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 Qo site in cytochrome b6f 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 b6f complexes. Spectroscopic analysis performed in anaerobic conditions in vivo revealed distinct alterations in cytochrome b6f 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 Qo site without a change in the affinity of Qo for reduced plastoquinones. The E78Q and E78N substitutions modified the characteristics of cytochrome b6f turnover under repetitive flash illumination. They caused a large increase in the electrogenicity of the electron-transfer reactions through the mutated cytochrome b6f 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 b6 became barely detectable in the mutants, unless inhibitors at the Qi site were present. We show that similar functional characteristics can be observed with the cytochrome b6f complex in the wild-type in anaerobic conditions, provided a single flash illumination regime is used. These observations suggest that cytochrome b6f turnover may involve a mechanism implying an extra proton pumping activity.
Zito
F
F
UPR9072 CNRS, Institut de Biologie Physico-Chimique, Paris, France.
Finazzi
G
G
Joliot
P
P
Wollman
F A
FA
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D002736:Chloroplasts / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism*; D045346:Cytochrome b6f Complex; D004579:Electron Transport; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D003573:Cytochrome b Group; D010446:Peptide Fragments; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D045346:Cytochrome b6f Complex; D007930:Leucine; D008239:Lysine