Role of the K-Channel in the pH-Dependence of the Reaction of Cytochrome c Oxidase with Hydrogen Peroxide

Abstract

The reaction of cytochrome c oxidase (COX) from Rhodobacter sphaeroides with hydrogen peroxide has been studied at alkaline (pH 8.5) and acidic (pH 6.5) conditions with the aid of a stopped-flow apparatus. Absorption changes in the entire 350-800 nm spectral range were monitored and analyzed by a global fitting procedure. The reaction can be described by the sequential formation of two intermediates analogous to compounds I and II of peroxidases: oxidized COX + H2O2 --> intermediate I --> intermediate II. At pH as high as 8.5, intermediate I appears to be a mixture of at least two species characterized by absorption bands at approximately 607 nm (P607) and approximately 580 nm (F-I580) that rise synchronously. At acidic pH (6.5), intermediate I is represented mainly by a component with an alpha-peak around 575 nm (F-I575) that is probably equivalent to the so-called F* species observed with the bovine COX. The data are consistent with a pH-dependent reaction branching at the step of intermediate I formation. To get further insight into the mechanism of the pH-dependence, the peroxide reaction was studied using two mutants of the R. sphaeroides oxidase, K362M and D132N, that block, respectively, the proton-conducting K- and D-channels. The D132N mutation does not affect significantly the Ox --> intermediate I step of the peroxide reaction. In contrast, K362M replacement exerts a dramatic effect, eliminating the pH-dependence of intermediate I formation. The data obtained allow us to propose that formation of the acidic form of intermediate I (F-I575, F*) requires protonation of some group at/near the binuclear site that follows or is concerted with peroxide binding. The protonation involves specifically the K-channel. Presumably, a proton vacancy can be generated in the site as a consequence of the proton-assisted heterolytic scission of the O-O bond of the bound peroxide. The results are consistent with a proposal [Vygodina, T. V., Pecoraro, C., Mitchell, D., Gennis, R., and Konstantinov, A. A. (1998) Biochemistry 37, 3053-3061] that the K-channel may be involved in the delivery of the first four protons in the catalytic cycle (starting from reduction of the oxidized form) including proton uptake coupled to reduction of the binuclear site and transfer of protons driven by cleavage of the dioxygen O-O bond in the binculear site. Once peroxide intermediate I has been formed, generation of a strong oxene ligand at the heme a3 iron triggers a transition of the enzyme to the "peroxidase conformation" in which the K-channel is closed and the binuclear site becomes protonically disconnected from the bulk aqueous phase.

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

“…The difference spectra are similar to those obtained for the reaction of oxidized mammalian and Rb. sphaeroides aa 3 -type oxidase with H 2 O 2 (49,50). When this experiment is conducted with the Y280H mutant, the difference spectrum (trace b) is slightly changed with the maxima at 593 and 610 nm.…”

How Hydrogen Peroxide Is Metabolized by Oxidized Cytochrome c Oxidase

“…27,29,51 The major feature of this dependence is the branching of the reaction at the level of P that is under the control of an acid–base group(s) characterized for bovine heart oxidase with a p K a of approximately 6.7–7.0. 25,29,52 Only at higher pH values, where the group is deprotonated, does the reaction of CcO with H 2 O 2 proceed with the dominant production of P .…”

Cytochrome c oxidase: Intermediates of the catalytic cycle and their energy‐coupled interconversion

“…Catalytic cycle of cytochrome c oxidase. The cycle going through the 2 electron reduced state is modified from the schemes in [12,61]. The intermediates at the peroxy level of heme a 3 ( P , ferric‐hydroperoxy, and P 0 , ferric‐dihydroperoxy) have not yet been observed experimentally, hence shown in dotted boxes.…”

“…Behaviour of the group is non‐trivial. It can be rapidly protonated via the K‐channel at the stage of Compound I formation during reaction of H 2 O 2 with the oxidized COX [61]. However, after Compound I has been formed at pH >8, acidification results in but very slow 607 → 580 nm conversion (k v ∼0.03 s −1 at pH 6.5 [60,61]) by virtue of proton leak through some unspecific pathway(s), as the rate it not affected by mutations in the proton channels [61].…”