Fig. 5. Reduction of hemes b_n/c_n by reduced ferredoxin/FNR.

The reaction solution contained 1.5 μM spinach b₆f complex with bound FNR and 3 μM spinach ferredoxin. Semi-anaerobic conditions were obtained through the addition of 10 mM glucose and 160 units of glucose oxidase. Decyl-plastoquinol (50 μM) was present to eliminate any contribution of cytochrome f to the difference spectra. Cytochrome b₆ /c_n reduction was initiated by addition of 0.3 mM NADPH. (A) Comparison of reduction of spinach b₆f complex by NADPH/ferredoxin ¹⁴ and dithionite. The NADPH/ferredoxin minus decyl-plastoquinol difference spectrum (blue trace) was measured 1 min after addition of NADPH. The spectrum of fully reduced cytochrome b₆ (pink) was obtained by addition of dithionite. (B) Dependence of reduction of hemes b_n/c_n reduction on the presence of FNR and ferredoxin. Reduction of spinach b₆f complex following addition of NADPH in the presence (blue) or absence (red) of ferredoxin (3 μM), and of M. laminosus b₆f complex, which does not have bound FNR, in the presence (green) or absence (purple) of ferredoxin. All spectra were measured 1 min after addition of NADPH. (C) Double difference spectrum, normalized at 563 nm, showing the Soret band spectrum of heme c_n (green), measured as the difference between the spectrum obtained in the presence and absence of NQNO. Difference spectra obtained in the absence (blue) and presence (pink) of NQNO (final concentration, 25 μM). (D) Proposed pathway of reduction of hemes b_n and c_n by reduced ferredoxin (Fd) in a Q cycle or cyclic pathway in which ferredoxin is reduced physiologically by the photosystem I reaction center and by NADPH in the present experiment. The requirement of FNR and Fd for the pathway of heme b_n reduction by NADPH is defined by the data shown in Fig. 5B. The ability of NQNO to block the pathway at the site of heme c_n is ascribed to NQNO binding as a ligand of this heme (Fig. 4B) and the NQNO-induced decrease in E_m7 of heme c_n ⁵⁵. The inference that hemes b_n and c_n, together with PQ, are part of a complex in which PQ serves as a ligand of heme c_n is based on the proximity of the two hemes ⁸; ⁹, their interaction measured by EPR ⁶⁶, and binding of the quinone analogue inhibitors, TDS (Fig. 3B) and NQNO (Fig. 4B).