Function-directed mutagenesis of the cytochrome b6f complex in Chlamydomonas reinhardtii: involvement of the cd loop of cytochrome b6 in quinol binding to the Q(o) site.
The FUD2 mutant from the green alga Chlamydomonas reinhardtii expresses a cytochrome b6 variant of higher apparent molecular mass [Lemaire et al. (1986) Biochim. Biophys. Acta 851, 239-248]. Here, we show that the mutation corresponds to a 36 base pair duplication in the chloroplast petB gene, which corresponds to a 12 amino acid duplication in the cd loop of cytochrome b6. The resulting protein still binds its heme cofactors and assembles into cytochrome b6f complexes, which accumulate in wild type amounts in exponentially growing cells of FUD2. However, these cytochrome b6f complexes show loosened binding of the Rieske protein and are more prone to degradation in aging cells. Electron transfer through the cytochrome b6f complexes is about 8 times slower in FUD2 than in wild type cells. This is due to a slower oxidation of plastoquinol at the Q(o) site, the folding of which is most likely altered by the duplication. By varying the redox state of the plastoquinone pool in vivo, we show that there is a dramatic decrease in the affinity of the Q(o) site for plastoquinols, which is about 100 times lower in FUD2 than in wild type cells. Our results show that the value of the binding constant of plastoquinol to the Q(o) site (2 x 10(4) M(-1)) derived in [Kramer et al. (1994) Biochim. Biophys. Acta 1184, 251-262] may be extrapolated to in vivo conditions.
Finazzi
G
G
CNRS UPR 9072, Institut de Biologie Physico-Chimique, Paris, France.
Büschlen
S
S
de Vitry
C
C
Rappaport
F
F
Joliot
P
P
Wollman
F A
FA
D000818:Animals; D001483:Base Sequence; D016227:Benzoquinones / Q000494:pharmacology; D001665:Binding Sites; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045346:Cytochrome b6f Complex; D003580:Cytochromes / Q000378:metabolism; D045348:Cytochromes f; D017931:DNA Primers; D004579:Electron Transport; D014450:Electron Transport Complex III; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics; D010971:Plastoquinone / Q000031:analogs & derivatives* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011804:Quinolines / Q000378:metabolism
D016227:Benzoquinones; D003573:Cytochrome b Group; D003580:Cytochromes; D017931:DNA Primers; D007506:Iron-Sulfur Proteins; C104657:PetB protein, plant; D010940:Plant Proteins; D011804:Quinolines; C037989:Rieske iron-sulfur protein; C003165:plastoquinol; C004532:quinone; D045346:Cytochrome b6f Complex; D045348:Cytochromes f; D014450:Electron Transport Complex III; D010971:Plastoquinone