Zinc Is a Potent Inhibitor of Thiol Oxidoreductase Activity and Stimulates Reactive Oxygen Species Production by Lipoamide Dehydrogenase

• Published in: The Journal of biological chemistry Vol. 277; no. 12; pp. 10064 - 10072
• Main Authors: Gazaryan, Irina G., Krasnikov, Boris F., Ashby, Gillian A., Thorneley, Roger N.F., Kristal, Bruce S., Brown, Abraham M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22-03-2002; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Dihydrolipoamide Dehydrogenase - metabolism; Disulfides; Dose-Response Relationship, Drug; Kinetics; Models, Chemical; Myocardium - enzymology; NAD - metabolism; Oxygen - metabolism; Oxygen Consumption; Protein Binding; Protein Disulfide Reductase (Glutathione) - chemistry; Protein Disulfide Reductase (Glutathione) - metabolism; Reactive Oxygen Species; Substrate Specificity; Swine; Time Factors; Zinc - pharmacology; Zinc - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M108264200

Submicromolar zinc inhibits α-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the α-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 α-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+(Ki ∼0.15 μm) 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 μm. 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.