Refine
Year of publication
- 2006 (2) (remove)
Document Type
- Article (2)
Language
- English (2) (remove)
Has Fulltext
- yes (2)
Is part of the Bibliography
- no (2)
Institute
- Medizin (2) (remove)
Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H+/2e−. This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.
Activation by diazoxide and inhibition by 5-hydroxydecanoate are the hallmarks of mitochondrial ATP-sensitive K+ (K(ATP)) channels. Opening of these channels is thought to trigger cytoprotection (preconditioning) through the generation of reactive oxygen species. However, we found that diazoxide-induced oxidation of the widely used reactive oxygen species indicator 2',7'-dichlorodihydrofluorescein in isolated liver and heart mitochondria was observed in the absence of ATP or K+ and therefore independent of K(ATP) channels. The response was blocked by stigmatellin, implying a role for the cytochrome bc1 complex (complex III). Diazoxide, though, did not increase hydrogen peroxide (H2O2) production (quantitatively measured with Amplex Red) in intact mitochondria, submitochondrial particles, or purified cytochrome bc1 complex. We confirmed that diazoxide inhibited succinate oxidation, but it also weakly stimulated state 4 respiration even in K+-free buffer, excluding a role for K(ATP) channels. Furthermore, we have shown previously that 5-hydroxydecanoate is partially metabolized, and we hypothesized that fatty acid metabolism may explain the ability of this putative mitochondrial K(ATP) channel blocker to inhibit diazoxide-induced flavoprotein fluorescence, commonly used as an assay of K(ATP) channel activity. Indeed, consistent with our hypothesis, we found that decanoate inhibited diazoxide-induced flavoprotein oxidation. Taken together, our data question the "mitochondrial K(ATP) channel" hypothesis of preconditioning. Diazoxide did not evoke superoxide (which dismutates to H2O2) from the respiratory chain by a direct mechanism, and the stimulatory effects of this compound on mitochondrial respiration and 2',7'-dichlorodihydrofluorescein oxidation were not due to the opening of K(ATP) channels.