Spectroscopic and oxidation–reduction properties of Rhodobacter capsulatus cytochrome c1 and its M183K and M183H variants

Abstract

Two variants of the cytochrome c 1 component of the Rhodobacter capsulatus cytochrome bc 1 complex, in which Met 183 (an axial heme ligand) was replaced by lysine (M183K) or histidine (M183H), have been analyzed. Electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectra of the intact complex indicate that the histidine/methionine heme ligation of the wild-type cytochrome is replaced by histidine/lysine ligation in M183K and histidine/histidine ligation in M183H. Variable amounts of histidine/histidine axial heme ligation were also detected in purified wild-type cytochrome c 1 and its M183K variant, suggesting that a histidine outside the CSACH heme-binding domain can be recruited as an alternative ligand. Oxidation–reduction titrations of the heme in purified cytochrome c 1 revealed multiple redox forms. Titrations of the purified cytochrome carried out in the oxidative or reductive direction differ. In contrast, titrations of cytochrome c 1 in the intact bc 1 complex and in a subcomplex missing the Rieske iron–sulfur protein were fully reversible. An E m7 value of −330 mV was measured for the single disulfide bond in cytochrome c 1. The origins of heme redox heterogeneity, and of the differences between reductive and oxidative heme titrations, are discussed in terms of conformational changes and the role of the disulfide in maintaining the native structure of cytochrome c 1.