Role of the K-channel in the pH-dependence of the reaction of cytochrome coxidase with hydrogen peroxide

The reaction of cytochrome c oxidase (COX) from Rhodobacter sphaeroides wit h 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 gl obal fitting procedure. The reaction can be described by the sequential for mation 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 char acterized by absorption bands at similar to 607 nm (P-607) and similar to 5 80 nm (F-I-580) that rise synchronously. At acidic pH (6.5), intermediate I is represented mainly by a component with an alpha -peak around 575 nm (F- I-575) that is probably equivalent to the so-called F-. species observed wi th the bovine COX. The data are consistent with a pH-dependent reaction bra nching at the step of intermediate I formation. To get further insight into the mechanism of the pH-dependence, the peroxide reaction was studied usin g two mutants of the R. sphaeroides oxidase. K362M and D132N, that block, r espectively, the proton-conducting K- and D-channels. The D132N mutation do es not affect significantly the Ox --> intermediate I step of the peroxide reaction. In contrast, K362M replacement exerts a dramatic effect, eliminat ing the pH-dependence of intermediate I formation. The data obtained allow us to propose that formation of the acidic form of intermediate I (F-I-575, F-.) requires protonation of some group at/near the binuclear site that fo llows or is concerted with peroxide binding. The protonation involves speci fically 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 re duction 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 a(3) iron triggers a transition of the enzyme to the "peroxidase conformation" in whi ch the K-channel is closed and the binuclear site becomes protonically disc onnected from the bulk aqueous phase.