Functional interaction between complex I and mitochondrial NOS (mtNOS).

BOMBICINO SS; IGLESIAS DE; ZAOBORNYJ T; BOVERIS A ; VALDEZ LB

Freiburg

Congreso; 17th European Bioenergetics Conference 2012; 2012

European Bioenergetics Conference (EBEC)

Mitochondrial complex I catalyses electron transfer from NADH to ubiquinone and it is the major entry point of substrates to the respiratory chain. Complex I is sensitive to oxidants and reactive nitrogen species. Complex I produces superoxide anion (O2-) through the auto-oxidation reaction of flavin-semiquinone (FMNH•) with O2. Nitric oxide (NO) inhibits mitochondrial complex I activity by S-nitrosylation or Fe-nitrosation. Mitochondrial NO production is carried out by the mitochondrial nitric oxide synthase (mtNOS), an isoenzyme of the NOS family located in mitochondrial inner membrane. Persichini and co-workers (2005) reported that mtNOS is associated to Va subunit of cytochrome oxidase. Franco et al. (2006) showed that not only complex IV but also complex I proteins immunoprecipitate with mtNOS, suggesting a direct physical interactions between mtNOS and complexes I and IV proteins. Therefore, the aim of this work was to characterize the functional interaction between complex I and mtNOS using phosphorylating electron transfer particles (ETPH-Mg2+), i.e. inside-out vesicles, that exposed NADH dehydrogenase of complex I and mtNOS to the surrounding medium. ETPH-Mg2+ showed a high NAD+ reductase activity (14.4 ± 0.9 nmol/min.mg protein) sustained by reversal electron flow of the respiratory chain at expense of ATP when succinate was added. This activity was inhibited by the addition of rotenone (88%), oligomycin (98%), antimycin (77%) and m-CCCP (93%). Nitric oxide generation by ETPH-Mg2+ resulted 0.62 ± 0.03 nmol/min.mg protein, in optimal experimental condition (0.5 mM MgCl2, 0.3 mM KCN). The mtNOS activity was still detectable (99%) in the absence of an exogenous electron donor (NADPH) suggesting that the NO production could be supported by electrons derived from the respiratory chain. Rotenone inhibited mtNOS activity (86%) supported by reversal electron flow, but it did not inhibit the activity of isolated nNOS indicating that its inhibitory effect on NO production by ETPH-Mg2+ is due to an electron flow inhibition and not by a direct action on mtNOS structure. The dependence of mtNOS activity on metabolic state and membrane potential, and the physical interaction among mtNOS, cytochrome oxidase and complex I proteins, support the hypothesis that mtNOS could interact with complex I proteins using electrons derived from the respiratory chain for its enzymatic activity.