The stoichiometry of the redox H+ pumps at site II and site II+III of the respiratory chain of rat-liver mitochondria has been determined with three different e− donors: succinate, duroquinol and NADH with duroquinone by-pass of rotenone inhibition. Succinate provides reducing equivalents to the cytochrome b-c1 complex from the matrix side. Duroquinol, either added from the outside or obtained from the reduction of exogenous duroquinone by the NADH dehydrogenase, presumably is an intramembrane e− donor to cytochrome b or to the endogenous ubiquinone. The stoichiometries were determined at steady-state rates of ion translocation and e− transfer. The ion translocation was followed on the basis of the rates of H+ extrusion and of K+ and Ca2+ uptake. The e− transfer was followed either with the polarographic or with the spectrophotometric technique, i. e. absorbance changes during oxidation of duroquinol to duroquinone. In this latter case N-ethylmaleimide was used to inhibit the NADH dehydrogenase activity responsible for the re-reduction of the duroquinone formed during duroquinol oxidation. During operation of sites II+III, with oxygen as e− acceptor, all three substrates tested gave H+/2e− and charge/2e− ratios close to 8. 0. During operation of site II, with ferricyanide as e− acceptor, the H+/2e− ratios were about 4. 0 and the charge/2e− ratios close to 2. 0. The present results support previous reports from our laboratory on the stoichiometries of the redox H+ pumps at sites II and III. Furthermore the equality of the H+ and charge stoichiometry, with substrates having different sides of interaction with complex III suggests that e− donors located on the matrix side or intramembrane (and possibly on the cytoplasmic side) share the same pathway in order to feed electrons into the H+ pump at site II.
Sidedness of e− Donation and Stoichiometry of H+ Pumps at Sites II + III in Mitochondria from Rat Liver
DI VIRGILIO, Francesco;
1981
Abstract
The stoichiometry of the redox H+ pumps at site II and site II+III of the respiratory chain of rat-liver mitochondria has been determined with three different e− donors: succinate, duroquinol and NADH with duroquinone by-pass of rotenone inhibition. Succinate provides reducing equivalents to the cytochrome b-c1 complex from the matrix side. Duroquinol, either added from the outside or obtained from the reduction of exogenous duroquinone by the NADH dehydrogenase, presumably is an intramembrane e− donor to cytochrome b or to the endogenous ubiquinone. The stoichiometries were determined at steady-state rates of ion translocation and e− transfer. The ion translocation was followed on the basis of the rates of H+ extrusion and of K+ and Ca2+ uptake. The e− transfer was followed either with the polarographic or with the spectrophotometric technique, i. e. absorbance changes during oxidation of duroquinol to duroquinone. In this latter case N-ethylmaleimide was used to inhibit the NADH dehydrogenase activity responsible for the re-reduction of the duroquinone formed during duroquinol oxidation. During operation of sites II+III, with oxygen as e− acceptor, all three substrates tested gave H+/2e− and charge/2e− ratios close to 8. 0. During operation of site II, with ferricyanide as e− acceptor, the H+/2e− ratios were about 4. 0 and the charge/2e− ratios close to 2. 0. The present results support previous reports from our laboratory on the stoichiometries of the redox H+ pumps at sites II and III. Furthermore the equality of the H+ and charge stoichiometry, with substrates having different sides of interaction with complex III suggests that e− donors located on the matrix side or intramembrane (and possibly on the cytoplasmic side) share the same pathway in order to feed electrons into the H+ pump at site II.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
