A FLASH-PHOTOLYSIS STUDY OF THE REACTIONS OF A CAA(3)-TYPE CYTOCHROME-OXIDASE WITH DIOXYGEN AND CARBON-MONOXIDE

The time course of absorbance changes following flash photolysis of th e fully-reduced carboxy-cytochrome oxidase from Bacillus PS3 in the pr esence of O-2 has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cy tochrome oxidase. The PS3 enzyme has a covalently bound cytochrome c s ubunit and the fully-reduced species therefore accommodates five elect rons instead of four as in the bovine enzyme. In the bovine enzyme, fo llowing CO dissociation, four phases were observed with time constants of about 10 mu s, 30 mu s, 100 mu s, and 1 ms at 445 nm. The initial, 10-mu s absorbance change at 445 nm is similar in the two enzymes. Th e subsequent phases involving heme a and Cu-A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the co valently bound cytochrome c, as indicated by absorbance changes at 550 nm, A reaction scheme consistent with the experimental observations i s presented. In addition, internal electron-transfer reactions in the absence of O-2 were studied following flash-induced CO dissociation fr om the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more el ectrons were transferred from heme a(3) to a in PS3 oxidase compared t o the bovine enzyme.