New insights on the proton pump associated with cytochrome b6f turnovers from the study of H/D substitution effects on the electrogenicity and electron transfer reactions.

We have studied the effect of protium/deuterium substitution on different kinetics associated with the turnovers of cytochrome b(6)f complex in whole cells of Chlamydomonas reinhardtii. Both the oxidation of cytochrome f and the reduction of hemes b were only little affected by the isotopic substitution. Contrasting with this, the initial slope of the electrogenic phase associated with cytochrome b(6)f turnover was slowed by a factor of 4 by H(2)O/D(2)O substitution. Whereas in the presence of H(2)O the electrogenic phase developed concomitantly with cytochrome b reduction, it lagged for a few hundreds of microseconds after cytochrome b reduction in the presence of D(2)O. We propose that a proton pump is triggered by the oxidation of plastoquinol at the Q(o) site. The proton transfer is specifically delayed upon isotopic substitution, accounting for the lack of significant effect on the electron-transfer reaction as well as for the strong decrease of the initial rate of the electrogenic phase.