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Zinc Switch in Pig Heart Lipoamide Dehydrogenase: Steady-State and Transient Kinetic Studies of the Diaphorase Reaction

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Abstract

Elevation of intracellular Zn2+ following ischemia contributes to cell death by affecting mitochondrial function. Zn2+ is a differential regulator of the mitochondrial enzyme lipoamide dehydrogenase (LADH) at physiological concentrations (Ka = 0.1 µM free zinc), inhibiting lipoamide and accelerating NADH dehydrogenase activities. These differential effects have been attributed to coordination of Zn2+ by LADH active-site cysteines. A detailed kinetic mechanism has now been developed for the diaphorase (NADH-dehydrogenase) reaction catalyzed by pig heart LADH using 2,6-dichlorophenol-indophenol (DCPIP) as a model quinone electron acceptor. Anaerobic stopped-flow experiments show that two-electron reduced LADH is 15-25-fold less active towards DCPIP reduction than four-electron reduced enzyme, or Zn2+-modified reduced LADH (the corresponding values of the rate constants are (6.5 ± 1.5) × 103 M–1·s–1, (9 ± 2) × 104 M–1·s–1, and (1.6 ± 0.5) × 105 M–1·s–1, respectively). Steady-state kinetic studies with different diaphorase substrates show that Zn2+ accelerates reaction rates exclusively for two-electron acceptors (duroquinone, DCPIP), but not for one-electron acceptors (benzoquinone, ubiquinone, ferricyanide). This implies that the two-electron reduced form of LADH, prevalent at low NADH levels, is a poor two-electron donor compared to the four-electron reduced or Zn2+-modified reduced LADH forms. These data suggest that zinc binding to the active-site thiols switches the enzyme from one- to two-electron donor mode. This zinc-activated switch has the potential to alter the ratio of superoxide and H2O2 generated by the LADH oxidase activity.

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Abbreviations

DCPIP:

2,6-dichlorophenol-indophenol

LADH:

dihydrolipoyl dehydrogenase (=lipoamide dehydrogenase)

KGDHC:

α-ketoglutarate dehydrogenase complex

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Acknowledgements

The authors thank Mrs. Gill A. Ashby and Prof. Roger N. F. Thorneley (UK) for their invaluable help with transient kinetics experiments.

Funding

The work in part was supported by the Russian Foundation for Basic Research (projects Nos. 20-04-00921, 17-54-33027, and 18-29-09154).

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Authors and Affiliations

  1. Department of Cell Biology and Anatomy, New York Medical College, 15 Dana Road, Valhalla, NY, 10605, USA

    I. G. Gazaryan & A. M. Brown

  2. Department of Chemistry and Physical Sciences, Dyson College of Arts and Sciences, Pace University, 861 Bedford Road, Pleasantville, NY, 10570, USA

    I. G. Gazaryan & S. V. Kazakov

  3. Department of Chemical Enzymology, Lomonosov Moscow State University, 119899, Moscow, Russia

    I. G. Gazaryan, V. A. Shchedrina & N. L. Klyachko

  4. Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences, 119071, Moscow, Russia

    I. G. Gazaryan & A. A. Zakhariants

  5. Derzhavin Tambov State University, 392000, Tambov, Russia

    N. L. Klyachko

  6. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Russia

    A. A. Zakhariants

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  1. I. G. Gazaryan
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Correspondence to A. M. Brown.

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Gazaryan, I.G., Shchedrina, V.A., Klyachko, N.L. et al. Zinc Switch in Pig Heart Lipoamide Dehydrogenase: Steady-State and Transient Kinetic Studies of the Diaphorase Reaction. Biochemistry Moscow 85, 908–919 (2020). https://doi.org/10.1134/S0006297920080064

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