Resolution of proton and electron transfer events in the photosynthetic reaction center and the cytochrome-bc1 complex of phototrophic bacteria

Abstract

We studied proton circuiting in the inner membrane vesicles (chromatophores) from phototrophic bacteria Rb. sphaeroides and Rb. capsulatus. To resolve the proton transfer steps, we monitored the flash induced Dy (by electrochromic bandshift), DpH (by pH-dyes), redox changes, and the ATP yield (by luciferin-luciferase). Specific inhibitors allowed to separate partial reactions in the individual enzymes: 1) In the photosynthetic reaction centers of Rb. sphaeroides, the secondary quinone QB is reduced and protonated to an ubiquinol QBH2. Two protonation steps were arranged with the twists of the quinone ring in the binding pocket yielding a mechanistic scheme of the QB turnover [1-3]. 2) Divalent cations were found to inhibit the oxidation of heme bh in the cytochrome-bc1 complex (bc1) of Rb. capsulatus. The block was "leaky" (in contrast to those imposed by antimycin A), because bc1 generated, although slowly, Dy that was as large as in the absence of the cations. Thereby we resolved the turnover of the bc1 into (i) a faster non-electrogenic reaction of the QH2 oxidation and bh reduction and (ii) a slower electrogenic reaction coupled with the oxidation of bh. 3) We revealed that chromatophore vesicles contain, on the average, one ATP synthase molecule. This finding allowed to study the proton flow through FOF1 in a single-flash, quasi-single-enzyme mode. 4) We used different means to switch off the Dy and DpH components of the protonmotive force. The flash-induced ATP yield turned out to be sensitive to the changes in Dy and in the surface activity of protons but not to the changes in the bulk lumenal pH.