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

---

I. G. Gazaryan^1,2,3,4, V. A. Shchedrina³, N. L. Klyachko^3,5, A. A. Zakhariants^4,6, S. V. Kazakov², and A. M. Brown^1,a*

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

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

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

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

⁵Derzhavin Tambov State University, 392000 Tambov, Russia

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

^* To whom correspondence should be addressed.

Received May 27, 2020; Revised June 11, 2020; Accepted June 14, 2020

---

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 (K_a = 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.

---

KEY WORDS: ubiquinone, 2,6-dichlorophenol indophenol, duroquinone, ferricyanide, enzyme kinetics, dehydrolipoyl dehydrogenase

DOI: 10.1134/S0006297920080064

---