Observation of a Novel Transient Ferryl Complex with Reduced CuB in Cytochrome c Oxidase

The reaction between mixed-valence (MV) cytochrome c oxidase from beef heart with H2O2 was investigated using the flow-flash technique with a high concentration 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 (τ ≅ 25 μs) results from the binding of one molecule of H2O2 to reduced heme a 3 and the formation of an intermediate which is heme a 3 oxoferryl (Fe4+O2-) with reduced CuB (plus water). During the second phase (τ ≅ 90 μs), the electron transfer from CuB + to the heme oxoferryl takes place, yielding the oxidized form of cytochrome oxidase (heme a 3 Fe3+ and CuB 2+, plus hydroxide). During the third phase (τ ≅ 4 ms), an additional molecule of H2O2 binds 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 reaction of the mixed-valence form of the enzyme with H2O2 yields the same compound P as does the reaction with dioxygen, as indicated by the final absorbance at 436 nm, which is the same in both cases. This experimental approach allows the investigation of the form of cytochrome c oxidase which has the heme a 3 oxoferryl intermediate but with reduced CuB. This state of the enzyme cannot be obtained from the reaction with dioxygen and is potentially useful to address questions concerning the role of the redox state in CuB in the proton pumping mechanism.