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 a3 and the formation of an intermediate which is heme a3 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 a3 Fe3+ and CuB2+, 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 f...