Vanadate-mediated oxidation of NADH: description of an in vitro system requiring ascorbate and phosphate

Abstract

Oxidation of NADH has been observed in an in vitro system requiring NADH, vanadate, ascorbate, and phosphate. Similar results were observed with NADPH. Ascorbate provides the reducing equivalents necessary to reduce vanadate to vanadyl. Vanadyl autoxidizes producing superoxide which initiates a free radical chain reaction resulting in oxidation of NADH. Oxidation is inhibited by superoxide dismutase but not by catalase or ethanol. Ascorbate functions to initiate the free radical chain reaction but is not required in stoichiometric concentrations. At higher concentrations, ascorbate inhibits NADH oxidation. Inorganic phosphate was required for NADH oxidation. Dialysis of phosphate buffers against solutions containing apoferritin or conalbumin or addition of transition metal cations or chelators to the reaction medium did not alter dependence on phosphate. Phosphate and vanadate were interchangeable in their effects on kinetic parameters of NADH oxidation except that vanadate was 100 times more potent than phosphate. Vanadate participates directly in the initiating and propagating redox reactions of NADH oxidation. Phosphate may be important in lowering the energy of activation for the necessary transfer of hydronium ion and water in the transition state between vanadate anion and vanadyl cation.