Interaction of nitric oxide with the activity of cytosolic NADH/cytochrome c electron transport system.

Nitric oxide ((.)NO) generated by the dissociation of S-nitrosoglutathione or added as gaseous solution, inhibits the oxidation of exogenous NADH supported by the activity of the cytosolic NADH/cyto-c electron transport pathway. The inhibition is immediate, very strong, higher at lower oxygen concentration, independent on the (.)NO concentration and remains constant as long as (.)NO is no more available and then is spontaneously removed. The data obtained, not in contrast with those reported with isolated cytochrome oxidase (Cox), strengthen a new concept: reduced cytochrome c (cyto-c) and (.)NO behave as two substrates of Cox, which promotes their oxidation with molecular oxygen as a co-substrate. In the presence of (.)NO, Cox exhibits the property of switching from cyto-c oxidase to (.)NO oxidase activity. With an "all or nothing" process Cox becomes an efficient (.)NO scavenger. The persistence of membrane potential, even in the presence of high inhibition of oxygen uptake, could be tentatively correlated to the protective effect of (.)NO on the ischaemic-reperfusion injury.

Effect of magnesium ions on the activity of the cytosolic NADH/cytochrome c electron transport system.

Cytochrome c (cyto-c), added to isolated mitochondria, activates the oxidation of extramitochondrial NADH and the generation of a membrane potential, both linked to the activity of the cytosolic NADH/cyto-c electron transport pathway. The data presented in this article show that the protective effect of magnesium ions on the permeability of the mitochondrial outer membrane, supported by previously published data, correlates with the finding that, in hypotonic but not isotonic medium, magnesium promotes a differential effect on both the additional release of endogenous cyto-c and on the increased rate of NADH oxidation, depending on whether it is added before or after the mitochondria. At the same time, magnesium prevents or almost completely removes the binding of exogenously added cyto-c. We suggest that, in physiological low-amplitude swelling, magnesium ions may have the function, together with other factors, of modulating the amount of cyto-c molecules transferred from the mitochondrial intermembrane space into the cytosol, required for the correct execution of the apoptotic programme and/or the activation of the NADH/cyto-c electron transport pathway.

Protective effect of magnesium and potassium ions on the permeability of the external mitochondrial membrane.

The data reported are fully consistent with the well-known observation that exogenous cytochrome c (cyto-c) molecules do not permeate through the outer membrane of mitochondria (MOM) incubated in isotonic medium (250 mM sucrose). Cyto-c is unable to accept electrons from the sulfite/cyto-c oxido-reductase (Sox) present in the intermembrane space, unless mitochondria are solubilized. Mitochondria incubated in a very high hypotonic medium (25 mM sucrose), in contrast to any expectation, continue to be not permeable to added cyto-c even if Sox and adenylate kinase are released into the medium. The succinate/exogenous cyto-c reductase activity, very low in isotonic medium, is greatly increased decreasing the osmolarity of the medium but in both cases remains insensitive to proteolysis by added trypsin. In hypotonic medium, magnesium and potassium ions have a protective effect on the release of enzymes and on the reactivity of cyto-c as electron acceptor from both sulfite and succinate; results which are consistent with the view that MOM preserves its identity and remains not permeable to exogenous cyto-c. This report strengthens the proposal, supported by previously published data that in isotonic medium the exogenous NADH/cyto-c electron transport system is catalyzed by intact mitochondria, not permeable to added cyto-c.