Evidence for a ligand CO that is required for catalytic activity of CO dehydrogenase from Rhodospirillum rubrum

Radiolabeling studies support the existence of a nonsubstrate CO ligand (CO L) to the Fe atom of the proposed [FeNi] cluster of carbon monoxide dehydro genase (CODH) from Rhodospirillum rubrum. Purified CODH has variable amount s of COL dissociated depending on the extent of handling of the proteins. T his dissociated COL can be restored by incubation of CODH with CO, resultin g in a 30-40% increase in initial activity relative to as-isolated purified CODH, A similar amount of COL binding is observed when as-isolated purifie d CODH is incubated with (CO)-C-14: approximately 0.33 mol of CO binds per 1 mol of CODH. Approximately 1 mol of CO was released from CO-preincubated CODH upon denaturation of the protein. No CO could be detected upon denatur ation of CODH that had been incubated with cyanide. COL binds to both Ni-co ntaining and Ni-deficient CODH, indicating that COL is liganded to the Fe a tom of the proposed [FeNi] center. Furthermore, the Ni in the COL-deficient CODH can be removed by treatment with a Ni-specific chelator, dimethylglyo xime. CO proincubation protects the dimethylglyoxime-labile Ni, indicating that COL is also involved in the stability of Ni in the proposed [FeNi] cen ter.