HETEROGENEOUS NICKEL ENVIRONMENTS IN CARBON-MONOXIDE DEHYDROGENASE FROM CLOSTRIDIUM-THERMOACETICUM
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Carbon monoxide dehydrogenase from Clostridium thermoaceticum has an ()3 quaternary protein structure and contains a novel Ni-and-Fe-containing complex (the NiFe complex) that exhibits an EPR signal (the NiFeC signal) of unusually low spin intensity. The Ni in the NiFe complex can be removed by reaction with 1,10-phenanthroline, yielding enzyme devoid of CO/acetyl-CoA exchange activity and unable to exhibit the NiFeC signal. On average, each CODH dimer was found to react rapidly and stoichiometrically with as few as 1.0 0.2 phenanthrolines. Metal analyses of the enzyme before and after phenanthroline treatment, and of the phenanthroline-containing products of the reaction, revealed that only 0.3 Ni per were removed. Incubation of phenanthroline-treated enzyme with radioactive 63Ni2+ followed by chromatographic separation of the 63Ni-containing enzyme from unreacted 63Ni2+ demonstrated that only 0.3 Ni per could be reinserted into the empty labile sites. These results indicate that the enzyme is heterogeneous; 30% of the protein subunits contain a labile Ni ion while the remaining 70% do not. Only those subunits with labile Ni ions can exhibit the NiFeC EPR signal and contain the NiFe complex in the form commonly recognized as such. Enzyme solutions lacking labile Ni are completely devoid of CO/acetyl-CoA exchange activity, suggesting that only subunits with labile Ni ions are capable of catalyzing CO/acetyl-CoA exchange. However, this activity may only be afforded to ()3 molecular assemblies that include both types of subunits, thereby precluding assignment of activity to a particular type of subunit. This analysis explains the low-spin intensity of the NiFeC signal and suggests that the NiFe complex contains significantly more irons than previously thought. The unusually mild conditions required for removal of the labile Ni suggests that this Ni may be coordinatively unsaturated. 1993, American Chemical Society. All rights reserved.