Confirmation of the involvement of protein domain movement during the catalytic cycle of the cytochrome bc1 complex by the formation of an intersubunit disulfide bond between cytochrome b and the iron-sulfur protein

To study the essentiality of head domain movement of the Rieske iron-sulfur protein (ISP) during bc(1) catalysis, Rhodobacter sphaeroides mutants expr essing His-tagged cytochrome be, complexes with three pairs of cysteines en gineered (one cysteine each) on the interface between cytochrome b and ISP, A185C(cytb)/K70C(ISP), I326C(cytb)/G165C(ISP), and T386C(cytb)/K164C(ISP), were generated and characterized. Formation of an intersubunit disulfide b ond between cytochrome b and ISP is detected in membrane (intracytoplasmic membrane and air-aged chromatophore), and purified be, complex was prepared from the A185C(cytb)/K70C(ISP) mutant cells. Formation of the intersubunit disulfide bond in this cysteine pair mutant complex is concurrent with the loss of its be, activity. Reduction of this disulfide bond by P-mercaptoet hanol restores activity, indicating that mobility of the head domain of ISP is functionally important in the cytochrome be, complex. The rate of intra molecular electron transfer, between 2Fe2S and heme c(1), in the A185C(cytb )/K70C(ISP) mutant complex is much lower than that in the wild type or in t heir respective single cysteine mutant complexes, indicating that formation of an intersubunit disulfide bond between cytochrome b and ISP arrests the head domain of ISP in the "fixed state" position, which is too far for ele ctron transfer to heme c(1).