Mitochondria are critical loci of cellular respiration, biosynthesis and metabolism of partially reduced oxygen species and participate in intracellular signaling including calcium homeostasis, oxygen sensing and the initiation of apoptosis. Nitric oxide (NO) is unique amongst signaling molecules in that it affects its target sites by both redox chemistry (S-nitrosylation of thiols) and coordination (especially to Fe2+). Consequently biomolecules affected by NO (and peroxynitrite) tend to have critical metalloregulatory and cysteine-enriched active sites. Accordingly, it has been well known since the discovery of the L-arginine biosynthetic pathway that NO can inhibit mitochondrial respiration. Nonetheless neither the precise targets nor the mechanism by which NO affects mitochondrial function are apparent. We hypothesize that NADH dehydrogenase (complex I) with it's multiple non-heme iron-sulfur (Fe/S) centers and cytochrome c reductase (complex III), an hemoprotein, are targets for NO and peroxynitrite. We propose a series of biophysical and biochemical studies that will reveal the molecular mechanism by which NO, partially reduced oxygen species (PROS) and ONO2 affect mitochondrial respiratory chain function.
Aim 1 : Investigate the molecular mechanism by which peroxynitrite (ONO2) and other oxidants irreversibly inhibit complex I and complex III.
Aim 2 : Investigate the mechanism by which complex IV (cytochrome c oxidase) catalyses the detoxification of ONO2 and H2O2.
Aim 3 : Identify the molecular targets of peroxynitrite and ferrous iron in Keilin-Hartee (K-H, submitochondrial) particles and intact mitochondria.
Aim 4 : describe the oxidant (NO, superoxide anion, ONO2, etc) induced changes (mitochondrial membrane potential) that occur in mitochondria of intact cultured endothelial cells. These studies, using a combination of low temperature electron paramagnetic resonance spectroscopy and magnetic circular and linear dichroism in isolated bovine heart mitochondria and voltage sensitive dyes in intact bovine pulmonary artery endothelial cells, will provide mechanistic information to support a role for NO induced mitochondrial changes in health and disease.
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