A redox study of the electron transport pathway responsible for generation of the slow electrochromic phase in chloroplasts

The amplitude of the slow phase of the electrochromic bandshift and the dark redox state of cytochrome b₆, as well as its flash-induced turnover, have been measured as a function of ambient redox potential between +200 and -200 mV. Formation of a quinol-like donor with an E_m,7 = +100 ± 10 mV is required for generation of the slow phase. 80-100% of the amplitude of this signal with a t _{1 2} = 3-4 ms is observed at -200 mV where cytochrome b₆ was almost fully reduced (E_m,7 of dark and flash-induced photoreduction was -30 mV and -75 mV, respectively). The change in the photoreduction of cytochrome b₆ above 0 mV had an E_m,7 of +50 mV, about 50 mV more negative than the midpoint at this pH for the onset of the slow electrochromic change. At potentials below -140mV the amplitude of b₆ photoreduction becomes small or negligible. The nature of the cytochrome b₆ photoresponse is changed at potentials below -140 mV from a net photoreduction with a t _{1 2} = ≲ 1 ms to a photooxidation with a t _{1 2} = 15-20 ms that is substantially slower than the electrochromic band-shift with a t _{1 2} = 3-4 ms. It is concluded that the slow electrochromic phase probably does not arise from a mechanism involving a turnover of cytochrome b₆. From consideration of the possible flash-induced electron-transfer steps and alternative mechanisms for generation of the slow phase, it is suggested that it may arise from a redox-linked H⁺ pump involving the high potential iron-sulfur protein.