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

• Published in: Biochemistry (Moscow) 2020-08, Vol.85 (8), p.908-919
• Main Authors: Gazaryan, I. G., Shchedrina, V. A., Klyachko, N. L., Zakhariants, A. A., Kazakov, S. V., Brown, A. M.
• Format: Article
• Language: English
• Subjects: 2,6-Dichloroindophenol - metabolism; 2,6-Dichlorophenol; Analysis; Animals; Benzoquinone; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Catalytic Domain; Cell death; Dehydrogenase; Dehydrogenases; Dihydrolipoamide Dehydrogenase - metabolism; Electrons; Enzymes; Escherichia coli - enzymology; Ferricyanide; Hydrogen peroxide; Hydrogen Peroxide - metabolism; Ischemia; Kinetics; Life Sciences; Microbiology; Mitochondria; Myocardium - metabolism; NADH; NADH dehydrogenase; NADH Dehydrogenase - metabolism; Nicotinamide adenine dinucleotide; Oxidases; Oxidation-Reduction; Physiological aspects; Quinone; Quinones; Rate constants; Steady state; Substrates; Superoxide; Superoxides - metabolism; Swine; Switches; Thiols; Thioredoxin-Disulfide Reductase - metabolism; Ubiquinone; Zinc; Zinc - metabolism; Zinc compounds
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297920080064

Elevation of intracellular Zn 2+ following ischemia contributes to cell death by affecting mitochondrial function. Zn 2+ is a differential regulator of the mitochondrial enzyme lipoamide dehydrogenase (LADH) at physiological concentrations ( K a = 0.1 µM free zinc), inhibiting lipoamide and accelerating NADH dehydrogenase activities. These differential effects have been attributed to coordination of Zn 2+ 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 Zn 2+ -modified reduced LADH (the corresponding values of the rate constants are (6.5 ± 1.5) × 10 3 M –1 ·s –1 , (9 ± 2) × 10 4 M –1 ·s –1 , and (1.6 ± 0.5) × 10 5 M –1 ·s –1 , respectively). Steady-state kinetic studies with different diaphorase substrates show that Zn 2+ 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 Zn 2+ -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 H 2 O 2 generated by the LADH oxidase activity.