flavin-adenine-dinucleotide and ubiquinol

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is the only respiratory enzyme that operates as a Na(+) pump. This redox-driven Na(+) pump is amenable to experimental approaches not available for H(+) pumps, providing an excellent system for mechanistic studies of ion translocation. An understanding of the internal electron transfer steps and their Na(+) dependence is an essential prerequisite for such studies. To this end, we analyzed the reduction kinetics of the wild type Na(+)-NQR, as well as site-directed mutants of the enzyme, which lack specific cofactors. NADH and ubiquinol were used as reductants in separate experiments, and a full spectrum UV-visible stopped flow kinetic method was employed. The results make it possible to define the complete sequence of redox carriers in the electrons transfer pathway through the enzyme. Electrons flow from NADH to quinone through the FAD in subunit F, the 2Fe-2S center, the FMN in subunit C, the FMN in subunit B, and finally riboflavin. The reduction of the FMN(C) to its anionic flavosemiquinone state is the first Na(+)-dependent process, suggesting that reduction of this site is linked to Na(+) uptake. During the reduction reaction, two FMNs are transformed to their anionic flavosemiquinone in a single kinetic step. Subsequently, FMN(C) is converted to the flavohydroquinone, accounting for the single anionic flavosemiquinone radical in the fully reduced enzyme. A model of the electron transfer steps in the catalytic cycle of Na(+)-NQR is presented to account for the kinetic and spectroscopic data.

Topics: Bacterial Proteins; Cation Transport Proteins; Electron Transport; Electron Transport Complex I; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Kinetics; Mutagenesis, Site-Directed; Mutation, Missense; NAD; Oxidation-Reduction; Sodium; Spectrophotometry, Ultraviolet; Ubiquinone; Vibrio cholerae

Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synthesized lipid-soluble antioxidant) to ubiquinol, the form in which it functions as an antioxidant. The reduction of ubiquinone was linear with time and exhibited turnover numbers of 5 and 1.2 min(-1) in the presence and absence of zinc, respectively. The reaction was stimulated by zinc and cadmium but not by the other divalent ions tested. The zinc/cadmium-dependent stimulation of the reaction increased rapidly and linearly up to a concentration of 0.1 mM and was even further increased at 0.5 mM. At pH 6, the activity was three times higher than at physiological pH. Alteration of the NADPH : NADP(+) ratio revealed that the reaction is inhibited by higher concentrations of the oxidized cofactors. FAD reduced ubiquinone in a dose-dependent manner at a considerably lower rate, suggesting that the reduction of ubiquinone by lipoamide dehydrogenase involves the FAD moiety of the enzyme.

Topics: Animals; Antioxidants; Cadmium; Cations, Divalent; Chromatography, High Pressure Liquid; Coenzymes; Dihydrolipoamide Dehydrogenase; Flavin-Adenine Dinucleotide; Heart; Hydrogen-Ion Concentration; Kinetics; Lipid Peroxidation; NAD; NADP; Oxidation-Reduction; Swine; Ubiquinone; Zinc

Several studies concerning the distribution of ubiquinone (UQ) in the cell report a preferential accumulation of this biogenic quinone in mitochondria, plasma membranes, Golgi vesicles, and lysosomes. Except for mitochondria, no recent comprehensive experimental evidence exists on the particular function of UQ in these subcellular organelles. The aim of a recent study was to elucidate whether UQ is an active part of an electron-transfer system in lysosomes. In the present work, a lysosomal fraction was prepared from a light mitochondrial fraction of rat liver by isopycnic centrifugation. The purity of our preparation was verified by estimation of the respective marker enzymes. Analysis of lysosomes for putative redox carriers and redox processes in lysosomes was carried out by optical spectroscopy, HPLC, oxymetry, and ESR techniques. UQ was detected in an amount of 2.2 nmol/mg of protein in lysosomes. Furthermore, a b-type cytochrome and a flavin-adenine dinucleotide (FAD) were identified as other potential electron carriers. Since NADH was reported to serve as a substrate of UQ redox chains in plasma membranes, we also tested this reductant in lysosomes. Our experiments demonstrate a NADH-dependent reduction of UQ by two subsequent one-electron-transfer steps giving rise to the presence of ubisemiquinone and an increase of the ubiquinol pool in lysosomes. Lysosomal NADH oxidation was accompanied by an approximately equimolar oxygen consumption, suggesting that O(2) acts as a terminal acceptor of this redox chain. DMPO/(*)OH spin adducts were detected by ESR in NADH-supplemented lysosomes, suggesting a univalent reduction of oxygen. The kinetic analysis of redox changes in lysosomes revealed that electron carriers operate in the sequence NADH > FAD > cytochrome b > ubiquinone > oxygen. By using the basic spin label TEMPAMINE, we showed that the NADH-related redox chain in lysosomes supports proton accumulation in lysosomes. In contrast to the hypothesis that UQ in lysosomes is simply a waste product of autophagy in the cell, we demonstrated that this lipophilic electron carrier is a native constituent of a lysosomal electron transport chain, which promotes proton translocation across the lysosomal membrane.

Topics: Animals; Coenzymes; Cyclic N-Oxides; Cytochrome b Group; Electron Spin Resonance Spectroscopy; Electron Transport; Flavin-Adenine Dinucleotide; Intracellular Membranes; Kinetics; Lysosomes; Male; Mitochondria, Liver; Models, Biological; NAD; Oxidation-Reduction; Oxygen; Protons; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reducing Agents; Ubiquinone