Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria

TLDR

The generation of O₂⁻ by rotenone, the uncoupler carbonyl cyanide ρ‑trifluoromethoxyphenylhydrazone, and oligomycin occurs at concentrations similar to those needed to inhibit energy‑dependent succinate‑NAD reductase. Submitochondrial particles produce ~0.9 nmol O₂⁻ min⁻¹ mg⁻¹ protein via NADH dehydrogenase, with rotenone, antimycin, and cyanide modulating production biphasically, cyanide inhibiting the ubiquinone‑cytochrome b site, and O₂⁻ generation showing pH‑dependent additivity and an NADH‑ and adrenaline‑dependent autocatalytic process.

Abstract

Submitochondrial particles from bovine heart in which NADH dehydrogenase is reduced by either addition of NADH and rotenone or by reversed electron transfer generate 0.9 +/- 0.1 nmol of O2-/min per mg of protein at pH 7.4 and at 30 degrees C. When NADH is used as substrate, rotenone, antimycin and cyanide increase O2- production. In NADH- and antimycin-supplemented submitochondrial particles, rotenone has a biphasic effect: it increases O2- production at the NADH dehydrogenase and it inhibits O2- production at the ubiquinone-cytochrome b site. The generation of O2- by the rotenone, the uncoupler carbonyl cyanide rho-trifluoromethoxyphenylhydrazone and oligomycin at concentrations similar to those required to inhibit energy-dependent succinate-NAD reductase. Cyanide did not affect O2- generation at the NADH dehydrogenase, but inhibited O2- production at the ubiquinone-cytochrome b site. Production of O2- at the NADH dehydrogenase is about 50% of the O2- generation but the ubiquinone-cytochrome b area at pH 7.4. Additivity of the two mitochondrial sites of O2- generation was observed over the pH range from 7.0 to 8.8. AN O2–dependent autocatalytic process that requires NADH, submitochondrial particles and adrenaline is described.