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

Time-resolved electron transfer and electrogenic H⁺ translocation have been compared in a bd-type quinol oxidase from Escherichia coli and its E445A mutant. The high-spin heme b₅₉₅ is found to be retained by the enzyme in contrast to the original proposal, but it is not reducible even by excess of dithionite. When preincubated with the reductants, both the WT (b₅₅₈²⁺, b₅₉₅²⁺, d²⁺) and E445A mutant oxidase (b₅₅₈²⁺, b₅₉₅³⁺, d²⁺) bind O₂ rapidly, but formation of the oxoferryl state in the mutant is ≈100-fold slower than in the WT enzyme. At the same time, the E445A substitution does not affect intraprotein electron re-equilibration after the photolysis of CO bound to ferrous heme d in the one-electron-reduced enzyme (the so-called "electron backflow"). The backflow is coupled to membrane potential generation. Electron transfer between hemes d and b₅₅₈ is electrogenic. In contrast, electron transfer between hemes d and b₅₉₅ is not electrogenic, although heme b₅₉₅ is the major electron acceptor for heme d during the backflow, and therefore is not likely to be accompanied by net H⁺ uptake or release. The E445A replacement does not alter electron distribution between hemes b₅₉₅ and d in the one-electron reduced cytochrome bd [E_m(d) > E_m(b₅₉₅), where E_m is the midpoint redox potential]; however, it precludes reduction of heme b₅₉₅, given heme d has been reduced already by the first electron. Presumably, E445 is one of the two redox-linked ionizable groups required for charge compensation of the di-heme oxygen-reducing site (b₅₉₅, d) upon its full reduction by two electrons.