Electrogenic Reactions of Cytochrome bd

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Abstract
Cytochrome bd is one of the two terminal quinol oxidases in the respiratory chain of Escherichia coli. The enzyme catalyzes charge separation across the bacterial membrane during the oxidation of quinols by dioxygen but does not pump protons. In this work, the reaction of cytochrome bd with O2 and related reactions has been studied by time-resolved spectrophotometric and electrometric methods. Oxidation of the fully reduced enzyme by oxygen is accompanied by rapid generation of membrane potential (ΔΨ, negative inside the vesicles) that can be described by a two-step sequence of (i) an initial oxygen concentration-dependent, electrically silent, process (lag phase) corresponding to the formation of a ferrous oxy compound of heme d and (ii) a subsequent monoexponential electrogenic phase with a time constant d, the product of the reaction of O2 with the 3-electron reduced enzyme. No evidence for generation of an intermediate analogous to the “peroxy” species of heme−copper oxidases could be obtained in either electrometric or spectrophotometric measurements of cytochrome bd oxidation or in a spectrophotometric study of the reaction of H2O2 with the oxidized enzyme. Backflow of electrons upon flash photolysis of the singly reduced CO complex of cytochrome bd leads to transient generation of a ΔΨ of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b558 and backward. The amplitude of the ΔΨ produced by the backflow process, when normalized to the reaction yield, is close to that observed in the direct reaction during the reaction of fully reduced cytochrome bd with O2 and is apparently associated with full transmembrane translocation of approximately one charge.