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

Abstract

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 (c_½) for half-maximum effect of 1.7, 0.1 and 5 µM, respectively. The valinomycin-stimulated proton uptake, which has been attributed to Q-cycle activity in thylakoids, was more sensitive to HQNO and NQNO, with c_½ of 0.6 and < 0.05 µM 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 c_½ being 0.05 µM. 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 c_½ of 0.05 µM. The diminution of the oxidation rate of cyt b6 was similarly related to [NQNO] but that of the reduction rate had a c_½ of -1 µM.

The findings on proton uptake are seen as consistent with HQNO and NQNO inhibiting at the Q_c sites on cyt b/f complexes, at Q_B 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 Q_c. Electron transport from H₂O to methyl purple was more sensitive to NQNO for the first few turnovers (c_½ 0.1 µM) than in the steady state (c_½ - 1 µM).