Oxidative Damage to Mitochondrial Complex I Due to Peroxynitrite

Abstract

There is growing evidence that oxidative phosphorylation (OXPHOS) generates reactive oxygen and nitrogen species within mitochondria as unwanted byproducts that can damage OXPHOS enzymes with subsequent enhancement of free radical production. The accumulation of this oxidative damage to mitochondria in brain is thought to lead to neuronal cell death resulting in neurodegeneration. The predominant reactive nitrogen species in mitochondria are nitric oxide and peroxynitrite. Here we show that peroxynitrite reacts with mitochondrial membranes from bovine heart to significantly inhibit the activities of complexes I, II, and V (50–80%) but with less effect upon complex IV and no significant inhibition of complex III. Because inhibition of complex I activity has been a reported feature of Parkinson's disease, we undertook a detailed analysis of peroxynitrite-induced modifications to proteins from an enriched complex I preparation. Immunological and mass spectrometric approaches coupled with two-dimensional PAGE have been used to show that peroxynitrite modification resulting in a 3-nitrotyrosine signature is predominantly associated with the complex I subunits, 49-kDa subunit (NDUFS2), TYKY (NDUFS8), B17.2 (17.2-kDa differentiation associated protein), B15 (NDUFB4), and B14 (NDUFA6). Nitration sites and estimates of modification yields were deduced from MS/MS fragmentograms and extracted ion chromatograms, respectively, for the last three of these subunits as well as for two co-purifying proteins, the β and the d subunits of the F1F0-ATP synthase. Subunits B15 (NDUFB4) and B14 (NDUFA6) contained the highest degree of nitration. The most reactive site in subunit B14 was Tyr122, while the most reactive region in B15 contained 3 closely spaced tyrosines Tyr46, Tyr50, and Tyr51. In addition, a site of oxidation of tryptophan was detected in subunit B17.2 adding to the number of post-translationally modified tryptophans we have detected in complex I subunits (Taylor, S. W., Fahy, E., Murray, J., Capaldi, R. A., and Ghosh, S. S. (2003) J. Biol. Chem. 278, 19587–19590). These sites of oxidation and nitration may be useful biomarkers for assessing oxidative stress in neurodegenerative disorders. There is growing evidence that oxidative phosphorylation (OXPHOS) generates reactive oxygen and nitrogen species within mitochondria as unwanted byproducts that can damage OXPHOS enzymes with subsequent enhancement of free radical production. The accumulation of this oxidative damage to mitochondria in brain is thought to lead to neuronal cell death resulting in neurodegeneration. The predominant reactive nitrogen species in mitochondria are nitric oxide and peroxynitrite. Here we show that peroxynitrite reacts with mitochondrial membranes from bovine heart to significantly inhibit the activities of complexes I, II, and V (50–80%) but with less effect upon complex IV and no significant inhibition of complex III. Because inhibition of complex I activity has been a reported feature of Parkinson's disease, we undertook a detailed analysis of peroxynitrite-induced modifications to proteins from an enriched complex I preparation. Immunological and mass spectrometric approaches coupled with two-dimensional PAGE have been used to show that peroxynitrite modification resulting in a 3-nitrotyrosine signature is predominantly associated with the complex I subunits, 49-kDa subunit (NDUFS2), TYKY (NDUFS8), B17.2 (17.2-kDa differentiation associated protein), B15 (NDUFB4), and B14 (NDUFA6). Nitration sites and estimates of modification yields were deduced from MS/MS fragmentograms and extracted ion chromatograms, respectively, for the last three of these subunits as well as for two co-purifying proteins, the β and the d subunits of the F1F0-ATP synthase. Subunits B15 (NDUFB4) and B14 (NDUFA6) contained the highest degree of nitration. The most reactive site in subunit B14 was Tyr122, while the most reactive region in B15 contained 3 closely spaced tyrosines Tyr46, Tyr50, and Tyr51. In addition, a site of oxidation of tryptophan was detected in subunit B17.2 adding to the number of post-translationally modified tryptophans we have detected in complex I subunits (Taylor, S. W., Fahy, E., Murray, J., Capaldi, R. A., and Ghosh, S. S. (2003) J. Biol. Chem. 278, 19587–19590). These sites of oxidation and nitration may be useful biomarkers for assessing oxidative stress in neurodegenerative disorders. Parkinson's disease (PD) 1The abbreviations used are: PD, Parkinson's disease; MPTP, 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine; 3NT, 3-nitrotyrosine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; LC, liquid chromatography; MS, mass spectrometry; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.1The abbreviations used are: PD, Parkinson's disease; MPTP, 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine; 3NT, 3-nitrotyrosine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; LC, liquid chromatography; MS, mass spectrometry; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. is a multifactorial, late-onset, neurodegenerative disease with both genetic and acquired etiologies and is characterized by the degeneration of dopaminergic neurons of the substantia nigra. There is growing evidence that mitochondrial damage, particularly to respiratory chain complex I (NADH:ubiquinone oxidoreductase), underlies the pathology of this and other neurological disorders. In rodent model systems, Parkinsonian symptoms have been observed as a result of the inhibition of complex I by the specific inhibitors rotenone (1Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2863) Google Scholar) and 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP) (2Heikkila R.E. Manzino L. Cabbat F.S. Duvoisin R.C. Nature. 1984; 311: 467-469Crossref PubMed Scopus (892) Google Scholar). Moreover, a 30% decrease in complex I activity has been detected in studies on post-mortem brain tissue from PD patients (3Schapira A.H. Cooper J.M. Dexter D. Clark J.B. Jenner P. Marsden C.D. J. Neurochem. 1990; 54: 823-827Crossref PubMed Scopus (1617) Google Scholar), and evidence of complex I subunit depletion has also been reported (4Mizuno Y. Ohta S. Tanaka M. Takamiya S. Suzuki K. Sato T. Oya H. Ozawa T. Kagawa Y. Biochem. Biophys. Res. Commun. 1989; 163: 1450-1455Crossref PubMed Scopus (645) Google Scholar). The extent of damage to complex I and other key enzymes of energy metabolism that triggers the onset of neurodegeneration and the characteristic symptoms of Parkinson's disease is not known. However, it is pertinent to note that the threshold for complex I activity in brain is the highest of any tissue, such that it requires only a modest degree of inhibition of oxidative phosphorylation to cause cellular dysfunction (5Davey G.P. Peuchen S. Clark J.B. J. Biol. Chem. 1998; 273: 12753-12757Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Increasingly, the evidence points to oxidative stress as the major cause of age-related mitochondrial damage. In line with the free radical hypothesis of neurodegeneration, depletion of glutathione, a critical antioxidant in cellular defenses against free radical damage, has been observed to precede mitochondrial damage and cell apoptosis of the neurons of the substantia nigra in presymptomatic PD patients (6Sian J. Dexter D.T. Lees A.J. Daniel S. Agid Y. Javoy-Agid F. Jenner P. Marsden C.D. Ann. Neurol. 1994; 36: 348-355Crossref PubMed Scopus (939) Google Scholar, 7Sofic E. Lange K.W. Jellinger K. Riederer P. Neurosci. Lett. 1992; 142: 128-130Crossref PubMed Scopus (481) Google Scholar). In accord with the above studies, glutathione depletion has been found to lead to complex I inhibition and reduced ATP production in a dopaminergic neuronal cell line (8Jha N. Jurma O. Lalli G. Liu Y. Pettus E.H. Greenamyre J.T. Liu R.M. Forman H.J. Andersen J.K. J. Biol. Chem. 2000; 275: 26096-26101Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). It has been known for 30 years that complexes I, II, and III of the respiratory chain produce superoxide (O2·¯) as an unwanted byproduct of oxidative phosphorylation, when partially reduced electron carriers such as flavins, non-heme iron centers and ubisemiquinone persist long enough to react with molecular oxygen (9Turrens J.F. Boveris A. Biochem. J. 1980; 191: 421-427Crossref PubMed Scopus (1342) Google Scholar, 10Boveris A. Chance B. Biochem. J. 1973; 134: 707-716Crossref PubMed Scopus (2065) Google Scholar). Peroxynitrite (ONOO–), formed by the reaction of O2·¯ with nitric oxide (NO·), is implicated as one of the major oxidants responsible for mitochondrial damage (11Jenner P. Ann. Neurol. 2003; 53 (discussion S36–S28): S26-S36Crossref PubMed Scopus (1661) Google Scholar). NO·, present in mitochondria in around 1 μm amounts (12Brown G.C. FEBS Lett. 1995; 369: 136-139Crossref PubMed Scopus (500) Google Scholar), is generated by the mitochondrial nitric oxide synthase (13Ghafourifar P. Richter C. FEBS Lett. 1997; 418: 291-296Crossref PubMed Scopus (525) Google Scholar, 14Elfering S.L. Sarkela T.M. Giulivi C. J. Biol. Chem. 2002; 277: 38079-38086Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 15Lopez-Figueroa M.O. Caamano C. Morano M.I. Ronn L.C. Akil H. Watson S.J. Biochem. Biophys. Res. Commun. 2000; 272: 129-133Crossref PubMed Scopus (108) Google Scholar), and this second messenger functions as a physiological regulator of the respiratory chain by reversibly binding and inhibiting complex IV (16Torres J. Darley-Usmar V. Wilson M.T. Biochem. J. 1995; 312: 169-173Crossref PubMed Scopus (185) Google Scholar). The short-lived but highly reactive ONOO– damages mitochondria by modifying DNA (17Byun J. Henderson J.P. Mueller D.M. Heinecke J.W. Biochemistry. 1999; 38: 2590-2600Crossref PubMed Scopus (98) Google Scholar), lipids (18Radi R. Beckman J.S. Bush K.M. Freeman B.A. Arch. Biochem. Biophys. 1991; 288: 481-487Crossref PubMed Scopus (2024) Google Scholar), and proteins within the organelle. Protein modification can occur by oxidation to produce for example N-formylkynurenine residues from tryptophans (19Taylor S.W. Fahy E. Murray J. Capaldi R.A. Ghosh S.S. J. Biol. Chem. 2003; 278: 19587-19590Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), by nitration of tyrosines to generate 3-nitrotyrosine (3NT) and by S-nitrosylation of cysteines (20Viner R.I. Williams T.D. Schoneich C. Biochemistry. 1999; 38: 12408-12415Crossref PubMed Scopus (211) Google Scholar). Recently the formation of 3NT has been observed in the MPTP-induced PD animal model (21Ferrante R.J. Hantraye P. Brouillet E. Beal M.F. Brain Res. 1999; 823: 177-182Crossref PubMed Scopus (75) Google Scholar, 22Pennathur S. Jackson-Lewis V. Przedborski S. Heinecke J.W. J. Biol. Chem. 1999; 274: 34621-34628Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Furthermore, 3NT α-synuclein has been detected in the Lewy body inclusions found in the substantia nigra cells of PD patients (23Giasson B.I. Duda J.E. Murray I.V. Chen Q. Souza J.M. Hurtig H.I. Ischiropoulos H. Trojanowski J.Q. Lee V.M. Science. 2000; 290: 985-989Crossref PubMed Scopus (1354) Google Scholar). The effect of ONOO– on mitochondrial function has been studied in vitro mainly by exposure of cultured cells to NO·. Complex I inhibition was observed in all cases but there is no consensus on whether reversible S-nitrosylation via thiols (24Borutaite V. Budriunaite A. Brown G.C. Biochim. Biophys. Acta. 2000; 1459: 405-412Crossref PubMed Scopus (160) Google Scholar, 25Orsi A. Beltran B. Clementi E. Hallen K. Feelisch M. Moncada S. Biochem. J. 2000; 346: 407-412Crossref PubMed Google Scholar, 26Clementi E. Brown G.C. Feelisch M. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7631-7636Crossref PubMed Scopus (733) Google Scholar) or irreversible inactivation by nitration of tyrosines (27Yamamoto T. Maruyama W. Kato Y. Yi H. Shamoto-Nagai M. Tanaka M. Sato Y. Naoi M. J. Neural. Transm. 2002; 109: 1-13Crossref PubMed Scopus (93) Google Scholar, 28Riobo N.A. Clementi E. Melani M. Boveris A. Cadenas E. Moncada S. Poderoso J.J. Biochem. J. 2001; 359: 139-145Crossref PubMed Scopus (252) Google Scholar), or both, is the cause of this inhibition. Here we have used direct addition of ONOO– to examine the oxidative damage induced in beef heart mitochondrial membranes by this reagent. The effect of exposure to ONOO– was assessed by activity measurements on all of the complexes of the OXPHOS system, and the sites of modification of complex I by the reagent were examined using mass spectrometry. The reactive tyrosines identified here may serve as candidates for early biomarkers of Parkinson's disease. Preparation of Bovine and Human Mitochondria—Bovine heart mitochondria were prepared according to Smith (29Smith A.L. Methods Enzymol. 1967; 10: 81-86Crossref Scopus (466) Google Scholar). Briefly, ventricles were homogenized, and particulate material was removed by centrifugation at 1,000 × g. Mitochondria were collected from the supernatant by spinning down at 12,000 × g and resuspended in the isotonic buffer, 10 mm Tris-HCl, pH 7.8, 0.25 m sucrose, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride. Human heart mitochondria, prepared from a 47-year-old male who died of brain cancer, were obtained from Analytical Biological Services. Protein concentration was determined by the BCA method (Pierce). Peroxynitrite Modification of Mitochondrial Proteins—ONOO– (77 mm) and degraded ONOO– (Upstate) were diluted in 0.3 m sodium hydroxide. The concentration of ONOO– was determined prior to titration by absorbance at 302 nm (extinction coefficient 1670 m–1 cm–1). For ONOO– modification, mitochondria at 5 mg/ml in 25 mm potassium phosphate, pH 7.2, 5 mm MgCl2 were placed on ice. ONOO– was deposited on the side of the tube above the mitochondria; vortexing results in rapid mixing of the mitochondrial membranes and ONOO– that then decays in a few seconds in the neutral pH of the buffer. The ONOO–-induced 3NT subunit modification (Fig. 1, Fig. 2) and inhibition of enzymatic activity (Fig. 1) were highly reproducible, suggesting that thorough mixing was achieved before ONOO– decay.Fig. 2Sucrose gradient separation of 3NT-modified mitochondrial complexes. After reaction with ONOO–, bovine heart mitochondria were solubilized, and the complexes were separated by sucrose gradient centrifugation. After one-dimensional SDS-PAGE gels were blotted and probed with an anti-3NT polyclonal antibody (A) or Coomassie Brilliant Blue stained (B). C, fraction 2 is highly enriched in complex I, and this fraction contains five complex I subunits that are 3NT-modified, labeled 1–5, and three non-complex I proteins, labeled a–c. Their relative abundance of modification is estimated by blot densitometry. D, ONOO– modification and separation of complexes in human heart mitochondria. Note the similar patterns of 3NT incorporation into the bovine and human respiratory chain complexes and ATP synthase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Determination of Enzymatic Activity of Each of the Respiratory Chain Complexes I–V—The mitochondrial preparation had been frozen and thawed once. The activity of complex I was determined by monitoring the reduction of NADH at 340 nm and at 30 °C according to the procedure by Birch-Machin and Turnbull (30Birch-Machin M.A. Turnbull D.M. Methods Cell Biol. 2001; 65: 97-117Crossref PubMed Google Scholar), using ubiquinone2 as an electron acceptor. This activity was 99% inhibited by rotenone. Complex II activity was measured by succinate-dependent reduction of 2,6-dichlorophenolindophenol. The reaction was monitored at 600 nm and at 30 °C using ubiquinone2 as an electron acceptor (30Birch-Machin M.A. Turnbull D.M. Methods Cell Biol. 2001; 65: 97-117Crossref PubMed Google Scholar). Complex III activity was measured by reduction of cytochrome c (III) at 550 nm and at 30 °C using ubiquinol2 as an electron donor, essentially as described by Birch-Machin and Turnbull (30Birch-Machin M.A. Turnbull D.M. Methods Cell Biol. 2001; 65: 97-117Crossref PubMed Google Scholar) but in the absence of dodecyl-β-d-maltoside, which was found to be inhibitory. Activity was determined as the initial rate of reduction of cytochrome c above the slower background rate of reduction of cytochrome c. This rate was inhibited by 99% using antimycin A. The activity of complex IV was determined by monitoring the oxidation of cytochrome c (II) at 550 nm and at 30 °C according to Birch-Machin and Turnbull (30Birch-Machin M.A. Turnbull D.M. Methods Cell Biol. 2001; 65: 97-117Crossref PubMed Google Scholar). This rate was greater than 90% inhibited by KCN. The ATP hydrolysis rate was performed according to Lotscher et al. (31Lotscher H.R. deJong C. Capaldi R.A. Biochemistry. 1984; 23: 4140-4143Crossref PubMed Scopus (99) Google Scholar) at 37 °C and was 98% inhibited by oligomycin. Finally, the activity of citrate synthase was performed according to Maneiro et al. (32Maneiro E. Martin M.A. de Andres M.C. Lopez-Armada M.J. FernandezSueiro J.L. del Hoyo P. Galdo F. Arenas J. Blanco F.J. Arthritis Rheum. 2003; 48: 700-708Crossref PubMed Scopus (178) Google Scholar) following the cleavage of acetyl coenzyme A in the presence of 5,5′-dithiobis(2-nitrobenzoic acid), monitored at 420 nm and at 30 °C. Sucrose Gradient Subfractionation of Mitochondrial Complexes and Electrophoresis—Subfractionation of mitochondria was performed by sucrose gradient density centrifugation according to Hanson et al. (33Hanson B.J. Schulenberg B. Patton W.F. Capaldi R.A. Electrophoresis. 2001; 22: 950-959Crossref PubMed Scopus (87) Google Scholar). Electrophoresis of NADH Dehydrogenase Complex—For one-dimensional electrophoresis, 5 μl of the sucrose gradient samples were separated on 10–22% acrylamide gels containing 0.05% SDS, 0.375 m Tris-HCl, pH 8.6. One-dimensional gels were stained with Coomassie Brilliant Blue (Sigma). For two-dimensional electrophoresis, 100-μl sucrose gradient samples were denatured in 350 μl of rehydration buffer (7 m urea, 2 m thiourea, 0.8% pH 3–10 ampholyte (Fluka), or 0.5% pH 6–11 IPG buffer (Amersham Biosciences), 2% CHAPS (Sigma), 1% Zwittergent 310 (Sigma), 0.1% SDS) for 15 min at room temperature. Each sample was used to hydrate 18 cm of immobiline pH gradient strips with pH ranges of 3–10 or 6–11 (Amersham Biosciences) for 12 h. Isoelectric focusing was performed in three stages of applied potential difference; 500 V for 1 h, 1000 V for 1 h, and 8000 V for up to 10 h, until 60,000 Vh were achieved. Focused strips were soaked in SDS-PAGE buffer (50 mm Tris-HCl, pH 8.8, 6 m urea, 30% glycerol, 2% SDS, 0.01% bromphenol blue) for 15 min at room temperature and then applied to 15% acrylamide gels for SDS-PAGE. Two-dimensional gels were stained with silver nitrate by the method of Schevchenko et al. (34Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7736) Google Scholar). Immunological Detection of Proteins Containing 3NT—One-dimensional or two-dimensional gels were transferred in CAPS buffer from gels to polyvinylidene difluoride membranes (0.45-μm pore size) according to Triepels (35Triepels R.H. Hanson B.J. van den Heuvel L.P. Sundell L. Marusich M.F. Smeitink J.A. Capaldi R.A. J. Biol. Chem. 2001; 276: 8892-8897Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Proteins containing 3NT were detected by an anti-3NT polyclonal antibody (Upstate), followed by a secondary goat anti-mouse polyclonal antibody conjugated to alkaline phosphatase (two-dimensional) or horseradish peroxidase (one-dimensional), then detected by the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad) or ECL+ (Amersham Biosciences) detection methods, respectively. Sample Preparation for Analysis by Mass Spectrometry—Gel bands and spots, corresponding to areas of 3NT immunoreactivity on blots, were manually excised from gels with a razor. Destaining of silver-stained samples and digestions were performed as described previously (36Murray J. Zhang B. Taylor S.W. Oglesbee D. Fahy E. Marusich M.F. Ghosh S.S. Capaldi R.A. J. Biol. Chem. 2003; 278: 13619-13622Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) but without reduction and alkylation to avoid the possibility of any partial reduction of the 3NT moieties. Digests were prepared for MALDI-TOF peptide mass and were acquired on a as reported previously (36Murray J. Zhang B. Taylor S.W. Oglesbee D. Fahy E. Marusich M.F. Ghosh S.S. Capaldi R.A. J. Biol. Chem. 2003; 278: 13619-13622Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). mass from and were then using the Protein P. A.L. Anal. Chem. 1999; PubMed Scopus Google Scholar) with and without the from as described previously P. A.L. Anal. Chem. 1999; PubMed Scopus Google Scholar, S.W. Zhang B. Fahy E. Capaldi R.A. Ghosh S.S. J. Res. 2002; PubMed Scopus Google Scholar). of Proteins by were a to by of a cell The had been previously with A 1% 0.5% The was to the liquid of a on that described in the A.J. Anal. Biochem. 1998; PubMed Scopus Google Scholar) by the of of and and an II by of an This at the from silver-stained gels A.J. Anal. Biochem. 1998; PubMed Scopus Google Scholar). The rate was to a a gradient of A to 0.5% 1% to followed by a with was used to and into the of a Mass Mass were acquired in the the following temperature and tube V. After one of the was and two MS/MS of the most and second most were the with an of and energy was to the number of peptide from the After MS/MS were acquired for a the of ion was placed on an with a for 1 Each was with the A. J.K. R. Protein Sci. 1998; PubMed Scopus Google Scholar) using the bovine or human of the that had been for with oxidation mass and nitration or tryptophan as modifications using The for a was at 1 peptide for a an of for a of for a or of for a In all cases be greater than as had to the following of upon a with 1) be N.A. Williams T.D. Schoneich C. Anal. Biochem. 2002; PubMed Scopus Google Scholar) and 2) MS/MS fragmentograms be mainly by mass of mass to the corresponding and 1 was by the ion for the 0.5 mass and 2 was by of the MS/MS for the and modified ion and MS/MS for the most peptide corresponding to subunit extracted ion for the peptide 0.5 mass extracted ion for the peptide 0.5 mass to the the for from the of a major peptide In addition a for a of than and to the and is by an C, MS/MS for the D, MS/MS for the The and ion to that by K. Methods Enzymol. 1990; PubMed Scopus Google Scholar). In this and all a mass in the MS/MS of the peptide relative to that of the that the in the second is the site of ONOO– Large Image Figure ViewerDownload Hi-res image Download ion and MS/MS for a peptide containing two residues corresponding to subunit extracted ion for the peptide 0.5 mass extracted ion for the peptide 0.5 mass C, MS/MS for the and D, MS/MS for the In this and all a mass in the MS/MS of the peptide relative to that of the Because for the peptide with the second is from for the peptide with the the two sites are on this Large Image Figure ViewerDownload Hi-res image Download (PPT) of of ion for and the corresponding were to using the of the and manually areas corresponding to the of the areas of the for relative that the species have ionization This was to be upon the that nitration no on the and for a in the molecular of the Furthermore, et al. N.A. R.J. Williams T.D. J.L. Chem. Res. 2003; PubMed Scopus Google Scholar) have that estimated from relative for a and peptide from were using absorbance at nm Biochemistry. PubMed Scopus Google Scholar). The method is of ionization of the Respiratory Chain Complexes by heart mitochondrial were with and μm ONOO– (Fig. μm ONOO– was of the was from the reduced of the Complexes I, II, and V ATP were inhibited and at μm ONOO–, complex IV was inhibited to and complex III was at this reagent of inhibition were for complexes I, II, and V at μm ONOO–, and of these complexes was inhibited at μm with μm ONOO–, complex IV