Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002.
The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.
Lee
T
T
Metzger
S U
SU
Cho
Y S
YS
Whitmarsh
J
J
Kallas
T
T
D000483:Alleles; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045346:Cytochrome b6f Complex; D003580:Cytochromes / Q000037:antagonists & inhibitors; D045348:Cytochromes f; D003991:Dibromothymoquinone / Q000737:chemistry / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction
D003573:Cytochrome b Group; D003580:Cytochromes; D004791:Enzyme Inhibitors; C104657:PetB protein, plant; D010940:Plant Proteins; D003991:Dibromothymoquinone; D045346:Cytochrome b6f Complex; D045348:Cytochromes f