Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase

Submicromolar zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration. This was attributed to in. hibition of the a-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B.S., Effron, M.S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem 275,13441-13447). Lipoamide dehydrogenase, a component of the alpha-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD(+). This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD(+). The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn2+ (K-i similar to0.15 muM) in both directions. Steady-state kinetic studies revealed that Zn2+ competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn2+ with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn2+ accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 +/- 0.02 muM. Activation is a consequence of Zn2+ binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn2+ on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn2+ can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.