Cytochrome b 6 arginine 214 of synechococcus sp. PCC 7002, a key residue for quinone-reductase site function and turnover of the cytochrome bf complex

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 ∼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. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.