in its EPR line width upon Ni substitution, indicating that nickel is a component of the EPR-detectable center. EXAFS studies of the R. rubrum enzyme indicate that the Ni—Fe distance is greater than 3.4 A, indicating that this center is not a NiFe_3S_4 cubane cluster.