Nickel is required for the transfer of electrons from carbon monoxide to the iron-sulfur center(s) of carbon monoxide dehydrogenase from Rhodospirillum rubrum.

The role of nickel in CO oxidation and electron flow was investigated in carbon monoxide dehydrogenase from Rhodospirillum rubrum. The Fe-S centers of oxidized, nickel-containing (holo) CO dehydrogenase were completely reduced within 1 min of exposure to CO. The Fe-S centers of oxidized, nickel-deficient (apo) CO dehydrogenase were not reduced during a 35-min incubation in the presence of CO. Apo-CO dehydrogenase Fe-S centers were reduced by dithionite. The Fe-S centers of cyanide-inhibited, holo-CO dehydrogenase were not reduced in the presence of CO but were reduced by dithionite. Treatment of apo-CO dehydrogenase with cobalt(II), zinc(II), and iron(II) resulted in association of these metal ions (0.70, 1.2, and 0.86 mol of M2+/mol, respectively) with the protein but no increase in specific activity. Purified holo-CO dehydrogenase contained 1.1 mol of nickel/mol of protein and could not be further activated upon addition of NiCl2, suggesting the presence of one catalytic nickel site on the enzyme. The M2+-treated enzymes could not be further activated by addition of NiCl2 as opposed to the untreated apoenzyme, whose activity was stimulated 50-100-fold to the level of holoenzyme upon addition of NiCl2. When placed under CO, the Fe-S centers of the cobalt-treated enzyme became reduced over a 35-min time course, as opposed to the zinc- and iron-treated enzymes, which remained oxidized. We conclude that nickel, or an appropriate nickel analogue in the nickel site, mediates electron flow from CO to the Fe-S centers of CO dehydrogenase.