Biochemical characterization of mitochondrial complex III inhibition by nitric oxide.

IGLESIAS DE,; BOMBICINO SS,; BOVERIS A,; VALDEZ LB

San Pedro, San Pablo

Congreso; Free Radicals in Brazil 2011. VII Meeting of the Society for Free Radical Biology and Medicine. South American Group.; 2011

Free RSociety for Free Radical Biology and Medicine. South American Group.

It is worth noting that NO acts in signaling by cGMP-dependent pathways and in the regulation of mitochondrial respiration through a cGMP-independent way. At sub-micromolar concentrations, NO exhibits two main effects on the mitochondrial respiratory chain: the competitive inhibition of cytochrome oxidase and the inhibition of electron transfer at complex III. Little is known about how NO interacts with the NO-reactive component of the ubiquinone-cytochrome b area of the mitochondrial respiratory chain. The aim of this work was to study the NO inhibitory effect on electron transfer between cyt. b and cyt. c. Succinate-cytochrome c reductase activity, and H2O2 and O2- production rates were measured using beef heart inside-out particles and S-nitrosoglutathione (GSNO; 0-500 mM) as NO donor. NO inhibited succinate-cytochrome c reductase activity with a maximal effect (55%) at 500 mM GSNO. This inhibition was independent on [O2], suggesting that the mechanism involved in the inhibition of complex III is different to the one observed for complex IV. The effective [NO] was estimated using a NO-sensitive electrode: 500 mM GSNO released 250 nM NO to the reaction medium when the assay was performed during 2 min. NO produced a hyperbolic increase in O2- and H2O2 production with a maximal rate at 500 mM GSNO (1.1 ± 0.2 nmol O2-/min. mg protein; 0.55 ± 0.05 nmol H2O2/min. mg protein). The ratio O2-/H2O2 was 2.0 in accordance to the stoichiometry of the O2- dismutation reaction. The effect of 500 ìM GSNO on the absorbance spectrum (between 500 nm and 650 nm) of mitochondrial membranes suspensions was followed in the presence of 20 mM and 230 mM O2. At low [O2] the characteristics peaks of cyt. b (562 nm), cyt. c (550 nm) and cyt. aa3 (605 nm) were obtained, while at air-saturated condition only cyt. b signal was detected. This spectrum showed a similar pattern to the one observed for the antimycin inhibition. To conclude, the interaction of NO with complex III leads to the inhibition of mitochondrial electron transfer, at an independent [O2] manner, with an enhancement in O2- and H2O2 production rates. This work was supported by the following research grants: B005, B813 and B393 from UBA, PICT 1138 from ANPCyT; and PIP 688 from CONICET.