Single-electron reduction of the oxidized state is coupled to proton uptake via the K pathway in Paracoccus denitrificans cytochrome c oxidase

Abstract

The reductive part of the catalytic cycle of cytochrome c oxidase from Paracoccus denitrificans was examined by using time-resolved potential measurements on black lipid membranes. Proteoliposomes were adsorbed to the black lipid membranes and Ru II (2,2-bipyridyl)3 2؉ was used as photoreductant to measure flash-induced membrane potential generation. Single-electron reduction of the oxidized wild-type cytochrome c oxidase reveals two phases of membrane potential generation (1 Ϸ 20 s and 2 Ϸ 175 s) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from CuA to heme a. The slower phase is inhibited completely by cyanide and shows a kinetic deuterium isotope effect by a factor of 2-3. Although two enzyme variants mutated in the so-called D pathway of proton transfer (D124N and E278Q) show the same time constants and relative amplitudes as the wild-type enzyme, in the K pathway variant K354M, 2 is increased to 900 s. This result suggests uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. After the second laser flash under anaerobic conditions, a third electrogenic phase with a time constant of Ϸ1 ms appears. The amplitude of this phase grows with increasing flash number. We explain this growth by injection of a second electron into the single-electron reduced enzyme. On multiple flashes, both D pathway mutants behave differently compared with the wild type and two additional slow phases of 3 Ϸ 2 ms and 4 Ϸ 15 ms are observed. These results suggest that the D pathway is involved in proton transfer coupled to the uptake of the second electron.

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

“…There is evidence that the stoichiometry should be 2H + /2e -upon reduction during the O to R transition, rather than 1.4, with only 1 proton bound into the BNC, as found here (Table 1). Using photoexcited ruthium bispyridyl to deliver single electrons to the P. denitrificans oxidase BNC, electrometric measurements of wild type and Lys I-362 to Met mutants showed that one electrometric phase is inhibited in the mutants, suggesting that the first reduction is coupled to the proton uptake via the K channel (15,16). Multiple excitation accumulates the doubly reduced enzyme, which is associated with a slower electrometric phase, attributed to a second proton uptake (16).…”

Redox-induced Protein Structural Changes in Cytochrome bo Revealed by Fourier Transform Infrared Spectroscopy and [13C]Tyr Labeling

“…Protonation State of Cross-linked Tyr-288 in Oxidized Form-Tyr-288 is located at the end of the K-channel (17)(18)(19)(20)(21)(22) and delivers one (or two) chemical protons to the heme-Cu B binuclear center (12,(25)(26)(27)(28). The presence of a peculiar C ⑀ -N ⑀ covalent bond between Tyr-288 and His-284 (19 -21, 43), one of three histidine ligands of Cu B , lowers the pK a of the phenol moiety (44 -46).…”

“…Upon two-electron reduction of the enzyme, two chemical protons are taken up from the cytoplasm to compensate for an increased negative charge at the binuclear center (12,33), and at least the first proton is delivered to the binuclear center by Tyr-288 at the end of the K-channel (27). If deprotonated at the oxidized (O) state (20), Tyr-288 serves as one of proton acceptors (26).…”

“…They proposed that the D-channel translocates four pumped protons, whereas the K-channel delivers four chemical protons to the binuclear center (18). However, it is now assumed that the K-channel delivers one or two chemical protons to the binuclear center at the initial reductive phase of dioxygen reduction and that the D-channel translocates all other chemical and pumped protons (12,(25)(26)(27)(28).…”

“…They proposed that the D-channel translocates four pumped protons, whereas the K-channel delivers four chemical protons to the binuclear center (18). However, it is now assumed that the K-channel delivers one or two chemical protons to the binuclear center at the initial reductive phase of dioxygen reduction and that the D-channel translocates all other chemical and pumped protons (12,(25)(26)(27)(28).In the vicinity of the binuclear center, Tyr-288 (the E. coli cytochrome bo numbering: Tyr-280 in the soil bacterium P. denitrificans and Tyr-244 in bovine cytochrome c oxidase) is highly conserved in the SoxMtype terminal oxidase and has been proposed to be involved in dioxygen reduction (25, 26, 29 -32). Upon two-electron reduction of the enzyme, two chemical protons are taken up from the cytoplasm to compensate for an increased negative charge at the binuclear center (12,33), and at least the first proton is delivered to the binuclear center by Tyr-288 at the end of the K-channel (27).…”