Expression of a cytochrome c2 isozyme restores photosynthetic growth of Rhodobacter sphaeroides mutants lacking the wild-type cytochrome c2 gene.
Deletion of the cytochrome c2 gene in the purple bacterium Rhodobacter sphaeroides renders it incapable of phototrophic growth (strain cycA65). However, suppressor mutants which restore the ability to grow phototrophically are obtained at relatively high frequency (1-10 in 10(7)). We examined two such suppressors (strains cycA65R5 and cycA65R7) and found the expected complement of electron transfer proteins minus cytochrome c2: SHP, c', c551.5, and c554. Instead of cytochrome c2 which elutes from DEAE-cellulose between SHP and cytochrome c', at about 50 mM ionic strength in wild-type extracts, we found a new high redox potential cytochrome c in the mutants which elutes with cytochrome c551.5 at about 150 mM ionic strength. The new cytochrome is more acidic than cytochrome c2, but is about the same size or slightly smaller (13,500 Da). The redox potential of the new cytochrome from strain cycA65R7 (294 mV) is about 70 mV lower than that of cytochrome c2. The 280 nm absorbance of the new cytochrome is smaller than that of cytochrome c2, which suggests that there is less tryptophan (the latter has two residues). In vitro kinetics of reduction by lumiflavin and FMN semiquinones show that the reactivity of the new cytochrome is similar to that of cytochrome c2, and that there is a relatively large positive charge (+2.6) at the site of reduction, despite the overall negative charge of the protein. This behavior is characteristic of cytochromes c2 and unlike the majority of bacterial cytochromes examined. Fourteen out of twenty-four of the N-terminal amino acids of the new cytochrome are identical to the sequence of cytochrome c2. The N-termini of the cycA65R5 and cycA65R7 cytochromes were the same. The kinetics and sequence data indicate that the new protein may be a cytochrome c2 isozyme, which is not detectable in wild-type cells under photosynthetic growth conditions. We propose the name iso-2 cytochrome c2 for the new cytochrome produced in the suppressor strains.
Fitch
J
J
Department of Biochemistry, University of Arizona, Tucson 85721.
Cannac
V
V
Meyer
T E
TE
Cusanovich
M A
MA
Tollin
G
G
Van Beeumen
J
J
Rott
M A
MA
Donohue
T J
TJ
D000595:Amino Acid Sequence; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003574:Cytochrome c Group / Q000235:genetics* / Q000302:isolation & purification; D045362:Cytochromes c2; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D005786:Gene Expression Regulation; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010788:Photosynthesis; D012242:Rhodobacter sphaeroides / Q000235:genetics* / Q000254:growth & development; D013489:Suppression, Genetic
D003574:Cytochrome c Group; D005415:Flavins; C001922:lumiflavin; D005486:Flavin Mononucleotide; D045362:Cytochromes c2