Structure of the yeast cytochrome bc1 complex with a hydroxyquinone anion Qo site inhibitor bound

Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc(1) complex catalysis. Binding of the competitive inhibitor 5-n-heptyl-6- hydroxy-4,7-dioxobenzothiazole to the Q(o) site of the cytochrome bc(1) complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pK(a) = 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His(181) and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr(279). The hydroxy group of the latter contributes to stabilization of the Rieske protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His(181) with a pK(a) of 7.5. Glu(272), a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme b(L) propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Q(o) site.