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

Discovery of the True Peroxy Intermediate in the Catalytic Cycle of Terminal Oxidases by Real-time Measurement

“…3, dashed trace. It consists of an initial non-electrogenic phase (the lag) followed by the electrogenic phase, in agreement with previous reports (37,39). The fit of the electrometric trace with the rate constants obtained in spectrophotometric measurements revealed that the first two processes, which were assigned to the formation of compound A followed by the transition from A to P, are electrically silent.…”

“…The A formation is followed by electron transfer from heme b 595 to form P. The A 3 P transition occurs with k ϭ 2.2 ϫ 10 5 s Ϫ1 and is also non-electrogenic. The finding that electron transfer from heme b 595 to heme d is not coupled with membrane potential generation is fully consistent with a recent study (37). The chemical structure of P needs to be established.…”

“…The reaction of the three electron-reduced enzyme with oxygen stalls at the F state, as expected (38,39). However, under specific conditions of the enzyme isolation, it is possible to purify cytochrome bd from E. coli that retains some quantity of bound quinol (37). Such a reduced enzyme contains five reducing equivalents, and the O 2 reaction proceeds beyond F. Our experiments with this enzyme preparation show an additional phase with ϭ 1.1 ms.…”

“…1C). In contrast, the spectrum of the 4.5-s phase corresponding to the A 3 P transition has minima at 560 and 595 nm, which are typical features of the oxidized-minus-reduced spectrum of heme b 595 (37,53,54). Further support for the conclusion that heme b 595 gets oxidized during the A 3 P transition comes from the fact that the difference spectrum of the 4.5-s phase in the Soret is dominated by the bleaching at 440 nm (Fig.…”

“…coli cells were obtained from GO105/pTK1 strain according to Ref. 46, and cytochrome bd oxidase was isolated from cell membranes as described (37,47). Cytochrome bd from A. vinelandii strain MK8 was isolated as reported in Ref.…”