Redox-Coupled Proton Pumping Activity in Cytochromeb6f, As Evidenced by the pH Dependence of Electron Transfer in Whole Cells ofChlamydomonas reinhardtii

Abstract

The pH dependence of cytochrome b(6)f catalytic activity has been measured in whole cells of the green alga Chlamydomonas reinhardtii over the 5-8 range. An acid pH slowed the reactions occurring at the lumenal side of the complex (cytochrome b(6) and f reduction) and affected also the rate and amplitude of the slow electrogenic reaction (phase b), which is supposed to reflect transmembrane electron flow in the complex. On the other hand, a direct measurement of the transmembrane electron flow from the kinetics of cytochrome b(6) oxidation revealed no pH sensitivity. This suggests that a substantial fraction of the electrogenicity associated with cytochrome b(6)f catalysis is not due to electron transfer in the b(6) hemes but to a plastoquinol-oxidation-triggered charge movement, in agreement with previous suggestions that a redox-coupled proton pump operates in cytochrome b(6)f complex. The pH dependence of cytochrome b(6)f activity has also been measured in two mutant strains, where the glutamic 78 of the conserved PEWY sequence of subunit IV has been substituted for a basic (E78K) and a polar (E78Q) residue [Zito, F., Finazzi, G., Joliot, P., and Wollman, F.-A. (1998) Biochemistry 37, 10395-10403]. Their comparison with the wild type revealed that this residue plays an essential role in plastoquinol oxidation at low pH, while it is not required for efficient activity at neutral pH. Its involvement in gating the redox-coupled proton pumping activity is also shown.