Cytochrome b sub(6) Arginine 214 of Synechococcus sp. PCC 7002, a Key Residue for Quinone-reductase Site Function and Turnover of the Cytochrome bf Complex

Quinone-reductase (Q sub(i)) domains of cyanobacterial/chloroplast cytochrome bf and bacterial/mitochondrial bc complexes differ markedly, and the cytochrome bf Q sub(i) site mechanism remains largely enigmatic. To investigate the bf Q sub(i) domain, we constructed the mutation R214H, which substitutes histidine for a conserved arginine in the cytochrome b sub(6) polypeptide of the cyanobacterium Synechococcus sp. SPCC 7002. At high light intensity, the R214H mutant grew [approx]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 sub(6) reduction and oxidation, the R214H mutation partially blocked electron transfer to the Q sub(i) site, mimicking the effect of the Q sub(i) site inhibitor 2-N-4-hydroxyquinoline- N-oxide. The kinetics of cytochrome b sub(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 sub(6) oxidation at the Q sub(i) site in the wild type, electron flow limited this reaction in the R214H mutant. Redox titration of membranes revealed midpoint potentials (E sub(m,7)) of the two b hemes similar to those in the wild type. Our data define cytochrome b sub(6) Arg super(214) as a key residue for Q sub(i) site catalysis and turnover of the cytochrome bf complex. In the recent cytochrome bf structures, Arg super(214) lies near the Q sub(i) pocket and the newly discovered c sub(i) or x heme. We propose a model for Q sub(i) site function and a role for Arg super(214) in plastoquinone binding.