Abstract
Quinone-reductase (Q(i)) domains of cyanobacterial/chloroplast cytochrome bf and bacterial/mitochondrial bc complexes differ markedly, and the cytochrome bf Q(i) site mechanism remains largely enigmatic. To investigate the bf Q(i) domain, we constructed the mutation R214H, which substitutes histidine for a conserved arginine in the cytochrome b(6) polypeptide of the cyanobacterium Synechococcus sp. SPCC 7002. At high light intensity, the R214H mutant grew approximately 2.5-fold more slowly than the wild type. Slower growth arose from correspondingly slower overall turnover of the bf complex. Specifically, as shown in single flash turnover experiments of cytochrome b(6) reduction and oxidation, the R214H mutation partially blocked electron transfer to the Q(i) site, mimicking the effect of the Q(i) site inhibitor 2-N-4-hydroxyquinoline-N-oxide. The kinetics of cytochrome b(6) oxidation were largely unaffected by hydrogen-deuterium exchange in the mutant but were slowed considerably in the wild type. This suggests that although protonation events influenced the kinetics of cytochrome b(6) oxidation at the Q(i) site in the wild type, electron flow limited this reaction in the R214H mutant. Redox titration of membranes revealed midpoint potentials (E(m,7)) of the two b hemes similar to those in the wild type. Our data define cytochrome b(6) Arg(214) as a key residue for Q(i) site catalysis and turnover of the cytochrome bf complex. In the recent cytochrome bf structures, Arg(214) lies near the Q(i) pocket and the newly discovered c(i) or x heme. We propose a model for Q(i) site function and a role for Arg(214) in plastoquinone binding.
Highlights
The cytochrome bf complex of oxygenic photosynthesis transfers electrons between the photosystem II and I reaction cen
Mutant and control cultures were insensitive to antimycin (30 M) under all conditions. These data indicate that the R214H mutation did not alter the antimycin insensitivity of the cytochrome bf Qi site but did suggest a partial blockage of electron flow to this site resulting in slower growth under conditions of high electron flux
Electron Flow through Inhibited Qi Sites in Cytochrome bf Complexes—We have shown that the Synechococcus R214H mutation had an effect like that of NQNO, which slows b6 heme oxidation and partially slows the turnover of the cytochrome bf complex [29]
Summary
The cytochrome bf complex of oxygenic photosynthesis transfers electrons between the photosystem II and I reaction cen-. The recent x-ray crystal structures of cytochrome bf complexes have revealed a previously undetected and unique heme designated heme x in the cyanobacterium Mastigocladus laminonsus [9] or heme ci in the alga Chlamydomonas reinhardtii [10]. The most widely accepted model to explain the mechanism of bc-bf complexes is the “Q cycle” hypothesis of Mitchell [13] This scheme, as modified by Crofts et al [14] and others (for example see Ref. 15), postulates both an oxidation of plastoquinol and a reduction of plastoquinone at two distinct sites within the protein, the Qo and Qi sites, located on opposite, positive and negative sides of the membrane.
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