Biochemical and functional interaction between mitochondrial complex I and mtNOS.

BOMBICINO SS ; ZAOBORNYJ T; IGLESIAS DE; GELPI RJ; 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

Society for Free Radical Biology and Medicine, South American Group

The aim of this work was to study the functional interaction between complex I and mtNOS in (1) physiological situations, using beef heart inside-out particles (ETPH-Mg2+); and in (2) pathological conditions, using a rabbit myocardial stunning model. (1) ETPH(Mg2+) showed NAD+ reductase activity (17.3 ± 0.7 nmol/min.mg protein) in the presence of succinate and ATP to establish a reversal electron flow. This activity was inhibited by rotenone (96%), m-CCCP (94%) and oligomycin (98%). A 0.5 mM Mg2+ concentration permits an acceptable reversal electron flow (11.8 ± 0.7 nmol NAD+/min. mg protein) without a significant impairment in the NO production detection. Bovine ETPH-Mg2+ produced NO at a rate of 0.83 ± 0.02 nmol/min.mg protein, in optimal experimental conditions (0.5 mM MgCl2; 0.3 mM KCN). This activity was still detectable (77%) in the absence of an exogenous electron donor (NADPH). Rotenone inhibited mtNOS activity supported by reversal electron flow (90%), but it did not inhibit the activity of isolated nNOS. These data suggest that mtNOS could physically and functionally interact with complex I, using electrons derived from the respiratory chain for its enzymatic activity. (2) In the myocardial stunning model, 15 min-ischemia/30 min-reperfusion selectively decreased malate-glutamate supported mitochondrial state 3 respiration (32%), and complex I and mtNOS activities (28%). The pattern observed for the decline of complex I activity was also observed for the reduction of mtNOS biochemical and functional activities, in accordance to the reported physical and functional interaction between complex I and mtNOS. This impairment was accompanied by an enhancement in malate-glutamate supported state 4 H2O2 production (78%), mitochondrial phospholipid oxidation products (42%) and proteins tyrosine nitration (50%), leading to a mitochondrial dysfunction named “complex I syndrome”. Thus, in physiological situations as well as in a pathological condition, a functional interaction between mtNOS and complex I was observed. These results are in agreement with the data reported by Franco et al. (2006) who showed that not only complex IV but also complex I proteins immunoprecipitate with mtNOS, and by Valdez and Boveris (2007) and by Parihar et al. (2008) who have proposed that mtNOS is structurally adjacent to complex I.