Mechanism of Superoxide Anion Generation in Intact Mitochondria in the Presence of Lucigenin and Cyanide

I. S. Yurkov^1*, A. G. Kruglov¹, Yu. V. Evtodienko¹, and L. S. Yaguzhinsky²

¹Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Institutskaya ul. 3, Pushchino 142290, Russia; fax: (0967) 79-0553; E-mail: yag@genebee.msu.su

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119894, Russia

^* To whom correspondence should be addressed.

Received February 22, 2003; Revision received July 7, 2003

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In the presence of cyanide and various respiratory substrates (succinate or pyruvate + malate) addition of high concentrations of lucigenin (400 µM; Luc²⁺) to rat liver mitochondria can induce a short-term flash of high amplitude lucigenin-dependent chemiluminescence (LDCL). Under conditions of cytochrome oxidase inhibition by cyanide the lucigenin-induced cyanide-resistant respiration (with succinate as substrate) was not inhibited by uncouplers (FCCP) and oligomycin. Increase in transmembrane potential (Deltaphi) value by stimulating F₀F₁-ATPase functioning (induced by addition of MgATP to the incubation medium) caused potent stimulation of the rate of cyanide-resistant respiration. At high Deltaphi values (in the presence of MgATP) cyanide resistant respiration of mitochondria in the presence of succinate or malate with pyruvate was insensitive to tenoyltrifluoroacetone (TTFA) or rotenone, respectively. However, in both cases respiration was effectively inhibited by myxothiazol or antimycin A. Mechanisms responsible for induction of LDCL and cyanide resistant mitochondrial respiration differ. In contrast to cyanide-resistant respiration, generation of LDCL signal, that was suppressed only by combined addition of Complex III inhibitors, antimycin A and myxothiazol, is a strictly potential-dependent process. It is observed only under conditions of high Deltaphi value generated by F₀F₁-ATPase functioning. The data suggest lucigenin-induced intensive generation of superoxide anion in mitochondria. Based on results of inhibitor analysis of cyanide-resistant respiration and LDCL, a two-stage mechanism of autooxidizable lucigenin cation-radical (Luc^·+) formation in the respiratory chain is proposed. The first stage involves two-electron Luc²⁺ reduction by Complexes I and II. The second stage includes one-electron oxidation of reduced lucigenin (Luc(2e)). Reactions of Luc(2e) oxidation involve coenzyme Q-binding sites of Complex III. This results in formation of autooxidizable Luc^·+ and superoxide anion generation. A new scheme for lucigenin-dependent electron pathways is proposed. It includes formation of fully reduced form of lucigenin and two-electron-transferring shunts of the respiratory chain. Lucigenin-induced activation of superoxide anion formation in mitochondria is accompanied by increase in ion permeability of the inner mitochondrial membrane.

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KEY WORDS: mitochondria, lucigenin, cyanide resistant respiration, superoxide anion, respiratory chain Complexes I, II, and III, ion permeability of the inner mitochondrial membrane

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