Glu78, from the conserved PEWY sequence of subunit IV, has a key function in cytochrome b6f turnover

We have investigated the structure to function relationship at the Q(o), site in cytochrome b(6)f complexes in vivo. To this end, we created site-directed mutants of Chlamydomonas reinhardtii, at position 78 in the sequence of subunit IV. The target glutamic acid, present in the highly conserved 77PEWY80 sequence, was changed to residues of different polarities which did not prevent the functional assembly of cytochrome b(6)f complexes. Spectroscopic analysis performed in anaerobic conditions in vivo revealed distinct alterations in cytochrome b(6)f function, depending on the nature of the substituted residue. The semiconservative E78D substitution, in which only the length of the side chain is reduced, retained the functional features of the wild-type configuration. The E78K and E78L substitutions caused a significant decrease, by factors of 3 and 5, respectively, in the rate of the concerted oxidation process at the Q(o), site without a change in the affinity of Q(o), for reduced plastoquinones. The E78Q and E78N substitutions modified the characteristics of cytochrome b(6)f turnover under repetitive flash illumination. They caused a large increase in the electrogenicity of the electron-transfer reactions through the mutated cytochrome b(6)f complex. This increase was specifically sensitive to the electrical component of the proton-motive force. Surprisingly, despite the larger number of charges translocated across the membrane per charge injected in the high potential chain, the reduction phase for cytochrome bs became barely detectable in the mutants, unless inhibitors at the Q(i) site were present. We show that similar functional characteristics can be observed with the cytochrome b(6)f complex in the wild-type in anaerobic conditions, provided a single flash illumination regime is used. These observations suggest that cytochrome b(6)f turnover may involve a mechanism implying an extra proton pumping activity.