Models for structure and function in quinone-binding sites: the Escherichia coli quinol oxidase, cytochrome bo3.

Abstract

Quinone-binding sites are obligatory in quinoneutilizing respiratory and photosynthetic complexes. In addition to their importance in catalysis in electron-transfer systems involved in energy transduction, these loci are the sites of action of numerous inhibitors, including pesticides, herbicides and antibiotics [l]. The binding sites are of three types ; acceptor, donor and pair-splitting loci. In the first two types of site the potentials of the two half reactions have to be approximately equal, as the electrons are fed to/from the same electron donor/acceptor and this requires stabilization of the semiquinone. Since the stability constant of free ubisemiquinone at neutral pH is x lo-'', the binding of the semiquinone must be at least x lo5 times as tight as the other two species to achieve a stability constant of unity [2,3]. Tight binding of the semiquinone also allows the site to confine the reactive semiquinone species, conferring kinetic and thermodynamic stabilization. Pair-splitting loci, such as that found in the quinol cytochrome c reductase complex, do not stabilize the semiquinone, as this is unnecessary for the thermodynamics of the reaction [4]. The pair-splitting site for quinol oxidation (Q, or Q,) by quinol cytochrome c reductase facilitates the oxidation of quinol to quinone by two different electron carriers, with widely separated redox potentials, functioning as acceptors [4]. The spatial separation of the acceptors and their gross thermodynamic difference requires a site with different properties from the sites of quinone reduction. The x 300-400 mV separation in the potentials of the acceptors effectively removes the requirement for thermodynamic stabilization of the semiquinone state at this site. The amino acid sequences of many enzymes with quinone-binding sites have been published. In addition, a small number of crystal structures are available. No general sequence motif has been recognized for quinone-binding sites, but a structural motif is beginning to emerge (see below). The Escherichiu coli quinol oxidase cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome uu3, in all aspects of its structure and function, except that it lacks the cytochrome c binding and the Cu, prosthetic group and instead has quinone-binding site(s) [S]. The putative oxidation of quinol by ferrihaem (cytochrome b) in sequential one-electron steps would require the stabilization of semiquinone. However, mechanisms involving two quinol-binding sites have also been proposed [6-81. Experimentally, the presence of a locum that highly stabilizes ubisemiquinone with respect to free ubisemiquinone is well established in the complex [9]. If there is a second site, for which there is evidence, it is not one at which the semiquinone radical is thermodynamically stabilized. Structural information is presented below on a putative quinol-binding site in the bo, oxidase.