The oxidative inactivation of mitochondrial electron transport chain components and ATPase.

Y Zhang,O Marcillat,C Giulivi,L Ernster,K J Davies

doi:10.1016/s0021-9258(17)46227-2

Y Zhang, O Marcillat + Show 3 more

Open Access

https://doi.org/10.1016/s0021-9258(17)46227-2

Copy DOI

Abstract

Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (.OH) alone, superoxide anion radical (O2-) alone, or mixtures of .OH and O2-, by gamma radiolysis in the presence of 100% N2O (.OH exposure), 100% O2 + formate (O2- exposure), or 100% O2 alone (.OH + O2- exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by .OH (10% inactivation at 15-40 nmol of .OH/mg of SMP protein, 50-90% inactivation at 600 nmol of .OH/mg of SMP protein) and by .OH + O2- (10% inactivation at 20-80 nmol of .OH + O2-/mg of SMP protein, 45-75% inactivation at 600 nmol of .OH + O2-/mg of SMP protein). Importantly, O2- was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O2-/mg of SMP protein, 40% inactivation at 600 nmol of O2-/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O2-/mg of SMP protein, 30% inactivation at 600 nmol of O2-/mg of SMP protein), and a poor inactivator of succinate oxidase (less than 10% inactivation at 600 nmol of O2-/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at .OH, O2-, OH + O2-, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of .OH/mg of SMP protein. The .OH-dependent inactivations reported above were largely inhibitable by the .OH scavenger mannitol. In contrast, the O2(-)-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with .OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all.(ABSTRACT TRUNCATED AT 400 WORDS)

Highlights

Alone, superoxide anion radical (0;) alone, or mixtures of ‘OH and 02, by y radiolysis in the presence of 100%
Cytochrome oxidase activity was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at ‘OH, 02, ‘OH + 02, or HzOz concentrations of
24% decrease in activity was observed at 3000 nmol/mg of protein. We suggest that these results are extremely important because they demonstrate that the internal activity of the cytochrome oxidase complex is highly resistant to oxidative stress; the only one we have so far found

Summary

Introduction

Alone, superoxide anion radical (0;) alone, or mixtures of ‘OH and 02, by y radiolysis in the presence of 100%. H202 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least. 500-600 nmol of HzOz/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N’,N’-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at ‘OH, 02, ‘OH + 02, or HzOz concentrations of. Cytochrome c oxidase activity, (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of ‘OH/mg of SMP protein. The costs of publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact

Methods

Results

Conclusion