Mitochondrial nitric oxide synthase and the inhibition of oxygen uptake

BOVERIS A; VALDEZ LB; ZAOBORNYJ T

Buenos Aires

Congreso; VIII Meeting of the South American Group of the Society for Free Radical Biology and Medicine; 2013

South American Group of the Society for Free Radical Biology and Medicine

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-alpha 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 (electrochemical potential = 170-180 mV) is associated with higher mtNOS activities (2-3 nmol NO/min.mg protein) and the lower potential of state 3 (electrochemical potential = 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.