Further Studies of Proton Translocations in Chloroplasts After Single-Turnover Flashes. IV. Effects of Cytochrome b/f Complex Inhibitors on Proton Uptake and Cytochrome b6 Turnover

The effects of the substances 2-n-heptyl- and 2-n-nonyl-4-hydroxyquinoline N-oxide (HQNO, NQNO), and antimycin A (AMA) on proton uptake stimulated by a 10-20 Hz train of single-turnover flashes given to pea thylakoids were investigated. Electron transport was from H2O to ferricyanide ('oxidising conditions') and the pH indicator of proton uptake was phenol red. All three of HQNO, NQNO and AMA inhibited proton uptake in control conditions, with concentrations (c½) for half-maximum effect of 1.7, 0.1 and 5 �M, respectively. The valinomycin-stimulated proton uptake, which has been attributed to Q-cycle activity in thylakoids, was more sensitive to HQNO and NQNO, with c½ of 0.6 and < 0.05 �M respectively. AMA had the same or less relative effect on proton uptake in the presence of valinomycin as in its absence. In oxidising conditions the maximum extent of flash-induced cytochrome (cyt) b6 reduction was 7-9% of the total present (which was 2 molecules/620 chlorophylls), as an average during 10 flashes, valinomycin being always added to reduce interference from the electrochromic effect. The average half- time for this reduction was 3.4 ms, while that for oxidation was 420 ms. The amount of cyt b6 reduced was increased by NQNO to a maximum of 14-19%, the c½ being 0.05 �M. Reduction and oxidation rates were both diminished by NQNO. In reducing conditions [electrons from duroquinol to methyl viologen, 3-(3,4-dichlorophenyl)-l,l- dimethylurea added to inhibit photosystem II], the cytochrome b6 was 12-16% reduced during flashes at 0.5-1 Hz, with half-times of 3.1 and 21 ms for reduction and oxidation, respectively. NQNO increased the percentage reduced to a maximum of 34-45, with a c½ of 0.05 �M. The diminution of the oxidation rate of cyt b6 was similarly related to [NQNO] but that of the reduction rate had a c½ of -1 �M. The findings on proton uptake are seen as consistent with HQNO and NQNO inhibiting at the Qc sites on cyt b/f complexes, at QB sites near photosystem II with less specificity and possibly at Q2 sites during the first few turnovers. Data for AMA indicated that it does not inhibit at Qc. Electron transport from H2O to methyl purple was more sensitive to NQNO for the first few turnovers (c½ 0.1 �M) than in the steady state (c½ - 1 �M).