Nitric oxide interacts with mitochondrial complex III producing antimycin-like effects

The effect of NO between cytochromes b and c of the mitochondrial respiratory chain were studied using submitochondrial particles (SMP) from bovine heart and GSNO and SPER-NO as NO sources. Succinate-cytochrome c reductase (complex activity (222 +/- 4 nmol/min. mg protein) was inhibited by 51% in the presence of 500 mu M GSNO and by 48% in the presence of 30 mu M SPER-NO, in both cases at 1.25 mu M NO. Neither GSNO nor SPER-NO were able to inhibit succinate-Q reductase activity (complex II; 220 +/- 9 nmol/min. mg protein), showing that NO affects complex III. Complex II-III activity was decreased (36%) when SMP were incubated with L-arginine and mtNOS cofactors, indicating that this effect is also produced by endogenous NO. GSNO (500 mu M) reduced cytochrome b(562) by 71%, in an [O-2] independent manner. Hyperbolic increases in 0(2)(center dot-) (up to 1.3 +/- 0.1 nmol/min. mg protein) and H2O2 (up to 0.64 +/- 0.05 nmol/min. mg protein) productions were observed with a maximal effect at 500 mu M GSNO. The 0(2)(center dot-)/H2O2 ratio was 1.98 in accordance with the stoichiometry of the 0(2)(center dot-) disproportionation. Moreover, H2O2 production was increased by 72-74% when heart coupled mitochondria were exposed to 500 mu M GSNO or 30 mu M SPER-NO. SMP incubated in the presence of succinate showed an EPR signal (g=1.99) compatible with a stable semiquinone. This EPR signal was increased not only by antimycin but also by GSNO and SPER-NO. These signals were not modified under N-2 atmosphere, indicating that they are not a consequence to the effect of NOx species on complex III area. These results show that NO interacts with ubiquinone-cytochrome b area producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQF(center dot)](ss) which, in turn, leads to an increase in 0(2)(center dot-) and H2O2 mitochondrial production rates. (C) 2015 Elsevier Inc. All rights reserved.