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 gre at value in generating and characterizing oxygenated species of the enzyme that are identical or similar to these 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 a void limitations by the bimolecular reaction. The flow-flash method is used , in which laser photolysis of the CO adduct of the fully reduced enzyme in itiates the reaction following rapid mixing of the enzyme with peroxide, an d the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced f rom 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 re mains reduced in the product, along with Cu-A and heme a. (2) The second ph ase of the reaction is an internal rearrangement of electrons and protons i n which the heme a(3) oxoferryl is reduced to ferric hydroxide (Fe3+OH-). I n about 40% of the population, the electron comes from heme a, and in the r emaining 60% of the population, CUB is oxidized. This step has a time const ant of about 65 mu s. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in t he binuclear center reacts with a second molecule of peroxide, forming comp ound F. The population of the enzyme with the two electrons on heme a and C u-A must first transfer an electron to the binuclear center, followed by re action with a second molecule of peroxide, also yielding compound F. In eac h of these reaction pathways, the reaction time is 100-200 mu s, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzym e. Thus, hydrogen peroxide is an efficient trap for a single electron in th e binuclear center. (4) Compound F is then reduced by the final available e lectron, 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-), whi ch reacts with a third molecule of hydrogen peroxide, forming compound P. T he rate of this final reaction step saturates at high concentrations of per oxide (V-max = 250 s(-1), K-m = 350 mM). The data indicate a reaction mecha nism for the steady-state peroxidase activity of the enzyme which, at pH 7. 5, proceeds via the single-electron reduction of the binuclear center follo wed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electron-reduced stat e of the binuclear center. The results also suggest that the reaction of hy drogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme as ferric species. The reaction of h ydrogen peroxide with heme a(3) is very substantially accelerated by the av ailability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.