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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL061411-02
Application #
6390108
Study Section
Special Emphasis Panel (ZRG1-RAP (01))
Program Officer
Garfinkel, Susan J
Project Start
2000-04-01
Project End
2004-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
2
Fiscal Year
2001
Total Cost
$206,240
Indirect Cost
Name
Carnegie-Mellon University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Fago, Angela; Crumbliss, Alvin L; Hendrich, Michael P et al. (2013) Oxygen binding to partially nitrosylated hemoglobin. Biochim Biophys Acta 1834:1894-900
Pearce, Linda L; Martinez-Bosch, Sandra; Manzano, Elisenda Lopez et al. (2009) The resistance of electron-transport chain Fe-S clusters to oxidative damage during the reaction of peroxynitrite with mitochondrial complex II and rat-heart pericardium. Nitric Oxide 20:135-42
Pearce, Linda L; Lopez Manzano, Elisenda; Martinez-Bosch, Sandra et al. (2008) Antagonism of nitric oxide toward the inhibition of cytochrome c oxidase by carbon monoxide and cyanide. Chem Res Toxicol 21:2073-81
Basova, Liana V; Kurnikov, Igor V; Wang, Lei et al. (2007) Cardiolipin switch in mitochondria: shutting off the reduction of cytochrome c and turning on the peroxidase activity. Biochemistry 46:3423-34
Pearce, Linda L; Kanai, Anthony J; Epperly, Michael W et al. (2005) Nitrosative stress results in irreversible inhibition of purified mitochondrial complexes I and III without modification of cofactors. Nitric Oxide 13:254-63
Chen, Tracy; Pearce, Linda L; Peterson, Jim et al. (2005) Glutathione depletion renders rat hepatocytes sensitive to nitric oxide donor-mediated toxicity. Hepatology 42:598-607
Kanai, Anthony; Epperly, Michael; Pearce, Linda et al. (2004) Differing roles of mitochondrial nitric oxide synthase in cardiomyocytes and urothelial cells. Am J Physiol Heart Circ Physiol 286:H13-21
Peterson, Jim; Kanai, Anthony J; Pearce, Linda L (2004) A mitochondrial role for catabolism of nitric oxide in cardiomyocytes not involving oxymyoglobin. Am J Physiol Heart Circ Physiol 286:H55-8
Fago, Angela; Crumbliss, Alvin L; Peterson, Jim et al. (2003) The case of the missing NO-hemoglobin: spectral changes suggestive of heme redox reactions reflect changes in NO-heme geometry. Proc Natl Acad Sci U S A 100:12087-92
Peterson, Jim; Bominaar, Emile L (2002) Resolving molecular electronic spectra using magnetic linear dichroism. Anal Chem 74:527A-533A

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