The redox properties, the site of action of the inhibitor NQNO, and the question of interheme transfer in the chloroplast cytochrome b6 have been examined with regard to the role of the b6-f complex in quinol oxidation and H+ translocation, (i) The two hemes of the cytochrome, bn and bp, have similar (ΔEm ≤ 50 mV) oxidation-reduction midpoint potentials that are pH-independent in the range pH 6.5-8.0 (Em7 = -40 mV) but are pH dependent below this range with an estimated pK = 6.7. (ii) Only half of cytochrome b6, the stromal-side heme, bn, was reducible by NADPH and ferredoxin. (iii) The 2-3-fold increase (to 0.60 ± 0.09 heme/600 Chi) in the amplitude of flash-induced cytochrome reduction caused by NQNO was not affected when heme bn was initially reduced, implying that NQNO affects flash reduction at the site of heme bp. (iv) Multiple light flashes did not increase the amplitude of b6 reduction in the presence or absence of NQNO or show binary oscillations. Together with localization of a site of action of NQNO near heme bp, these data provide no evidence for efficient electron transfer from heme bp to heme bn as specified by the Q cycle model, (v) NQNO interaction with heme bp does not block its oxidation, since reoxidation of the flash-reduced cytochrome in its presence or absence was 4-5 times faster (t1/2 ≈ 30 ms) when heme bn was reduced. The faster oxidation of the photoreduced cytochrome after NADPH-Fd reduction of heme bn indicates that the oxidation of bn and bp may be cooperative.
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