Interactions Between Ascorbyl Free Radical and Coenzyme Q at the Plasma Membrane

Antonio Arroyo(1), Francisco Navarro(2), Consuelo Gómez-Díaz(3), Frederick L. Crane(4), Francisco J. Alcaín(1), Plácido Navas (3) and José M. Villalba (5)

1. Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Universidad de Córdoba, E-14071 Córdoba, Spain
2. Departamento de Ciencias Agroforestales, Universidad de Huelva, Huelva, Spain
3. Laboratorio Andaluz de Biología, Universidad Pablo de Olavide, Sevilla, Spain
4. Department of Biological Sciences, Purdue University, West Lafayette, Indiana, 47907
5. Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Universidad de Córdoba, E-14071 Córdoba, Spain

Abstract
A role for coenzyme Q in the stabilization of extracellular ascorbate by intact cells has been recently recognized. The aim of this work was to study the interactions between reduced ubiquinone in the plasma membrane and the ascorbyl free radical, as an approach to understand ubiquinone-mediated ascorbate stabilization at the cell surface. K-562 cells stabilized ascorbate and decreased the steady-state levels of the semi ascorbyl radical.

The ability of cells to reduce ascorbyl free radical was inhibited by the quinone analogs capsaicin and chloroquine and stimulated by supplementing cells with coenzyme Q10. Purified plasma membranes also reduced ascorbyl free radical in the presence of NADH. Free-radical reduction was not observed in quinone-depleted plasma membranes, but restored after its reconstitution with coenzyme Q10.

Addition of reduced coenzyme Q10 to depleted membranes allowed them to reduce the signal of the ascorbyl free radical without NADH incubation and the addition of an extra amount of purified plasma membrane quinone reductase further stimulated this activity. Reduction was abolished by treatment with the reductase inhibitor p-hydroximercuribenzoate and by blocking surface glycoconjugates with the lectin wheat germ agglutinin, which supports the participation of transmembrane electron flow.

The activity showed saturation kinetics by NADH and coenzyme Q, but not by the ascorbyl free radical in the range of concentrations used. Our results support that reduction of ascorbyl free radicals at the cell surface involves coenzyme Q reduction by NADH and the membrane-mediated reduction of ascorbyl free radical.