The cytochrome (cyt) c(1) heme of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) is covalently attached to two cysteine residues of the cyt c(1) polypeptide chain via two thioether bonds, and the fifth and sixth axial ligands of its iron atom are histidine (H) and methionine (M), respectively. The latter residue is M183 in Rhodobacter capsulatus cyt c(1), and previous mutagenesis studies revealed its critical role for the physicochemical properties of cyt c(1) [Gray, K. a., Davidson, E., and Daldal, F. (1992) Biochemistry 31, 11864-11873]. In the homologous chloroplast b(6)f complex, the sixth axial ligand is provided by the amino group of the amino terminal tyrosine residue. To further pursue our investigation on the role played by the sixth axial ligand in heme-protein interactions, novel cyt c(1) variants with histidine-lysine (K) and histidine-histidine axial coordination were sought. Using a R. capsulatus genetic system, the cyt c(1) mutants M183K and M183H were constructed by site-directed mutagenesis, and chromatophore membranes as well as purified bc(1) complexes obtained from these mutants were characterized in detail. The studies revealed that these mutants incorporated the heme group into the mature cyt c(1) polypeptides, but yielded nonfunctional bcr complexes with unusual spectroscopic and thermodynamic properties, including shifted optical absorption maxima (lambda(max)) and decreased redox midpoint potential values (E-m7) The availability and future detailed studies of these stable cyt cl mutants should contribute to our understanding of how different factors influence the physicochemical and folding properties of membrane-bound c-type cytochromes in general.
