Electrogenic Reactions of Cytochrome bd

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 O(2) 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 (delta psi, 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 <60 mus that matches the formation of ferryl-oxo heme d, the product of the reaction of O(2) 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 H(2)O(2) 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 delta psi of the opposite polarity (positive inside the vesicles) concurrent with electron flow from heme d to heme b(558) and backward. The amplitude of the delta psi 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 O(2) and is apparently associated with full transmembrane translocation of approximately one charge.

Time-resolved electrometric and optical studies on cytochrome bd suggest a mechanism of electron-proton coupling in the di-heme active site

“…3, trace 1 (A 3 F transition). In previous studies of the WT cytochrome bd (11), the electrogenic events stopped at this point, as expected for the three-electron-reduced enzyme (11,41). However, the preparation used in this work was modified (see Materials and Methods) so as to retain bound quinol, which was demonstrated by HPLC analysis (data not shown).…”

“…Both the WT and the E445A mutant cytochrome bd oxidases were purified as described (36), but the second (hydroxyapatite) chromatographic step was omitted as it can cause destabilization and substantial degradation of heme b 595 in the E445A mutant enzyme (35). Besides, in this work for solubilization of the membranes sucrose monolaurate detergent was used instead of N-dodecyl-N,N-dimethylammonio-3-propane-sulfonate used in an earlier report (11). Change of detergent allowed us to isolate the enzyme containing bound quinone.…”

“…Change of detergent allowed us to isolate the enzyme containing bound quinone. Reconstitution of cytochromes bd into liposomes was performed as described (11) except that the final concentration of the enzyme was increased by four times.…”

“…Unlike the heme-copper oxidases (4,5), cytochrome bd does not pump protons (6). However, the oxidation of ubiquinol by O 2 catalyzed by cytochrome bd is linked to the generation of transmembrane electric potential difference (⌬⌿) (7)(8)(9)(10)(11) by virtue of the fact that the protons resulting from the oxidation of ubiquinol are released into the bacterial periplasm, whereas the protons used to convert O 2 to H 2 O are taken from the bacterial cytoplasm. Cytochrome bd is a heterodimeric enzyme containing three hemes: b 558 , b 595 , and d (12)(13)(14) but no copper.…”

“…These approaches have been successfully applied to the WT cytochrome bd oxidase to study its reaction with O 2 (11). Oxidation of the three-electron-reduced WT cytochrome bd by O 2 has been found to occur in two steps: (i) the electrically silent process of oxygen binding to heme d with formation of compound A (heme d oxy-complex, d 2ϩ -O 2 ), followed by (ii) the monoexponential generation of ⌬⌿ with of Ϸ60 s that corresponds to the conversion of compound A to compound F (ferryl-oxo heme d, Fe 4ϩ ϭ O 2Ϫ ) (11).…”