Nitrite reduction and superoxide-dependent nitric oxide degradation by Arabidopsis mitochondria: influence of external NAD(P)H dehydrogenases and alternative oxidase in the control of nitric oxide levels.

Mitochondria recently have emerged as important sites in controlling NO levels within the cell. In this study, the synthesis of nitric oxide (NO) from nitrite and its degradation by mitochondria isolated from Arabidopsis thaliana were examined. Oxygen and NO concentrations in the reaction medium were measured with specific electrodes. Nitrite inhibited the respiration of isolated A. thaliana mitochondria, in competition with oxygen, an effect that was abolished or potentiated when electron flow occurred via alternative oxidase (AOX) or cytochrome c oxidase (COX), respectively. The production of NO from nitrite was detected electrochemically only under anaerobiosis because of a superoxide-dependent process of NO degradation. Electron leakage from external NAD(P)H dehydrogenases contributed the most to NO degradation as higher rates of Amplex Red-detected H(2)O(2) production and NO consumption were observed in NAD(P)H-energized mitochondria. Conversely, the NO-insensitive AOX diminished electron leakage from the respiratory chain, allowing the increase of NO half-life without interrupting oxygen consumption. These results show that the accumulation of nitric oxide derived from nitrite reduction and the superoxide-dependent mechanism of NO degradation in isolated A. thaliana mitochondria are influenced by the external NAD(P)H dehydrogenases and AOX, revealing a role for these alternative proteins of the mitochondrial respiratory chain in the control of NO levels in plant cells.

Nitric oxide degradation by potato tuber mitochondria: evidence for the involvement of external NAD(P)H dehydrogenases.

The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2(-)). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2(-) generation, besides complex III, stimulated NO degradation. Larger amounts of O2(-) were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2(-) production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2(-) production that favors NO degradation in potato tuber mitochondria.