Cytochrome c2 mutants of Rhodobacter capsulatus.
Although structurally related to other members of the class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology to the eukaryotic cytochromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have developed a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. capsulatus wild-type cytochrome c2 in cytochrome c2-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement for photosynthetic growth in R. sphaeroides. Further, we describe the generation, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overproduced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These findings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants.
Caffrey
M
M
Department of Biochemistry, University of Arizona, Tucson 85721.
Davidson
E
E
Cusanovich
M
M
Daldal
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D045362:Cytochromes c2; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010101:Oxygen Consumption; D010788:Photosynthesis; D016354:Rhodobacter capsulatus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D003574:Cytochrome c Group; D009838:Oligodeoxyribonucleotides; D045362:Cytochromes c2