Tyrosine modification of glucose dehydrogenase from Bacillus megaterium. Effect of tetranitromethane on the enzyme in the tetrameric and monomeric state

The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residue/subunit. The nitration does not influence the dissociation-reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP. The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr-254 in the covalent structure of the enzyme. After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer. Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr-254 which is essential for the catalytic activity and the other one is Tyr-160 which seems to be located in the subunit binding area.     

The reaction of tetranitromethane with human chorionic gonadotropin.

The reaction of tetranitromethane with human chorionic gonadotropin and its subunits has been investigated. The hormone consists of two subunits, α and β, containing four and three tyrosyl residues, respectively. Introduction of 1 nitrated tyrosine residue into the native hormone was accompanied by a 20% loss in immunological reactivity and a 50% loss in biological activity. This initial reaction occurred at α Tyr-88 and/or α Tyr-89. Exhaustive nitration of the hormone modified α tyrosines 65, 88, and 89 and resulted in 75% inactivation biologically and 50% immunologically. Either nitrated α subunit obtained by dissociation of the nitrated hormone recombined with the native β subunit to give a hormone whose activity was in reasonable agreement with that of the corresponding nitrated monomer. These results indicate involvement of α Tyr-88 and/or α Tyr 89 in binding of the hormone to its receptor. These residues are not required for binding to the β subunit, however. Tyr-65 of the α subunit is probably not involved in binding to either the β subunit or the hormone receptor. The β subunit obtained from the exhaustively nitrated hormone was unmodified and recombined with native α to give fully active hormone. About 25% of the protein was recovered as polymeric material following nitration; lesser amounts of crosslinked monomer were formed. Both were biologically inactive. The polymer products retained about 30% of the native immunological competence.Nitration of the isolated α subunit fully converted the remaining tyrosine (Tyr-37) to 3-nitrotyrosine in a two-step reaction. The fully nitrated α subunit did not recombine well with the native β subunit and the recombinant hormone has 10% or less of the native activity. Involvement of α Tyr-37 in binding to the β subunit is suggested by these data. However, exposure of this residue by a conformational change in the α subunit after dissociation of the native hormone, while it seems unlikely in view of the high disulfide content, is also consistent with the data. Reaction of the free β subunit with tetranitromethane resulted in complete nitration of Tyr-37, 85% nitration of Tyr-59, and 25% nitration of Tyr-82. The nitrated β subunit did not recombine well with native α but the isolated recombinant had two-thirds of the native activity. From these data we conclude that β Tyr-37 and/or β Tyr-59 are possibly involved in binding to the α subunit but do not have a role in the biological activity. Tyr-82 of β is apparently not involved in either subunit interactions or hormone-receptor binding.     

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. Complete tyrosine assignments in the 1H nuclear-magnetic-resonance spectrum of the phosphocarrier protein HPr.

Upon nitration of the phosphocarrier protein HPr three nitrated derivatives of the protein were isolated: mononitrated HPr, dinitrated HPr and trinitrated HPr. Tryptic digestion of the derivatives leads to nitrotyrosine-containing peptides which were isolated and characterized by amino acid analysis. This resulted in the determination of the positions of the nitrated tyrosyl residues in the amino acid sequence. In mononitrated HPr only Tyr-56 was modified, in dinitrated HPr both Tyr-56 and Tyr-37 had reacted with the nitrating agent; modification of all three tyrosyl residues in trinitrated HPr required more drastic reaction conditions. The nuclear magnetic resonance spectra of the three derivatives allowed the assignments of the tyrosine resonances as follows: Tyr-A and Tyr-B with pK values of 10.5 and 11.5 were designated Tyr-56 and Tyr-37 whereas Tyr-C, whose protons are not titratable before denaturation of the protein, was assigned to Tyr-6 in the amino acid sequence. The nitration studies, together with the titration behaviour of the three tyrosines, indicate the topology of the tyrosyl residues to be as follows: Tyr-56 is located at the surface, Tyr-37 is slightly buried, Tyr-6 is deeply buried. The nitrotyrosyl derivatives retain their biological activity.     

Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives

The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.     

The inactivation of plasma alpha 1-proteinase inhibitor by nitrous acid

Exposure of alpha 1-PI to nitrous acid resulted in a complete inactivation of either of its elastase or trypsin inhibitors activities. Amino acid analyses of the nitrous acid treated inhibitor revealed only losses of one tryphanyl and three lysyl residues. Reductive methylation of alpha 1-PI offered no protection against loss of activity by nitrous acid. Since no further loss of lysyl residues was observed upon exposure of fully active reductively methylated alpha 1-PI to nitrous acid, modification of one tryptophanyl residue appears to be responsible for the inhibitor's sensitivity to nitrous acid. Absorption spectral studies of the nitrous acid treated alpha 1-PI indicated that the tryptophanyl residue was modified to its N-nitroso derivative.     

Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues

Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.     

Localization of nitration and chlorination sites on apolipoprotein A-I catalyzed by myeloperoxidase in human atheroma and associated oxidative impairment in ABCA1-dependent cholesterol efflux from macrophages

We recently reported that apolipoprotein A-I (apoA-I), the major protein component of high density lipoprotein, is a selective target for myeloperoxidase (MPO)-catalyzed nitration and chlorination in both and serum of subjects with cardiovascular disease. We further showed that the extent of both apoA-I nitration and chlorination correlated with functional impairment in reverse cholesterol transport activity of the isolated lipoprotein. Herein we used tandem mass spectrometry to map the sites of MPO-mediated apoA-I nitration and chlorination in vitro and in vivo and to relate the degree of site-specific modifications to loss of apoA-I lipid binding and cholesterol efflux functions. Of the seven tyrosine residues in apoA-I, Tyr-192, Tyr-166, Tyr-236, and Tyr-29 were nitrated and chlorinated in MPO-mediated reactions. Site-specific liquid chromatography-mass spectrometry quantitative analyses demonstrated that the favored modification site following exposure to MPO-generated oxidants is Tyr-192. MPO-dependent nitration and chlorination both proceed with Tyr-166 as a secondary site and with Tyr-236 and Tyr-29 modified only minimally. Parallel functional studies demonstrated dose-dependent losses of ABCA1-dependent cholesterol acceptor and lipid binding activities with apoA-I modification by MPO. Finally tandem mass spectrometry analyses showed that apoA-I in human atherosclerotic tissue is nitrated at the MPO-preferred sites, Tyr-192 and Tyr-166. The present studies suggest that site-specific modifications of apoA-I by MPO are associated with impaired lipid binding and ABCA1-dependent cholesterol acceptor functions, providing a molecular mechanism that likely contributes to the clinical link between MPO levels and cardiovascular disease risk.     

Nitration of solvent-exposed tyrosine 74 on cytochrome c triggers heme iron-methionine 80 bond disruption. Nuclear magnetic resonance and optical spectroscopy studies

Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pKa = 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile Omega-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.     

Tyrosine 385 of prostaglandin endoperoxide synthase is required for cyclooxygenase catalysis

There are spectral and biochemical data suggesting that a tyrosine group(s) is involved in the cyclooxygenase reaction catalyzed by prostaglandin endoperoxide (PGH) synthase. Treatment with tetranitromethane, a reagent which nitrates tyrosine residues, abolishes cyclooxygenase activity, but this inactivation can be largely prevented by competitive cyclooxygenase inhibitors such as ibuprofen and indomethacin. To identify sites of nitration, native PGH synthase and indomethacin-pretreated PGH synthase were incubated with tetranitromethane, and the sequences of peptides containing nitrotyrosine were determined. Three unique tyrosines (Tyr-355, Tyr-385, and Tyr-417) were nitrated in the native enzyme but not in the indomethacin-treated PGH synthase. Using site-directed mutagenesis of sheep PGH synthase, each of these tyrosines, as well as two other tyrosine residues selected as controls (Tyr-254 and Tyr-262), were replaced with phenylalanine; cos-1 cells were transfected with constructs containing cDNAs coding for the native PGH synthase and each of the five phenylalanine mutants, and microsomes from these cells were assayed for cyclooxygenase and hydroperoxidase activities. The Phe-385 mutant of PGH synthase lacked cyclooxygenase activity but retained peroxidase activity; all other mutants expressed both enzyme activities. Our results establish that Tyr-385 is essential for the cyclooxygenase activity of PGH synthase and that nitration of this residue can be prevented by indomethacin. We conclude that Tyr-385 is at or near the cyclooxygenase active site of PGH synthase and could be the tyrosine residue proposed to be involved in the first step of the cyclooxygenase reaction, abstraction of the 13-proS hydrogen from arachidonate.     

Peroxynitrite treatment in vitro disables catalytic activity of recombinant p38 MAPK

Protein tyrosine nitration is a post-translational modification occurring under conditions of oxidative stress in a number of diseases. The causative agent of tyrosine nitration is the potent prooxidant peroxynitrite that results from the interaction of nitric oxide and superoxide. We have previously demonstrated existence of nitrotyrosine in placenta from pregnancies complicated by preeclampsia, which suggested the possibility of the existence of nitrated proteins. Nitration of various proteins has been demonstrated to more commonly result in loss of protein function. Potential nitration of p38 MAPK, a critical signaling molecule has been suggested and also tentatively identified in certain in vivo systems. In this study we demonstrate for the first time nitration of recombinant p38 MAPK in vitro and an associated loss of its catalytic activity. LC-MS data identified tyrosine residues Y132, Y245 and Y258 to be nitrated. Nitration of these specific residues was deduced from the 45.0-Da change in mass that these residues exhibited that was consistent with the loss of a proton and addition of the nitro group.     

The status of tyrosyl residues in a Formosan cobra cardiotoxin.

Spectrophotometric titration of Formosan cobra cardiotoxin showed that two of the three tyrosyl residues were titrated freely with a normal apparent pKa of 9.6 whereas the remaining one ionized at pH above 11.0. Nitration of cardiotoxin in Tris . HCl buffer with tetranitromethane resulted in the selective nitration of tyrosine 11 and tyrosine 22. It also revealed that tyrosine 51 was the abnormal one in the spectrophotometric titration. Complete nitration occurred in the presence of 6.0 M guanidine hydrochloride. Compared with the conformation of native cardiotoxin, the peptide conformation of the partially nitrated cardiotoxin did not change significantly but the conformation of the completely nitrated cardiotoxin changed remarkably. The biological activity of cardiotoxin was indeed affected by nitration, but the immunological activity was nearly intact even when all the tyrosine residues were nitrated.     

Identification of the nitration site of insulin by peroxynitrite.

Our previous investigation indicated that insulin can be nitrated by peroxynitrite in vitro. In this study, the preferential nitration site of the four tyrosine residues in insulin molecule was confirmed. Mononitrated and dinitrated insulins were purified by RP-HPLC. Following reduction of insulin disulfide bridges, Native-PAGE indicated that A-chain was preferentially nitrated. Combination of enzymatic digestion of mononitrated insulin with endoproteinase Glu-C, mass spectrometry confirmed that Tyr-A14 was the preferential nitration site when insulin was treated with peroxynitrite. Tyr-A19, maybe, was the next preferential nitration site. According to the crystal structure, Tyr-B26 between the two tyrosine residues in insulin B-chain was likely easier to be nitrated by peroxynitrite.     

The reactivity of a functional tyrosyl residue in carboxypeptidase B. Nitration of the cadmium enzyme

Cadmium-carboxypeptidase B was nitrated with tetranitromethane. The enzyme polymerized extensively during nitration. In the monomer nitrated Cd-carboxypeptidase B, 70% of the activity of Cd-carboxypeptidase B was retained. In order to identify the tyrosyl residues nitrated, the enzyme was digested with chymotrypsin and subtilisin and the nitrotyrosyl peptides were purified by affinity chromatography on antityrosyl-antibody-Sepharose conjugate followed by two-dimensional thin-layer chromatography. The major nitropeptides, representing 65% of the nitrotyrosyl label, were compatible with the segment of the sequence containing Tyr-240 and Tyr-259. Only 10% of the nitrotyrosyl label was found in the segment of Tyr-248. This indicates that the state of Tyr-248 in Cd-carboxypeptidase B differs from that in zinc-carboxypeptidase B where it shows chemical hyperreactivity due to its proximity to the metal ion.     

Oxidation of alpha 1-protease inhibitor: role of lipid peroxidation products

Previously we demonstrated that in vivo exposure of humans to NO2 resulted in significant inactivation of alpha 1-protease inhibitor (alpha 1-PI) in the bronchoalveolar lavage fluid. However, alpha 1-PI retains its elastase inhibitory activity in vitro when exposed to 10 times the concentration of NO2 used in vivo. We suggested exogenous oxidants such as O2 and NO2 exert their effect in vivo in part through lipid peroxidation. We investigated the mechanism of inactivation of alpha 1-PI in the presence or absence of lipids under oxidant atmosphere. alpha 1-PI in solutions containing phosphate buffer (control), 0.1 mM stearic acid (saturated fatty acid, 18:0), or 0.1 mM linoleic acid (polyunsaturated fatty acid, 18:2) was exposed to either N2 or NO2 (50 ppm for 4 h). Elastase inhibitory capacity of alpha 1-PI was significantly diminished in the presence of 0.1 mM linoleic acid and under NO2 atmosphere (75 +/- 8% of control, P less than 0.01), whereas there was no change in elastase inhibitory capacity of alpha 1-PI in the presence or absence (buffer only) of 0.1 mM stearic acid under a similar condition (109 +/- 11 and 94 +/- 6%, respectively). The inactivated alpha 1-PI as the result of peroxidized lipid could be reactivated by dithiothreitol and methionine sulfoxide peptide reductase, suggesting oxidation of methionine residue at the elastase inhibitory site. Furthermore the inhibitory effect of peroxidized lipid on alpha 1-PI could be prevented by glutathione and glutathione peroxidase and to some extent by alpha-tocopherol.     

The effect of reducing agents on proteolytic enzymes and oxidation of alpha 1-proteinase inhibitor

We have studied the effect of the mucolytic agent N-acetylcysteine and dithiothreitol on the oxidation of alpha 1-PI by hydrogen peroxide, and their effect on porcine pancreatic elastase and leukocyte elastase. In addition, the effect of S-(carboxymethyl)cysteine (= carbocisteine, a mucolytic agent which does not have reducing properties) was studied in vitro and in patients with chronic obstructive bronchitis. Following addition of 59.6mM N-acetylcysteine, the amidolytic activity of leukocyte elastase was decreased by 55.3% and that of porcine pancreatic elastase by 57.0%. Dithiothreitol (5.7 mM) caused the loss of 97.4% and 67.6% of amidolytic activity of leukocyte elastase and porcine pancreatic elastase respectively whereas S-(carboxymethyl)cysteine had no effect. Similar results were found for the effect on elastolytic activity. Oxidation of alpha 1-PI by 8.6mM H2O2 resulted in partial loss of inhibitory function (mean 68.7% activity of native alpha 1-PI). N-Acetylcysteine and dithiothreitol prevented oxidation of alpha 1-PI when pre-incubated with H2O2 or incubated with alpha 1-PI and H2O2 simultaneously (94.5% and 94.4% activity of native alpha 1-PI for N-acetylcysteine; 78.3% and 87.6% activity for dithiothreitol - p less than 0.025). S-(Carboxymethyl)cysteine, when pre-incubated with H2O2 or incubated concurrently with alpha 1-PI and H2O2, caused a further decrease in the porcine pancreatic elastase inhibitory capacity of alpha 1-PI (53.1% and 63.0% respectively - p less than 0.025). None of the agents reversed oxidative inactivation once it had occurred. S-(Carboxymethyl)cysteine had no effect on alpha 1-PI function in sputum at the dose used.     

Kinetic mechanism of chicken liver xanthine dehydrogenase.

Human inter-alpha-trypsin inhibitor (I alpha I) is a plasma proteinase inhibitor active against cathepsin G, leucocyte elastase, trypsin and chymotrypsin. It owes its broad inhibitory specificity to tandem Kunitz-type inhibitory domains within an N-terminal region. Sequence studies suggest that the reactive-centre residues critical for inhibition are methionine and arginine. Reaction of I alpha I with the arginine-modifying reagent butane-2,3-dione afforded partial loss of inhibitory activity against both cathepsin G and elastase but complete loss of activity against trypsin and chymotrypsin. Reaction of I alpha I with the methionine-modifying reagent cis-dichlorodiammineplatinum(II) resulted in partial loss of activity against cathepsin G and elastase but did not affect inhibition of either trypsin or chymotrypsin. Employment of both reagents eliminated inhibition of cathepsin G and elastase. These findings suggest that both cathepsin G and elastase are inhibited at either of the reactive centres of I alpha I. Trypsin and chymotrypsin, however, appear to be inhibited exclusively at the arginine reactive centre.     

The transferable tail: fusion of the N-terminal acidic extension of heparin cofactor II to alpha1-proteinase inhibitor M358R specifically increases the rate of thrombin inhibition

The conversion of the reactive center bond of the serpin alpha1-proteinase inhibitor (alpha1-PI, also known as alpha1-antitrypsin) from Met-Ser to Arg-Ser decreases the rate at which it inhibits neutrophil elastase and endows it with the ability to inhibit thrombin and activated protein C (APC). Another serpin, heparin cofactor II (HCII), contains a unique N-terminal extension that binds thrombin exosite 1. We fused residues 1-75 of HCII to the N-terminus of alpha1-PI M358R, forming an HCII-alpha1-PI chimera (HAPI M358R). It inhibited alpha-thrombin 21-fold faster than alpha1-PI M358R, with second-order rate constants of 2.3 x 10(8) M(-1) min(-1) versus 1.1 x 10(7) M(-1) min(-1), respectively. When gammaT-thrombin, which lacks an intact exosite 1, was substituted for alpha-thrombin, the kinetic advantage of HAPI M358R over alpha1-PI M358R was reduced to 9-fold, whereas APC and trypsin, proteases lacking exosite 1-like regions, were inhibited only 1.3- and 2-fold more rapidly by HAPI M358R than by alpha1-PI M358R, respectively. Maximal enhancement of alpha1-PI M358R activity required the acidic residues found between HCII residues 55 and 75, because no enhancement was observed either by fusion of residues 1-54 alone or by fusion of a mutated HCII acidic extension in which all Glu and Asp residues between positions 55 and 75 were neutralized by mutation. Fusing residues 55-75 to alpha1-PI M358R yielded a relative rate enhancement of only 6-fold, suggesting a need for the full tail region to achieve maximal enhancement. Our results suggest that transfer of the N-terminal acidic extension of HCII to alpha1-PI M358R enhanced its inhibition of thrombin by conferring the ability to bind exosite 1 on HAPI M358R. This enhancement may aid in efforts to tailor this inhibitor for therapeutic use.     

Cytochrome c nitration by peroxynitrite

Peroxynitrite (ONOO(-)), the product of superoxide (O(2)) and nitric oxide (.NO) reaction, inhibits mitochondrial respiration and can stimulate apoptosis. Cytochrome c, a mediator of these two aspects of mitochondrial function, thus represents an important potential target of ONOO(-) during conditions involving accelerated rates of oxygen radical and.NO generation. Horse heart cytochrome c(3+) was nitrated by ONOO(-), as indicated by spectral changes, Western blot analysis, and mass spectrometry. A dose-dependent loss of cytochrome c(3+) 695 nm absorption occurred, inferring that nitration of a critical heme-vicinal tyrosine (Tyr-67) promoted a conformational change, displacing the Met-80 heme ligand. Nitration was confirmed by cross-reactivity with a specific antibody against 3-nitrotyrosine and by increased molecular mass compatible with the addition of a nitro-(-NO(2)) group. Mass analysis of tryptic digests indicated the preferential nitration of Tyr-67 among the four conserved tyrosine residues in cytochrome c. Cytochrome c(3+) was more extensively nitrated than cytochrome c(2+) because of the preferential oxidation of the reduced heme by ONOO(-). Similar protein nitration patterns were obtained by ONOO(-) reaction in the presence of carbon dioxide, whereupon secondary nitrating species arise from the decomposition of the nitroso-peroxocarboxylate (ONOOCO(2)(-)) intermediate. Peroxynitrite-nitrated cytochrome c displayed significant changes in redox properties, including (a) increased peroxidatic activity, (b) resistance to reduction by ascorbate, and (c) impaired support of state 4-dependent respiration in intact rat heart mitochondria. These results indicate that cytochrome c nitration may represent both oxidative and signaling events occurring during .NO- and ONOO(-)-mediated cell injury.     

Reversible inhibition of neutrophil elastase by thiol-modified alpha-1 protease inhibitor

We have modified the single cysteine residue of alpha 1-protease inhibitor (alpha 1-PI) with HgCl2, methylmethane thiosulfonate, oxidized glutathione (GSSG), and N-(1-anilinonaphthyl-4)maleimide (ANM). Whereas native alpha 1-PI combines rapidly and quasi-irreversibly with neutrophil elastase, the thiol-modified alpha 1-PI derivatives are dissociable reversible competitive inhibitors of the enzyme, with values of Ki in the range of 6-7 nM. Removal of the thiol modifications restores the rapid irreversible mode of inhibition. Once native alpha 1-PI has combined with neutrophil elastase, the enzyme-inhibitor complex retains a reactive thiol group, but the two proteins can no longer be dissociated by subsequent reaction with ANM, even after exposure to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From kinetic measurements of fluorescence, ANM-modified alpha 1-PI combines with neutrophil elastase via an apparent biomolecular process with a second order rate constant on the order of 10(5) M-1 S-1. We estimate a dissociation rate constant on the order of 10(-3) S-1. The emission of ANM-modified alpha 1-PI is increased in intensity and blue shifted from the maximum in ANM-modified cysteine, consistent with a predominantly nonpolar environment. Association with neutrophil elastase results in an additional blue shift with further increase in intensity, consistent with a further decrease in polarity of the environment of the cysteine. Modification with methylmethane thiosulfonate or GSSG results in a small decrease in quantum yield and a red shift in the tryptophan emission spectrum of the modified inhibitor, suggestive of increased polarity of the environment of at least 1 of the 2 tryptophan residues in alpha 1-PI. These changes are reversed by dithiothreitol and are consistent with a conformational change which transforms the inhibitory activity from a rapid, irreversible mode in native alpha 1-PI to a dissociable competitive mode in the mixed disulfide derivatives.     

Exposed tyrosine residues of lambda cro repressor protein evidenced by nitration and photo CIDNP experiments

The tyrosine residues of lambda cro repressor were partially nitrated with tetranitromethane under mild conditions. After digestion by Achromobacter protease I, the extent of nitration was determined by HPLC and amino acid analysis. Tyr 26 was most easily nitrated and Tyr 51 followed it. Tyr 10 was resistant to nitration. By comparison of the proton magnetic resonance spectrum of the partially nitrated cro protein with the above result, the aromatic proton resonances of the tyrosine side chains could be assigned to individual tyrosine residues. The extent of nitration is parallel to the accessibility to a flavin dye as measured by photo CIDNP (chemically induced dynamic nuclear polarization).