Discovery of a labile nickel ion required for CO/acetyl-CoA exchange activity in the NiFe complex of carbon monoxide dehydrogenase

Abstract

The oxygen-sensitive, nickel and iron-containing enzyme carbon monoxide dehydrogenase (CODH) from clostridium thermoaceticum was purified without any significant loss of the activity present in the crude extract. The careful examinations of activities, metal contents, and EPR properties of CODH during its purification showed that the low spin intensities of CODH EPR signals were not coming from purification procedures, oxygen damage, or metal ion dissociation. If these low intensities arise from heterogeneous distributions of active sites, the heterogeneity should originate before the purification. Oxygen-damaged enzyme could be separated from active enzyme chromatographically. Exposure to oxygen during the purification lowered the overall yield but not the specific activity of the finally purified enzyme. Contrary to previous reports (Ragsdale et al., 1983a, Ramer et al., 1989), CODH does not require zinc or a third type of protein subunit for CO oxidation or CO/acetyl-CoA exchange activities. It has been shown that CODH has a low Hase activity. CO binding properties of the NiFe complex was examined by the titration of CO with dithionite-reduced CODH. One molecule of CO is needed to develop full NiFeC signal intensity (0.23 spin/αβ). CO binds tightly to the NiFe complex, with K[d] = 80 μM, similar to those of some well characterized nickel complexes. This suggests that the low spin intensity of the NiFeC signal is not caused by substoichiometric binding of CO. The addition of phen to CODH selectively destroyed the CO/acetyl-CoA exchange activity and eliminated the NiFeC signal completely. CO oxidation activity and other EPR signals were unaffected. Such behavior demonstrates that CODH has two distinct active sites and that the NiFe complex is only responsible for the CO/acetyl-CoA exchange activity. Phen caused the removal of only 30% of Ni in the NiFe complex (0.3 Ni/αβ). The phen-treated CODH could be reactivated fully by incubation in Ni2+. The amount of radioactively labeled Ni2+ .incorporated into phen-treated enzyme was the same as the removed amount by the phen treatment, 0.3 Ni/αβ...