Evidence for complexed plastocyanin as the immediate electron donor of P-700

The reduction of P-700 by its electron donors shows two fast phases with half-times of 20 and 200 mus in isolated spinach chloroplasts. We have studied this electron transfer and the oxidation kinetics of cytochrome f. Incubation of chloroplasts with KCN or HgCl2 decreased the amplitude of the 20 mus phase. This provides evidence for a function of plastocyanin as the immediate electron donor of P-700. At low concentrations of salt and sugar the fast phases of P-700+ reduction were largely inhibited. Increasing concentrations of MgCl2, KCl and sorbitol (up to 5, 150 and 200 mM, respectively) were found to increase the relative amplitudes of the fast phases to about one-third of the total P-700 signal. Addition of both 3 mM MgCl2 and 200 mM sorbitol increased the relative amplitude of the 20 mus phase to 70%. The interaction between P-700 and plastocyanin is concluded to be favoured by a low internal volume of the thylakoids and compensation of surface charges of the membrane. The half-time of 20 mus was not changed when the amplitude of this phase was altered either by salt and sorbitol, or by inhibition of plastocyanin. This is evidence for the existence of a complex between plastocyanin and P-700 with a lifetime long compared to the measuring time. The 200 mus phase exhibited changes in its half-time that indicated the participation of a more mobile pool of plastocyanin. Cytochrome f was oxidized with a biphasic time course with half-times of 70--130 mus and 440--860 mus at different salt and sorbitol concentrations. The half-time of the faster phase and a short lag of 30--50 mus in the beginning of the kinetics indicate an oxidation of cytochrome f via the 20 mus electron transfer to P-700. An inhibition of this oxidation by MgCl2 suggests that the electron transfer from cytochrome f to complexed plastocyanin is not controlled by negative charges in contrast to that from plastocyanin to P-700.