ascorbic-acid and duroquinol

The long-lived mutant of Caenorhabditis elegans, clk-1, is unable to synthesize ubiquinone, CoQ(9). Instead, the mutant accumulates demethoxyubiquinone(9) and small amounts of rhodoquinone(9) as well as dietary CoQ(8). We found a profound defect in oxidative phosphorylation, a test of integrated mitochondrial function, in clk-1 mitochondria fueled by NADH-linked electron donors, i.e. complex I-dependent substrates. Electron transfer from complex I to complex III, which requires quinones, is severely depressed, whereas the individual complexes are fully active. In contrast, oxidative phosphorylation initiated through complex II, which also requires quinones, is completely normal. Here we show that complexes I and II differ in their ability to use the quinone pool in clk-1. This is the first direct demonstration of a differential interaction of complex I and complex II with the endogenous quinone pool. This study uses the combined power of molecular genetics and biochemistry to highlight the role of quinones in mitochondrial function and aging.

Topics: Animals; Ascorbic Acid; Caenorhabditis elegans; Electron Transport Complex I; Electron Transport Complex II; Glutamic Acid; Hydroquinones; Malates; Mitochondria; Mutation; Oxidative Phosphorylation; Pyruvic Acid; Quinones; Substrate Specificity; Tetramethylphenylenediamine; Time Factors; Ubiquinone

The toxicity of hydrophilic (cholate) and lipophilic (deoxycholate, chenodeoxycholate, and lithocholate) bile acids on the function of the electron transport chain was investigated in intact and disrupted rat liver mitochondria. In intact mitochondria, lipophilic bile acids used at a concentration of 100 mumol/L (0.1 mumol/mg protein) inhibited state 3 and state 3u (dinitrophenol-uncoupled) oxidation rates for L-glutamate, succinate, duroquinol or ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine as substrates. In contrast, state 4 oxidation rates and ADP/oxygen ratios were not significantly affected. At a bile acid concentration of 10 mumol/L (0.01 mumol/mg protein), the state 3 oxidation rate for L-glutamate was decreased in the presence of deoxycholate, chenodeoxycholate or lithocholate, whereas only lithocholate inhibited state 3 oxidation for succinate or duroquinol. In broken mitochondria, inhibition of oxidative metabolism was found for NADH or duroquinol as substrate in the presence of 100 mumol/L lithocholate (0.2 mumol/mg protein) and for duroquinol in the presence of 100 mumol/L chenodeoxycholate. Direct assessment of the activities of the enzyme complexes of the electron transport chain revealed decreased activities of complex I and complex III in the presence of 100 mumol/L deoxycholate or chenodeoxycholate or 10 mumol/L lithocholate. Inhibition of complex IV required higher bile acid concentrations (300 mumol/L for chenodeoxycholate or 30 mumol/L for lithocholate), and complex II was not affected. Both chenodeoxycholate and lithocholate were incorporated into mitochondrial membranes. The phospholipid content of mitochondrial membranes decreased in incubations containing 100 mumol/L (0.1 mumol/mg protein) chenodeoxycholate but was not affected in the presence of 100 mumol/L lithocholate.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Ascorbic Acid; Bile Acids and Salts; Chenodeoxycholic Acid; Cholic Acid; Cholic Acids; Deoxycholic Acid; Electron Transport; Glutamates; Glutamic Acid; Hydroquinones; Lithocholic Acid; Male; Mitochondria, Liver; Oxidation-Reduction; Oxidoreductases; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Succinates; Succinic Acid; Tetramethylphenylenediamine

The triphasic course previously reported for the reduction of cytochrome b in the succinate-cytochrome c reductase by either succinate or duroquinol has been shown to be dependent on the redox state of the enzyme preparation. Prior reduction with increasing concentrations of ascorbate leads to partial reduction of cytochrome c1, and a gradual decrease in the magnitude of the oxidation phase of cytochrome b. At an ascorbate concentration sufficient to reduce cytochrome c1 almost completely, the reduction of cytochrome b by either succinate or duroquinol becomes monophasic. Owing to the presence of a trace amount of cytochrome oxidase in the reductase preparation employed, the addition of cytochrome c makes electron flow from substrate to oxygen possible. Under such circumstances, the addition of a limited amount of either succinate or duroquinol leads to a multiphasic reduction and oxidation of cytochrome b. After the initial three phases as described previously, cytochrome b becomes oxidized before cytochrome c1 when the limited amount of added substrate is being used up. However, at the end of the reaction when cytochrome c1 is being rapidly oxidized, cytochrome b becomes again reduced. The above observations support a cyclic scheme of electron flow in which the reduction of cytochrome b proceeds by two different routes and its oxidation controlled by the redox state of a component of the respiratory chain.

Topics: Animals; Antimycin A; Ascorbic Acid; Cytochrome b Group; Cytochromes c1; Hydroquinones; Kinetics; Oxidation-Reduction; Oxidoreductases; Succinate Cytochrome c Oxidoreductase