title: On the location of the H+-extruding steps in site 2 of the mitochondrial electron transport chain.
abstract: The location of the H+-translocating reactions within energy-conserving Site 2 of the mitochondrial electron transport chain was evaluated from two sets of data. In the first, the H+/2e- ejection ratios and Ca2+/2e- uptake ratios were compared for electron flow from succinate dehydrogenase, whose active site is on the matrix side of the inner membrane and from glycerol phosphate dehydrogenase, whose active site is on the cytosolic side. In intact rat liver mitochondria both substrates yielded H+/2e- ejection ratios close to 4.0 and Ca2+/2e- uptake ratios close to 1.0 during antimycin-sensitive reduction of ferricyanide. With rat liver mitoplasts and ferricytochrome c as electron acceptor, both substrates again gave the same stoichiometric ratios. The second approach involved determination of the sidedness of H+ formation during electron flow from succinate to ferricyanide via bypass of the antimycin block of the cytochrome b.c1 complex provided by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), under conditions in which the TMPD-TMPD+ couple does not act as a membrane-penetrating protonophore. Electron flow in this system was inhibited by 2-then-oyltrifluoroacetone, indicating that TMPD probably accepts electrons from ubiquinol. The 2 H+ formed in this system were not delivered into the matrix but appeared directly in the medium in the absence of a protonophore. To accommodate the available evidence on Site 2 substrates, it is concluded that the substrate hydrogens are first transferred to ubiquinone, 2 H+ per 2e then appear in the medium by protolytic dehydrogenation of a species of ubiquinol or ubiquinol-protein having the appropriate sidedness (designated Site 2A), and the other 2 H+ are translocated from the matrix to the medium on passage of 2e- through the cytochrome b x c1 complex (designated Site 2B).
mesh_terms: D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D004579:Electron Transport; D005292:Ferricyanides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006885:Hydroxybutyrates / Q000378:metabolism; D007700:Kinetics; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013386:Succinates / Q000378:metabolism
chemical_list: D005292:Ferricyanides; D006885:Hydroxybutyrates; D013386:Succinates; D000968:Antimycin A; D002118:Calcium