Role of the PEWY glutamate in hydroquinone-quinone oxidation-reduction catalysis in the Qo Site of cytochrome bc1.

The glutamic acid residue of the conserved PEWY motif of the Q(o) site of cytochrome bc(1) is widely discussed as central to reversible Q(o) site catalysis of two-electron, two-proton hydroquinone-quinone oxidation-reduction. Extensive mutation of this glutamate (E295) to A, V, F, H, K, and Q in purple photosynthetic Rhodobacter capsulatus results in hydroquinone oxidation rates that are between 5 and 50-fold slower than that in the wild type. However, the mutants show little or no detectable effects on hydroquinone or quinone exchange and binding at the Q(o) site nor on subsequent Q(o) site-mediated redox equilibria in the c-chain and b-chain from pH 5-10. Lack of effects of mutations on the E(m)/pH plots rules out involvement of E295 in the strong electron-proton coupling evident in either the FeS center or heme b(L). These detailed equilibrium and kinetic analyses demonstrate that E295 is not irreplaceable in the Q(o) site catalytic mechanism. Rather, E295 and several other Q(o) site residues that can also be widely varied and still support hydroquinone oxidation illustrate the considerable resilience of Q(o) site activity to mutational change in Q(o) site environs. Residues and water molecules appear to cooperate in providing a physical and chemical environment supporting hydroquinone oxidation rates comparable to those seen in nonprotein aqueous environments at electrodes. We suggest that residues at the Q(o) site (and, possibly, other respiratory and photosynthetic quinone and oxygen binding sites) are a product of natural selection primarily acting not to lower catalytic barriers according to the traditional view of enzymatic catalysis but rather to develop specificity by raising barriers in defense of semiquinone loss or energy wasting short-circuit reactions.