The involvement of serine 175 and alanine 185 of cytochrome b of Rhodobacter sphaeroides cytochrome bc1 complex in interaction with iron- sulfur protein

Abstract

An approach involving cysteine replacement of potentially noncritical amino acid residues, followed by chemical modification studies, was used to investigate structure-function of the 'cd helix' of cytochrome b from Rhodobacter sphaeroides. Three amino acid residues, Ser-155, Ser-175, and Ala-185, which span this region of cytochrome b, were selected for this study. The S155C substitution yields cells unable to support photosynthetic growth, indicating that Ser-155 is a critical amino acid residue. Further mutational studies of Ser-155 indicate that the size of the amino acid side chain at this position is critical for photosynthetic growth of R. sphaeroides. On the other hand, the S175C and A185C substitutions yield cells with photosynthetic growth rates and enzyme kinetics of the bc1 complexes very similar to those of the unmutated complex, indicating that Ser-175 and Ala-185 are noncritical residues. Thus, engineered cysteines at these two positions of cytochrome b are suitable for membrane topology and domain/subunit interaction studies. Cys-175 does not react with a sulfhydryl- modifying reagent, N-ethylmaleimide (NEM), either in sealed, inside-out chromatophores or in detergent-disrupted chromatophores, indicating that position 175 of cytochrome b is inaccessible from both sides of the membrane and is probably buried within the protein complex. Cys-185 reacts with NEM only after detergent disruption of the sealed, inside-out chromatophores, indicating that this position of cytochrome b is accessible on the outer (periplasmic) surface of the membrane. These results place the cd helix of cytochrome b on the periplasmic side of the chromatophore membrane. When purified A185C-substituted bc1 complex was treated with NEM, about 87% of the activity was abolished due to NEM modification of Cys-185. The signature of the Rieske iron-sulfur center is broadened upon NEM modification of A185C, with the g(x) signal shifting from g = 1.80 to g = 1.75, suggesting that Ala- 185 of cytochrome b interacts with the iron-sulfur protein. When purified S175C-substituted bc1 complex is treated with NEM, no change in the activity is observed, since Cys-175 is inaccessible to NEM. However, when the iron- sulfur protein is removed from the S175C-substituted bc1 complex, Cys-175 becomes accessible to NEM, indicating that Ser-175 of cytochrome b is shielded by the iron-sulfur protein in the bc1 complex.