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 (COL) to the Fe atom of the proposed [FeNi] cluster of carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum. Purified CODH has variable amounts of COL dissociated depending on the extent of handling of the proteins. This dissociated COL can be restored by incubation of CODH with CO, resulting 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 purified 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 denaturation of CODH that had been incubated with cyanide. COL binds to both Ni-containing and Ni-deficient CODH, indicating that COL is liganded to the Fe atom of the proposed [FeNi] center. Furthermore, the Ni in the COL-deficient CODH can be removed by treatment with a Ni-specific chelator, dimethylglyoxime. CO proincubation protects the dimethylglyoxime-labile Ni, indicating that COL is also involved in the stability of Ni in the proposed [FeNi] center.