Cytochrome c oxidase, the terminal enzyme of cellular respiration in mitochondria and many bacteria, reduces O(2) to water. This four-electron reduction process is coupled to translocation (pumping) of four protons across the mitochondrial or bacterial membrane; however, proton pumping is poorly understood. Proton pumping was thought to be linked exclusively to the oxidative phase, that is, to the transfer of the third and fourth electron. Upon re-evaluation of these data, however, this proposal has been questioned, and a transport mechanism including proton pumping in the reductive phase--that is, during the transfer of the first two electrons--was suggested. Subsequently, additional studies reported that proton pumping during the reductive phase can occur, but only when it is immediately preceded by an oxidative phase. To help clarify the issue we have measured the generation of the electric potential across the membrane, starting from a defined one-electron reduced state. Here we show that a second electron transfer into the enzyme leads to charge translocation corresponding to pumping of one proton without necessity for a preceding turnover.

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.

Using a combination of stopped-flow spectrophotometric proton pumping measurements and time-resolved potential measurements on black lipid membranes, we have investigated the effect of Zn 2+ ions on the proton transfer properties of Paracoccus denitrificans cytochrome c oxidase. When zinc was enclosed in the interior of cytochrome c oxidase containing liposomes, the H/e stoichiometry was found to gradually decrease with increasing Zn 2+ concentration. Halfinhibition of proton pumping was observed at [Zn 2+ ] i = 75 W WM corresponding to about 5^6 Zn 2+ ions per oxidase molecule. In addition, there was a significant increase in the respiratory control ratio of the proteoliposomes upon incorporation of Zn 2+ . Time-resolved potential measurements on a black lipid membrane showed that the electrogenic phases slowed down in the presence of Zn 2+ correspond to phases that have been attributed to proton uptake from the cytoplasmic side and to proton pumping. We conclude that Zn 2+ ions bind close to or within the two proton transfer pathways of the bacterial cytochrome c oxidase. ß

Hydrogenase and fumarate reductase isolated from Woli-nella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteolipo-somes. The proteoliposomes catalyzed fumarate reduction by H 2 which generated an electrical proton potential (Dw ¼ 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H + /e ratio brought about by fumarate reduction with H 2 in proteoliposomes in the presence of valinomycin and external K + was approximately 1. The same Dw and H + /e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H 2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaqui-none and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH 2 which did not generate a Dw. Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Dw of 0.13 V (negative inside). The H + /e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Dw generation is coupled to menaquinone reduction by H 2 or formate, but not to menaquinol oxidation by fumarate. Dw generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H 2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.