The effect of nitric oxide on mitochondrial electron transfer complexes was studied by comparing the activities of nitric oxide-treated and untreated, deoxygenated samples of purified beef heart succinate-cytochrome c reductase, succinate-ubiquinone reductase, and ubiquinol-cytochrome c reductase. More than 90% of succinate-cytochrome c reductase activity is lost during nitric oxide treatment. The activity of the succinate-ubiquinone reductase component of succinate-cytochrome c reductase decreases 95%, while the ubiquinol-cytochrome c reductase component is unaffected by nitric oxide. This inactivation is due primarily to the destruction of iron-sulfur clusters from succinate-ubiquinone reductase. When purified beef heart succinate-ubiquinone reductase was treated with nitric oxide, virtually all activity was irreversibly lost. The electron paramagnetic resonance (EPR) spectra of the treated complex showed typical iron-nitric oxide complex signals, confirming that inactivation is due to destruction of the iron-sulfur clusters. Similar results were obtained with purified Escherichia coli succinate-ubiquinone reductase. Pure beef heart ubiquinol-cytochrome c reductase treated with nitric oxide loses 40% of its initial activity, but regains most of it (90-100 % after 24 h of incubation at 0 degrees C in the absence of nitric oxide. This suggests that ubiquinol-cytochrome c reductase is protected from nitric oxide when complexed with succinate-ubiquinone reductase or that when split from succinate-ubiquinone reductase, ubiquinol-cytochrome c reductase undergoes a conformational change which allows access of nitric oxide to the Rieske iron-sulfur center. Such access is not possible when ubiquinol-cytochrome c reductase is complexed with succinate-ubiquinone reductase. The loss of ubiquinol-cytochrome c reductase activity correlates with a decrease in the Rieske protein EPR signal intensity without formation of any new EPR signal. The Rieske iron-sulfur cluster signal is recovered after 24 h incubation in the absence of nitric oxide.