Mechanism of Redox-linked Proton Pumping in Terminal Enzyme of Cellular Respiration Studied by Pulse Radiolysis

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Cytochrome bo-type ubiquinol oxidase from Escherichia coli belongs to the heme-copper terminal oxidase and serves as a redox-driven proton pump of the aerobic respiratory chain. To understand the molecular mechanism of proton pumping, we have been carrying out site-directed mutagenesis on subunit 1, where dioxygen reduction and proton translocation take place. We applied pulse radiolysis technique, one of the powerful methods for studying one-electron transfer processes, to subunit 1 mutants lacking the Cu_B center or having defects either in D-or K-channel for proton translocation. Upon pulse radiolysis of the wild-type enzyme in the presence of N-methyl nicotinamide as an electron mediator, we observed the generation of ubisemiquinone anion radical with a broad peak at 440 nm at the Q_H site and subsequent electron transfer to hemes b and o with a first-order of 1.5 x 10^3 s^<-1>. In His333ala, a biphasic reduction of the hemes with the rate constants of 1.1 x 10^5 s^<-1> and 8.9 x 10^2 s^<-1> was observed, indicating that the perturbation in heme-to heme electron transfer. The enzymes variants mutated in the D pathway of proton transfer (E286D, D135N) show the same time constants as the wild-type enzyme. In the K pathway variant K362Q and Y288F, on the other hand, the intramolecular electron transfer from heme b to o were decreased. These results suggest that uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. The E_m values for the K mutant enzymes were increased by 125 mV as the wild-type enzyme. Moreover, the difference in the redox potentials between heme b and o increased with the decrease of the intramolecular electron transfer. This suggests that electron transfer is controlled by the proton uptake.