Biochemical characterization of mitochondrial complex III inhibition by nitric oxide

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

Londres

Congreso; 16th Biennial Meeting of the Society for Free Radical Research International.; 2012

Society for Free Radical Research International

Little is known about how nitric oxide (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 inhibitory effect of NO on electron transfer between cyt. b and cyt. c using beef heart inside-out particles (ETPH-Mg2+). 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, 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.