دانلود رایگان مقاله تولید اکسید نیتریک میتوکندری توسط انتقال الکترون معکوس

Abstract

Heart phosphorylating electron transfer particles (ETPH) produced NO at 1.2 ± 0.1 nmol NO. min−1 mg protein−1 by the mtNOS catalyzed reaction. These particles showed a NAD+ reductase activity of 64 ± 3 nmol min−1 mg protein−1 sustained by reverse electron transfer (RET) at expenses of ATP and succinate. The same particles, without NADPH and in conditions of RET produced 0.97 ± 0.07 nmol NO. min−1 mg protein−1. Rotenone inhibited NO production supported by RET measured in ETPH and in coupled mitochondria, but did not reduce the activity of recombinant nNOS, indicating that the inhibitory effect of rotenone on NO production is due to an electron flow inhibition and not to a direct action on mtNOS structure. NO production sustained by RET corresponds to 20% of the total amount of NO released from heart coupled mitochondria. A mitochondrial fraction enriched in complex I produced 1.7 ± 0.2 nmol NO. min−1 mg protein−1 and reacted with anti-75 kDa complex I subunit and anti-nNOS antibodies, suggesting that complex I and mtNOS are located contiguously. These data show that mitochondrial NO production can be supported by RET, and suggest that mtNOS is next to complex I, reaffirming the idea of a functional association between these proteins.

4. Discussion

The results presented in this paper provide evidence that bovine heart submitochondrial particles (ETPH) produce NO both by using NADPH, the conventional electron donor of NOS [5,7,10], and also supported by mitochondrial reverse electron transfer (RET) derived from succinate. The NO production detected in the complex I enriched mitochondrial fraction, together with the reactivity of that fraction both with anti-complex I and anti-nNOS antibodies, suggest that complex I and mtNOS enzyme are located contiguously. In this study, the use of inside-out particles which expose the NADH binding site of complex I, the F1-ATPase and mtNOS to the surrounding medium, has been a useful experimental strategy because potential interferences of mitochondrial matrix, intermembrane space, or outer membrane are excluded. The reaction catalyzed by complex I is physiologically reversible because coupled mitochondria or submitochondrial particles are capable of DmH þ-dependent reduction of NADþ in the presence of the substrates that provide the reduction of ubiquinone, i.e. succinate. In our experimental conditions, RET was generated by succinate oxidation, in the presence of KCN to inhibit cytochrome oxidase, and energized by the proton-motive force coupled to ATP hydrolysis. Under these conditions the electrons are transferred from succinate to the FMN of complex I and are able to reduce NADþ or to participate in the production of NO by mtNOS (Scheme 1).