Metabolism of S-nitrosoglutathione in intact mitochondria.

S-nitrosation of protein thiol groups by nitric oxide (NO*) is a widely recognized protein modification. Only few intracellular S-nitrosated proteins have been identified and it has been reported that S-nitrosation/denitrosation can serve as a regulatory process in signal-transduction pathways. Given the potential physiological importance of S-nitrosothiols, and considering that mitochondria are endowed with high levels of thiols and the biochemical requisites for synthesizing NO*, we examined the occurrence of S-nitrosoglutathione (GSNO) in intact, coupled rat liver mitochondria. These organelles contained 0.34 nmol of GSNO/mg of protein, detected by HPLC with UV-visible and electrochemical detections. This concentration was dynamically modulated by the availability of NO*; its decay was affected mainly by GSH and superoxide dismutase in a reaction that entailed the generation of GSSG. On the basis of the relatively long half-life of GSNO and the negligible recovery of NO* during its decay, roles for GSNO as a storage and transport molecule for NO* are discussed. Moreover, the formation of GSNO and its reaction with GSH can be considered to be partly responsible for the catabolism of NO* via a complex mechanism that might result in the formation of hydroxylamine, nitrite or nitrous oxide depending upon the availability of oxygen, superoxide dismutase and glutathione. Finally, the high concentrations of GSH in the cytosol and mitochondria might favour the formation of GSNO by reacting with NO* 'in excess', thereby avoiding damaging side reactions (such as peroxynitrite formation), and facilitate the inactivation of NO* by generating other nitrogen-related species without the chemical properties characteristic of NO*.

The modulation of oxygen radical production by nitric oxide in mitochondria.

Biological systems that produce or are exposed to nitric oxide (NO radical) exhibit changes in the rate of oxygen free radical production. Considering that mitochondria are the main intracellular source of oxygen radicals, and based on the recently documented production of NO(radical) by intact mitochondria, we investigated whether NO(radical), produced by the mitochondrial nitric-oxide synthase, could affect the generation of oxygen radicals. Toward this end, changes in H(2)O(2) production by rat liver mitochondria were monitored at different rates of endogenous NO(radical) production. The observed changes in H(2)O(2) production indicated that NO(radical) affected the rate of oxygen radical production by modulating the rate of O(2) consumption at the cytochrome oxidase level. This mechanism was supported by these three experimental proofs: 1) the reciprocal correlation between H(2)O(2) production and respiratory rates under different conditions of NO(radical) production; 2) the pattern of oxidized/reduced carriers in the presence of NO(radical), which pointed to cytochrome oxidase as the crossover point; and 3) the reversibility of these effects, evidenced in the presence of oxymyoglobin, which excluded a significant role for other NO(radical)-derived species such as peroxynitrite. Other sources of H(2)O(2) investigated, such as the aerobic formation of nitrosoglutathione and the GSH-mediated decay of nitrosoglutathione, were found quantitatively negligible compared with the total rate of H(2)O(2) production.