Expression of a cytochrome c2 isozyme restores photosynthetic growth of Rhodobacter sphaeroides mutants lacking the wild-type cytochrome c2 gene

Abstract

Deletion of the cytochrome c 2 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 c 2: SHP, c′, c 551.5, and c 554. Instead of cytochrome c 2 which elutes from DEAE-cellulose between SHP and cytochrome c′, at about 50 m m ionic strength in wild-type extracts, we found a new high redox potential cytochrome c in the mutants which elutes with cytochrome c 551.5 at about 150 m m ionic strength. The new cytochrome is more acidic than cytochrome c 2, 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 c 2. The 280 nm absorbance of the new cytochrome is smaller than that of cytochrome c 2, 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 c 2, 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 c 2 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 c 2. 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 c 2 isozyme, which is not detectable in wild-type cells under photosynthetic growth conditions. We propose the name iso-2 cytochrome c 2 for the new cytochrome produced in the suppressor strains.