Exclusive CO binding to cytochrome oxidase.

CO added to dithionite-reduced cytochrome oxidase pretreated with azide, cyanide, or fluoride yielded CO-ferrous heme a3 trapping the unliganded reduced heme. Ferrous heme a3 was either an equilibrium species initially present, or provided by dissociation of ligand-bound ferric heme a3 followed by the reduction with dithionite. In the latter case the ligand dissociation was rate-limiting for the CO compound formation. Pretreatment of the enzyme with the inhibitory ligands affected neither photodissociation and reassociation of the CO compound thus formed, nor reaction with dioxygen initiated by the flow-flash method to any significant degree. Only the cyanide treatment slightly decreased the rate of intramolecular electron transfer. These results indicate that no inhibitory ligand but CO remains in the vicinity of the heme a3-CuB center in the CO compound of cytochrome oxidase.