On the Question of Interheme Electron Transfer in the Chloroplast Cytochrome b₆ in Situ

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, b_n and b_p, have similar (ΔE_m ≤ 50 mV) oxidation-reduction midpoint potentials that are pH-independent in the range pH 6.5-8.0 (E_m7 = -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, b_n, 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 b_n was initially reduced, implying that NQNO affects flash reduction at the site of heme b_p. (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 b_p, these data provide no evidence for efficient electron transfer from heme b_p to heme b_n as specified by the Q cycle model, (v) NQNO interaction with heme b_p does not block its oxidation, since reoxidation of the flash-reduced cytochrome in its presence or absence was 4-5 times faster (t_1/2 ≈ 30 ms) when heme b_n was reduced. The faster oxidation of the photoreduced cytochrome after NADPH-Fd reduction of heme b_n indicates that the oxidation of b_n and b_p may be cooperative.