The oxygen reactions of reduced cytochrome c oxidase. Position of a form with an unusual EPR signal in the sequence of early intermediates.

The order of appearance of intermediates in the reoxidation of reduced cytochrome c oxidase by oxygen has been examined. Particular emphasis was placed on determining where the intermediate with the EPR signal at g = 5, 1.78, 1.69 (Shaw, R.W., Hansen, R.E. and Beinert, H. (1978) J. Biol. Chem. 253, 6637--6640) appears in the sequence of events during reoxidation. Flash photolysis of reduced, CO-complexed samples of cytochrome c oxidase in the presence of oxygen in a buffer containing 30% (v/v) ethylene glycol at 77 K and 195 K has been used to generate states of partial reoxidation. The intermediate with the EPR signal at g = 5, 1.78, and 1.69 can be detected as a product of the photolysis and subsequent oxidation but does not appear until the photolyzed sample is incubated at temperatures well above 196 K. In the course of the reoxidation, the intermediate characterized by the g = 5, 1.78, 1.69 signal occurs in the reaction sequence after the states referred to as 'Compound A' and 'Compound B' (Chance, B., Saronio, C., and Leigh, J.S. (1975) J. Biol. Chem. 250, 9226--9237). Its appearance is within the time range reported for the formation of 'oxygenated' cytochrome c oxidase (Orii, Y. (1979) in Cytochrome Oxidase (King, T.E., Orii, Y., Chance, B. and Okunuki, K., eds.), pp. 331--340, Elsevier/North-Holland Biomedical Press, Amsterdam).