Observation of a novel transient ferryl complex with reduced Cu-B in cytochrome c oxidase

The reaction between mixed-valence (MV) cytochrome c oxidase from beef hear t with H2O2 was investigated using the flow-flash technique with a high con centration of H2O2 (1 M) to ensure a fast bimolecular interaction with the enzyme. Under anaerobic conditions the reaction exhibits 3 apparent phases. The first phase ( tau congruent to 25 mu s) results from the binding of on e molecule of H2O2 to reduced heme a(3) and the formation of an intermediat e which is heme a(3) oxoferryl (Fe4+=O2-) with reduced Cu-B (plus water). D uring the second phase (tau congruent to 90 mu s), the electron transfer fr om Cu-B(+) to the heme oxoferryl takes place, yielding the oxidized form of cytochrome oxidase (heme a(3). Fe3+ and Cu-B(2+), plus hydroxide). During the third phase (tau congruent to 4 ms), an additional molecule of H2O2 bin ds to the oxidized form of the enzyme and forms compound P, similar to the product observed upon the reaction of the mixed-valence (i.e., two-electron reduced) form of the enzyme with dioxygen. Thus, within about 30 ms the re action of the mixed-valence form of the enzyme with H2O2 yields the same co mpound P as does the reaction with dioxygen, as indicated by the final abso rbance at 436 nm, which is the same in both cases. This experimental approa ch allows the investigation of the form of cytochrome c oxidase which has t he heme a(3) oxoferryl intermediate but with reduced Cu-B. This state of th e enzyme cannot be obtained from the reaction with dioxygen and is potentia lly useful to address questions concerning the role of the redox state in C UB ill the proton pumping mechanism.