Cytochrome b6 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 ϳ2.5-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.

The Q cycle of cytochrome bc complexes: A structure perspective

“…There is a discrepancy between titrations of the b 6 f complex in vitro , for which a measurable ΔE m ≈ 100 mV is consistently resolved (251–254, 130), and a number of in situ (in membrane) titrations that do not show resolved b n and b l (ΔE m ≤ 50 mV) (255–257, 187, 229, 253). Two studies on in situ titrations report two resolved b heme components (258, 259), and a large ΔE m has been inferred in studies on the slow electrochromic phase (260–262, 192, 186, 79), and biphasic kinetics of heme reduction (105). Regarding the more negative E m values obtained for one of the b hemes titrated i n vitro , it could result from solvent exposure (263), as shown for cytochrome b -559 in the PS II reaction center (264, 265).…”

Thylakoid potassium channel is required for efficient photosynthesis in cyanobacteria

“…17, and chlorophyll fluorescence was measured using a Dual-PAM-100 (Walz). Cytochrome b 6 f kinetics were measured using a JTS-10 spectrophotometer (Biologic) as described previously (37). P 700 redox changes were assessed at 705 nm, using the same instrumental setup.…”

Quinone-dependent proton transfer pathways in the photosynthetic cytochrome b 6 f complex

“…1B). Although the presence of water molecules in the n-side pathway was predicted (19), the simpler Asp20→Arg207 pathway is devoid of water molecules. An alternate n-side Glu29→Asp35 (subunit IV) proton transfer pathway contains a water molecule.…”

“…Neither the composition nor the structure of the proton transfer chains has been solved in the b 6 f complex. On the basis of structure analogy with the bc 1 complex (18) and the effect of mutagenesis on redox properties, it has been inferred that on the negative (n)-side, Arg207 of cytochrome b 6 (Arg214 in Synechococcous PCC 7002) (19) and on the positive (p)-side, Glu78 (20) in the conserved Pro-Glu-Trp-Tyr (PEWY) sequence of subunit IV, are involved in pathways of proton transfer.…”