OBSERVATION AND ASSIGNMENT OF PEROXY AND FERRYL INTERMEDIATES IN THE REDUCTION OF DIOXYGEN TO WATER BY CYTOCHROME-C-OXIDASE

The reaction of fully reduced cytochrome c oxidase with oxygen has bee n studied in flow-flash experiments at -25 degrees C. Under these cond itions the time course of the reaction at 445 nm is qualitatively simi lar to that recorded at room temperature. In addition to heme redox ev ents, three intermediates in the oxygen reaction are observed: a ferro us-oxy species (A), a 607-nm species (P), and a 580-nm ferryl species (F). Formation of A is not resolved. Conversion of the ferrous-oxy int ermediate (A) into the 607-nm species (P) lakes place at the same time that an electron is transferred from the low-spin heme to the oxygen reduction center (k approximate to 1500 s(-1)). Subsequently, P decays into the 580-nm species F at the same time that the low-spin heme bec omes partially re-reduced by Cu-A (k approximate to 280 s(-1)). Althou gh the 607-nm species (P) has been produced ih other reactions of the enzyme, this is the first time that it has been observed as a transien t in the forward reaction of the fully reduced enzyme with its natural substrate, demonstrating that it is a true catalytic intermediate. Th e structures of both P and F are discussed in the light of these findi ngs.