Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine

The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreduct ase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues. In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome cl cont ains two additional cysteines, C144 and C167. Site-directed mutagenesis rev eals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c, but absent in higher organisms. Both single and double Cys to Ala substitutions drastically lower the +320 mV redox pot ential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1). In sharp contrast to the native protein, mutant cytochrome cl binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential. In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta -b ranched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern beta XM, naturally common in many other high-poten tial cytochromes c. Despite the unrepaired disulfide bond, the beta XM reve rtants are no longer vulnerable to CO binding and restore function by raisi ng the redox potential to +227 mV, which is remarkably close to the value o f the beta XM containing but loop-free mitochondrial cytochrome c(1). The d isulfide anchored loop and beta XM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.