Substitution of the sixth axial ligand of Rhodobacter capsulatus cytochrome c1 heme yields novel cytochrome c1 variants with unusual properties.

The cytochrome (cyt) c1 heme of the ubihydroquinone:cytochrome c oxidoreductase (bc1 complex) is covalently attached to two cysteine residues of the cyt c1 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 c1, and previous mutagenesis studies revealed its critical role for the physicochemical properties of cyt c1 Gray, K. A., Davidson, E., and Daldal, F. (1992) Biochemistry 31, 11864-11873]. In the homologous chloroplast b6f 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 c1 variants with histidine-lysine (K) and histidine-histidine axial coordination were sought. Using a R. capsulatus genetic system, the cyt c1 mutants M183K and M183H were constructed by site-directed mutagenesis, and chromatophore membranes as well as purified bc1 complexes obtained from these mutants were characterized in detail. The studies revealed that these mutants incorporated the heme group into the mature cyt c1 polypeptides, but yielded nonfunctional bc1 complexes with unusual spectroscopic and thermodynamic properties, including shifted optical absorption maxima (lambdamax) and decreased redox midpoint potential values (Em7). The availability and future detailed studies of these stable cyt c1 mutants should contribute to our understanding of how different factors influence the physicochemical and folding properties of membrane-bound c-type cytochromes in general.