Mitochondrial nitric oxide synthase and the inhibition of oxygen uptake

BOVERIS A; VALDEZ LB; ZAOBORNYJ T

Buenos Aires

Congreso; VIII Meeting of the Society for Free Radical Biology and Medicine. South American Group (VIII SFRBM-SAG); 2013

The activity of the mtNOS of mitochondrial membranes of mammalian tissues (heart, brain, kidney, thymus and diaphragm) is 0.7-1.5 nmol NO/min.mg protein, in agreement with the electrochemical determination of NO release after a Ca2+ pulse in a single mouse heart mitochondrion. Rat liver mtNOS was sequenced and found the transcript of nNOS- myristylated and phosphorylated. The mtNOS is an integral protein of the inner membrane with the reaction center facing the matrix and structural associations with complex I and complex IV. The biochemical activity is determined spectrophotometrically by formation of metHb and by the 14C-citrulline assay (50-times lower activities). The ?mtNOS functional activity assay? determines state 3 respiration in two parallel samples. The first sample (minimal NO level), is added with a NOS inhibitor (L-NMMA) and HbO2 and shows an increase in respiration. The second sample (maximal NO level), is added with L-arginine and SOD and shows a decrease in respiration. The difference between both samples is 25-35 % of state 3 respiration. The intramitochondrial NO steady-state concentration has been calculated as 200-360 nM NO. Interestingly, mtNOS is a voltage-dependent enzyme that is regulated by the inner membrane potential. In rat heart and liver mitochondria the higher state 4 electrical potential ( = 170-180 mV) is associated with higher mtNOS activities (2-3 nmol NO/min.mg protein) and the lower potential of state 3 ( = 155-160 mV) with an about 50% lower mtNOS activity. A model considering collisional second order reactions (Antunes et al., 2004) explains the properties of NO-inhibited of respiration: dependence of NO levels, competitively with O2, and absence of effect in state 4.