HETEROGENEOUS NICKEL ENVIRONMENTS IN CARBON-MONOXIDE DEHYDROGENASE FROM CLOSTRIDIUM-THERMOACETICUM

Carbon monoxide dehydrogenase from Clostridium thermoaceticum has an ( alphabeta)3 quaternary protein structure and contains a novel Ni-and-F e-containing complex (the NiFe complex) that exhibits an EPR signal (t he NiFeC signal) of unusually low spin intensity. The Ni in the NiFe c omplex can be removed by reaction with 1,10-phenanthroline, yielding e nzyme devoid of CO/acetyl-CoA exchange activity and unable to exhibit the NiFeC signal. On average, each CODH alphabeta dimer was found to r eact rapidly and stoichiometrically with as few as 1.0 +/- 0.2 phenant hrolines. Metal analyses of the enzyme before and after phenanthroline treatment, and of the phenanthroline-containing products of the react ion, revealed that only approximately 0.3 Ni per alphabeta were remove d. Incubation of phenanthroline-treated enzyme with radioactive Ni-63( 2+) followed by chromatographic separation of the Ni-63-containing enz yme from unreacted Ni-63(2+) demonstrated that only 0.3 Ni per alphabe ta could be reinserted into the empty labile sites. These results indi cate that the enzyme is heterogeneous; 30% of the alphabeta protein su bunits contain a labile Ni ion while the remaining 70% do not. Only th ose alphabeta subunits with labile Ni ions can exhibit the NiFeC EPR s ignal and contain the NiFe complex in the form commonly recognized as such. Enzyme solutions lacking labile Ni are completely devoid of CO/a cetyl-CoA exchange activity, suggesting that only alphabeta subunits w ith labile Ni ions are capable of catalyzing CO/acetyl-CoA exchange. H owever, this activity may only be afforded to (alphabeta)3 molecular a ssemblies that include both types of subunits, thereby precluding assi gnment of activity to a particular type of subunit. This analysis expl ains 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.