BPS2026 – Regulation of cytochrome c binding in the yeast bc1 complex

The cytochrome bc1 complex (Complex III, ubiquinol-cytochrome c oxidoreductase) is a highly conserved multi-subunit protein found in the mitochondria and is a key complex in the electron transport chain. During oxidative phosphorylation, cytochrome (cyt) c, a mobile electron carrier, binds to one cyt c1 subunit of a bc1 complex dimer and shuttles electrons from Complex III to Complex IV. X-ray crystallographic studies revealed that only one molecule of cyt c binds to one bc1 complex dimer, despite two cytochrome c1 subunits available for binding, pointing toward the existence of a regulation mechanism preventing the docking of a second cyt c substrate. However, the structural basis for such a mechanism of long-range (>30Å) regulation of substrate binding is not clear from static structural studies. We employed all-atom molecular dynamics simulations to uncover a possible mechanism of regulation. Our results reveal that a finger-like extended domain of the vacant cyt c1 subunit undergoes a conformational change with its tip moving toward cyt c, transferring mechanical motion and causing distortion of the vacant cyt c binding site. Using fluorescence anisotropy experiments with cyt c covalently labeled with a long-lived Ru(bpy)3 fluorophore, we experimentally measure cyt c binding stoichiometry and affinity to detergent solubilized yeast bc1 under a range of conditions and compare the results with our computational predictions. We also explore the impact of structural dynamics on electron transfer and heme redox potentials as well as implications for generation of reactive oxygen species.