Inhibition of mitochondrial complex III by nitric oxide.

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

Freiburg

Congreso; 17th European Bioenergetics Conference 2012; 2012

European Bioenergetics Conference (EBEC)

Nitric oxide (NO) plays central roles through its binding to the heme group of guanylate ciclase to produce cGMP, and it is able to regulate mitochondrial respiration through cGMP-independent ways. NO exhibits two main effects on the respiratory chain: the inhibition of cytochrome oxidase and of electron transfer at complex III. It is known that the inhibition of complex IV occurs by a competition between NO and O2 for the two-electron-reduced CuB/heme a3 center of the enzyme. However, little is known about how NO interacts with the NO-reactive component of the ubiquinone-cytochrome b area. The aim of this work was to study the inhibitory effect of NO on electron transfer between cyt. b and cyt. c, using beef heart inside-out particles (ETPH-Mg2+), with emphasis on the biochemical characteristics of this inhibition: the redox state of cytochromes, the production of reactive species and the steady state concentration of ubisemiquinone (UQH•). GSNO (0-500 µM) was used as NO donor. Succinate-cytochrome c reductase activity (222+-4 nmol/min.mg protein) was inhibited (51%) by 500 uM GSNO; this activity was also reduced (36%) when ETPH-Mg2+ had been incubated in the presence of L-arginine and mtNOS cofactors (Ca2+ and NADPH), suggesting that this inhibitory effect is caused by mtNOS-produced NO. GSNO (500 M) reduced cyt. b562 within mitochondrial membranes by 70%, similarly to the antimycin effect. The effective [NO] was assessed using a NO-sensitive electrode: 500 uM GSNO releases 240 nM NO to the reaction medium when the assay is achieved during 2 min. NO produced a hyperbolic increase in O2•- and H2O2 production rates with a maximal effect at 500 M GSNO (1.30.1 nmol O2•-/min.mg protein; 0.640.05 nmol H2O2/min.mg protein). The O2•-/H2O2 ratio was 2.0 in accordance to the stoichiometry of the O2•- dismutation reaction. EPR spectrum confirmed that the reactions of duroquinol with NO produces a stable semiquinone, with a signal centred at g~2.03. ETPH-Mg2+ incubated in the presence of succinate for 1 min showed an EPR signal at g~1.99, which was increased by GSNO or antimycin. When ETPH-Mg2+ were incubated in the presence of GSNO for 5 min EPR spectra showed an additional signal at g~2.02, suggesting that two ubiquinol pools are partially oxidized by NO. The interaction of NO with complex III leads to electron transfer inhibition in an [O2] independent manner, with a (UQH•)ss enhancement, which in turn generates, an increase in O2•- and H2O2 production rates.