Examination of the Reaction of Fully Reduced Cytochrome Oxidase with Hydrogen Peroxide by Flow-Flash Spectroscopy

The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a 3 oxoferryl (Fe4+O2- + H2O). Since the conversion of ferrous to ferryl heme a 3 (Fe2+ to Fe4+) is sufficient for this reaction, presumably CuB remains reduced in the product, along with CuA and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a 3 oxoferryl is reduced to ferric hydroxide (Fe3+OH-). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, CuB is oxidized. This step has a time constant of about 65 μs. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and CuA must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100−200 μs, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a 3 Fe3+OH-), which reacts with a third molecule of hy