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 been studied in flowflash experiments at -25 degrees C. Under these conditions the time course of the reaction at 445 nm is qualitatively similar to that recorded at room temperature. In addition to heme redox events, three intermediates in the oxygen reaction are observed: a ferrous-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 intermediate (A) into the 607-nm species (P) takes place at the same time that an electron is transferred from the low-spin heme to the oxygen reduction center (k approximately 1500 s-1). Subsequently, P decays into the 580-nm species F at the same time that the low-spin heme becomes partially re-reduced by CuA (k approximately 280 s-1). Although the 607-nm species (P) has been produced in other reactions of the enzyme, this is the first time that it has been observed as a transient in the forward reaction of the fully reduced enzyme with its natural substrate, demonstrating that it is a true catalytic intermediate. The structures of both P and F are discussed in the light of these findings.