The Interaction of Cytochrome Oxidase with Hydrogen Peroxide: The Relationship of Compounds P and F

Abstract

Upon reaction of cytochrome oxidase with hydrogen peroxide, the spectral changes are complete, with slightly less than 1 equiv of hydrogen peroxide per cytochrome oxidase. At pH 8 the product is a mixture of the P and F forms, while at pH 6 the product is exclusively the F form. These data are inconsistent with current interpretations of the structure of compounds P and F. Two stable radical species are detected by EPR; the relative amounts of these species are pH dependent. The MCD spectra of pure P and F are reported. It is suggested that compound F is a hydrogen peroxide adduct of cytochrome oxidase with cytochrome a3 in the low-spin state and that compound P is an oxyferryl state of cytochrome alpha 3 in support of the recent Raman data of Proshlyakov et al. [(1994) J. Biol. Chem. 269, 29385-29388]. We also suggest that copper B is in the trivalent state in compound P.

Direct Detection of Fe(IV)=O Intermediates in the Cytochrome aa3 Oxidase from Paracoccus denitrificans/H2O2 Reaction

“…Its population increases as the pH increases in the protocol used by the present authors and by Fabian and Palmer (24,26), but not in that used by Proshlyakov et al (23). The latter authors reported that in the pH 7.4 -10 range, the population of the 607-nm form decreases at higher pH, and the final distribution of the enzyme between the 607-and 580-nm forms at a given pH depends on the enzyme preparation.…”

“…It should be noted that Fabian and Palmer (24,25) reported the formation of 7% of a radical species in the oxidized CcO/H 2 O 2 reaction, whereas MacMillan et al (27) reported a yield of 20% for a tyrosine radical species produced in the same reaction. Interestingly, density functional calculations have shown that in the covalent bond between N ⑀ of His-276 and C ⑀ of Tyr-280, the neutral radical of the tyrosine Y ⅐ could place the spin density of N ␦ of the histidine, and thus, coupling between the copper and the organic radical, Y ⅐ -Cu(II) B 3 Y-Cu B (III) might occur (57).…”

“…The CcO/H 2 O 2 reaction has been a conundrum and subject of debate for a number of years (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). The reaction serves as an alternative view of the peroxy (2e Ϫ ) oxidation level.…”

“…Addition of a second equivalent of H 2 O 2 converts each of the above mentioned species to Fe(IV)ϭO Cu B 2ϩ -His-Y. On the other hand, it has been reported that the formation of the 580-nm form, but not that of the 607-nm form, results in a radical-like EPR signal (24). The involvement of Cu B as the alternative electron source in the O-O cleavage process through its Cu B (III) oxidation state has been suggested (25).…”

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

“…The peak-to-trough magnitude of the H 2 O 2 -induced difference spectra in the Soret band was typically 30-45 mM Ϫ1 cm Ϫ1 in the WT and in the K channel mutants as compared with ca. 50 mM Ϫ1 cm Ϫ1 observed for beef heart enzyme (29,(42)(43)(44) Before the flash, the sample was incubated for several minutes with 1 mM H2O2 to convert heme a3 to the ferryl-oxo state. After recording a trace (Wildtype), 1 mM potassium cyanide was added and the second trace (ϩKCN) was recorded.…”