Proton-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans plasma membranes catalyzes FMN-independent reverse electron transfer to hexaammineruthenium (III).

NADH-OH, the specific inhibitor of NADH-binding site of the mammalian complex I, is shown to completely block FMN-dependent reactions of P. denitrificans enzyme in plasma membrane vesicles: NADH oxidation (in a competitive manner with Ki of 1 nM) as well as reduction of pyridine nucleotides, ferricyanide and oxygen in the reverse electron transfer. In contrast to these activities, the reverse electron transfer to hexaammineruthenium (III) catalyzed by plasma membrane vesicles is insensitive to NADH-OH. To explain these results, we hypothesize the existence of a non-FMN redox group of P. denitrificans complex I that is capable of reducing hexaammineruthenium (III), which is corroborated by the complex kinetics of NADH: hexaammineruthenium (III)-reductase activity, catalyzed by this enzyme. A new assay procedure for measuring succinate-driven reverse electron transfer catalyzed by P. denitrificans complex I to hexaammineruthenium (III) is proposed.

Oxygen-dependence of mitochondrial ROS production as detected by Amplex Red assay.

The initial rates of superoxide plus hydrogen peroxide (ROS) generation by intact or permeabilized rat heart mitochondria and coupled inside-out bovine heart submitochondrial particles (SMP) oxidizing NAD-dependent substrates, NADH, and succinate were measured by detecting resorufin formation in the Amplex Red assay at various oxygen concentrations. Linear dependences of the initial rates on oxygen concentration within the range of ~125-750 µM were found for all significant mitochondrial generators, i.e. the respiratory complexes and ammonium-stimulated dihydrolipoamide dehydrogenase. At lower oxygen concentrations upon its decrease from air saturation level to zero, the time-course of resorufin formation by SMP catalyzing coupled oxidation of succinate (the total ROS production by respiratory complexes II and III and by the reverse electron transfer (RET)-mediated by complex I) also corresponds to the linear dependence on oxygen with the same first-order rate constant determined in the initial rate studies. Prolonged incubation of SMP generating succinate-supported complex I-mediated ROS affected neither their NADH oxidase nor ROS generating activity. In contrast to SMP significant deviation from the first-order oxygen dependence in the time-course kinetics during coupled oxidation of succinate by intact mitochondria was evident. Complex I catalyzes the NADH:resorufin oxidoreductase reaction resulting in formation of colorless reduced resorufin. Hydrogen peroxide oxidizes reduced resorufin in the presence of peroxidase, thus showing its dihydroresorufin peroxidase activity. Combined NADH:resorufin reductase and dihydroresorufin peroxidase activities result in underestimation of the amount of hydrogen peroxide generated by mitochondria. We conclude that only initial rates of the mitochondrial ROS production, not the amount of resorufin accumulated, should be taken as the reliable measure of the mitochondrial ROS-generating activity, because of the cycling of the oxidized and reduced resorufin during Amplex Red assays fed by NADH and other possible reductant(s) present in mitochondria.

Mitochondrial hydrogen peroxide production as determined by the pyridine nucleotide pool and its redox state.

The rates of NADH-supported superoxide/hydrogen peroxide production by membrane-bound bovine heart respiratory complex I, soluble pig heart dihydrolipoamide dehydrogenase (DLDH), and by accompanying operation of these enzymes in rat heart mitochondrial matrix were measured as a function of the pool of pyridine nucleotides and its redox state. Each of the activities showed nontrivial dependence on nucleotide pool concentration. The NAD(+)/NADH ratios required for their half maximal capacities were determined. About half of the total NADH-supported H(2)O(2) production by permeabilized mitochondria in the absence of stimulating ammonium could be accounted for by DLDH activity. The significance of the mitochondrial NADH-dependent hydrogen peroxide production under physiologically relevant conditions is discussed. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Molecular identification of the enzyme responsible for the mitochondrial NADH-supported ammonium-dependent hydrogen peroxide production.

A homogeneous protein with a subunit apparent molecular mass of ∼50 kDa that catalyzes the previously described mitochondrial NADH-supported ammonium-stimulated hydrogen peroxide production (Grivennikova, V.G., Gecchini, G. and Vinogradov, A.D. (2008) FEBS Lett. 583, 1287-1291) was purified from the mitochondrial matrix of bovine heart. Chromatography of partially purified protein showed that the peaks of ammonium-stimulated NADH-dependent H(2)O(2) production and that of NADH:lipoamide oxidoreductase activity coincided. The catalytic properties and mass spectrometry of the trypsin-digested protein revealed peptides that allowed identification of the protein as the Bos taurus dihydrolipoyl dehydrogenase.

What are the sources of hydrogen peroxide production by heart mitochondria?

Coupled rat heart mitochondria produce externally hydrogen peroxide at the rates which correspond to about 0.8 and 0.3% of the total oxygen consumption at State 4 with succinate and glutamate plus malate as the respiratory substrates, respectively. Stimulation of the respiratory activities by ADP (State 4-State 3 transition) decreases the succinate- and glutamate plus malate-supported H2O2 production 8- and 1.3-times, respectively. NH4+ strongly stimulates hydrogen peroxide formation with either substrate without any effect on State 4 and/or State 3 respiration. Rotenone-treated, alamethicin-permeabilized mitochondria catalyze NADH-supported H2O2 production at a rate about 10-fold higher than that seen in intact mitochondria under optimal (State 4 succinate-supported respiration in the presence of ammonium chloride) conditions. NADH-supported hydrogen peroxide production by the rotenone-treated mitochondria devoid of a permeability barrier for H2O2 diffusion by alamethicin treatment are only partially (approximately 50%) sensitive to the Complex I NADH binding site-specific inhibitor, NADH-OH. The residual activity is strongly (approximately 6-fold) stimulated by ammonium chloride. NAD+ inhibits both Complex I-mediated and ammonium-stimulated H2O2 production. In the absence of stimulatory ammonium about half of the total NADH-supported hydrogen peroxide production is catalyzed by Complex I. In the presence of ammonium about 90% of the total hydrogen peroxide production is catalyzed by matrix located, ammonium-dependent enzyme(s).