Involvement of protein radical, protein aggregation, and effects on NO metabolism in the hypochlorite-mediated oxidation of mitochondrial cytochrome c

Cytochrome c (cyt c)-derived protein radicals, radical adduct aggregates, and protein tyrosine nitration have been implicated in the pro-apoptotic event connecting inflammation to the development of diseases. During inflammation, one of the reactive oxygen species metabolized via neutrophil activation is hypochlorite (HOCl); destruction of the mitochondrial electron transport chain by hypochlorite is considered to be a damaging factor. Previous study has shown that HOCl induces the site-specific oxidation of cyt c at met-80. In this work, we have assessed the hypothesis that exposure of cyt c to physiologically relevant concentrations of HOCl leads to protein-derived radical and consequent protein aggregation, which subsequently affects cyt c's regulation of nitric oxide metabolism. Reaction intermediates, chemical pathways available for protein aggregation, and protein nitration were examined. A weak ESR signal for immobilized nitroxide derived from the protein was detected when a high concentration of cyt c was reacted with hypochlorite in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane. When a low concentration of cyt c was exposed to the physiologically relevant levels of HOCl in the presence of 5,5-dimethyl-pyrroline N-oxide (DMPO), we detected DMPO nitrone adducts derived from both protein and protein aggregate radicals as assessed by Western blot using an antibody raised against the DMPO nitrone adduct. The cyt c-derived protein radicals formed by HOCl were located on lysine and tyrosine residues, with lysine predominating. Cyt c-derived protein aggregates induced by HOCl involved primarily lysine residues and hydrophobic interaction. In addition, HOCl-oxidized cyt c (HOCl-cyt c) exhibited a higher affinity for NO and enhancement of nonenzymatic NO synthesis from nitrite reduction. Furthermore, HOCl-mediated cyt c oxidation also resulted in a significant elevation of cyt c nitration derived from either NO trapping of the cyt c-derived tyrosyl radical or cyt c-catalyzed one-electron oxidation of nitrite.     

Protein oxidation of cytochrome C by reactive halogen species enhances its peroxidase activity

Reactive halogen species (RHS; X(2) and HOX, where X represents Cl, Br, or I) are metabolites mediated by neutrophil activation and its accompanying respiratory burst. We have investigated the interaction between RHS and mitochondrial cytochrome c (cyt c) by using electrospray mass spectrometry and electron spin resonance (ESR). When the purified cyt c was reacted with an excess amount of hypochlorous acid (HOCl) at pH 7.4, the peroxidase activity of cyt c was increased by 4.5-, 6.9-, and 8.6-fold at molar ratios (HOCl/cyt c) of 2, 4, and 8, respectively. In comparison with native cyt c, the mass spectra obtained from the HOCl-treated cyt c revealed that oxygen is covalently incorporated into the protein as indicated by molecular ions of m/z = 12,360 (cyt c), 12,376 (cyt c + O), and 12,392 (cyt c + 2O). Using tandem mass spectrometry, a peptide (obtained from the tryptic digests of HOCl-treated cyt c) corresponding to the amino acid sequence MIFAGIK, which contains the methionine that binds to the heme, was identified to be involved in the oxygen incorporation. The location of the oxygen incorporation was unequivocally determined to be the methionine residue, suggesting that the oxidation of heme ligand (Met-80) by HOCl results in the enhancement of peroxidase activity of cyt c. ESR spectroscopy of HOCl-oxidized cyt c, when reacted with H(2)O(2) in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), yielded more immobilized MNP/tyrosyl adduct than native cyt c. In the presence of H(2)O(2), the peroxidase activity of HOCl-oxidized cyt c exhibited an increasing ability to oxidize tyrosine to tyrosyl radical as measured directly by fast flow ESR. Titration of both native cyt c and HOCl-oxidized cyt c with various amounts of H(2)O(2) indicated that the latter has a decreased apparent K(m) for H(2)O(2), implicating that protein oxidation of cyt c increases its accessibility to H(2)O(2). HOCl-oxidized cyt c also displayed an impaired ability to support oxygen consumption by the purified mitochondrial cytochrome c oxidase, suggesting that protein oxidation of cyt c may break the electron transport chain and inhibit energy transduction in mitochondria.     

Peroxynitrite induces long-lived tyrosyl radical(s) in oxyhemoglobin of red blood cells through a reaction involving CO2 and a ferryl species

Peroxynitrite-mediated oxidative chemistry is currently the subject of intense investigation owing to the toxic side effects associated with nitric oxide overproduction. Using direct electron spin resonance spectroscopy (ESR) at 37 degrees C, we observed that in human erythrocytes peroxynitrite induced a long-lived singlet signal at g = 2.004 arising from hemoglobin. This signal was detectable in oxygenated red blood cells and in purified oxyhemoglobin but significantly decreased after deoxygenation. The formation of the g = 2.004 radical required the presence of CO2 and pH values higher than the pKa of peroxynitrous acid (pKa = 6.8), indicating the involvement of a secondary oxidant formed in the interaction of ONOO- with CO2. The g = 2.004 radical yield leveled off at a 1:1 ratio between peroxynitrite and oxyhemoglobin, while CO-hemoglobin formed less radical and methemoglobin did not form the radical at all. These results suggest that the actual oxidant is or is derived from the ONOOCO2- adduct interacting with oxygenated FeII-heme. Spin trapping with 2-methyl-2-nitrosopropane (MNP) of the g = 2.004 radical and subsequent proteolytic digestion of the MNP/hemoglobin adduct revealed the trapping of a tyrosyl-centered radical(s). A similar long-lived unresolved g = 2.004 singlet signal is a common feature of methemoglobin/H2O2 and metmyoglobin/H2O2 systems. We show by spin trapping that these g = 2.004 signals generated by H2O2 also indicated trapping of radicals centered on tyrosine residues. Analysis of visible spectra of hemoglobin treated with peroxynitrite revealed that, in the presence of CO2, oxyhemoglobin was oxidized to a ferryl species, which rapidly decayed to lower iron oxidation states. The g = 2.004 radical may be an intermediate formed during ferrylhemoglobin decay. Our results describe a new pathway of peroxynitrite-dependent hemoglobin oxidation of dominating importance in CO2-containing biological systems and identify the g = 2.004 radical(s) formed in the process as tyrosyl radical(s).     

A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide

The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)O(2) were calculated using hyperfine coupling constants of a(H2,6) = 1.3 G and a(H3,5) = 7.0 G for the ring and a(beta)(H1) = 16.7 G and a(beta)(H2) = 14.2 G for the methylene protons, and are consistent with a highly constrained, conformationally unstable tyrosyl radical. Spectra obtained at later time points contained a mixture of the 3 s signal and another signal that was insufficiently resolved for simulation. Efficient spin trapping with 3, 5-dibromo-4-nitrosobenzenesulfonic acid was observed only when the spin trap was present at the time of H(2)O(2) addition. Spin trapping experiments with either 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or perdeuterated 2-methyl-2-nitrosopropane (MNP-d(9)), which have been shown to trap tyrosyl radicals, were nearly equally effective when the spin trap was added before or 10 min after the addition of H(2)O(2). The superhyperfine structure of the ESR spectra obtained from Pronase-treated MNP-d(9)/*metMb confirmed the assignment to a tyrosyl radical. Delayed spin trapping experiments with site-directed mutant myoglobins in which either Tyr-103 or Tyr-146 was replaced by phenylalanine indicated that radical adduct formation with either DMPO or MNP-d(9) requires the presence of Tyr-103 at all time points, implicating that residue as the radical site.     

Tyrosinase scavenges tyrosyl radical

Melanosomes scavenged tyrosyl radical that was generated by ultraviolet irradiation of tyrosine. Purified mushroom tyrosinase also removed tyrosyl radical in a dose-dependent manner. To elucidate the underlying mechanism, we analyzed the reaction of mushroom tyrosinase with tyrosyl radical generated by horseradish peroxidase and hydrogen peroxide. Resting tyrosinase, which contained a small amount of oxytyrosinase, did not oxidize tyrosine to DOPAchrome until horseradish peroxidase exhausted H(2)O(2) and thereafter the enzyme recovered its full activity. During the inhibition period most tyrosine was converted to dityrosine, suggesting that only a small amount of tyrosyl radical was enough to interact with a fraction of tyrosinase which was in the active oxy-form. When horseradish peroxidase and H(2)O(2) were added to oxytyrosinase, which was prepared by allowing it to turn over beforehand, DOPAchrome production was abolished with an accelerated consumption of H(2)O(2). Dityrosine formation was totally suppressed and tyrosine concentration stayed constant during the inhibition period with a concomitant production of O(2). The results are accounted for by a mechanism in which tyrosyl radical is reduced to tyrosine by oxytyrosinase and the resulting met-form reacts with H(2)O(2) to return to the oxy-form.     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

Intramolecular electron transfer between tyrosyl radical and cysteine residue inhibits tyrosine nitration and induces thiyl radical formation in model peptides treated with myeloperoxidase, H2O2, and NO2-: EPR SPIN trapping studies

We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H(2)O(2), and nitrite anion (NO(2)(-)) and with horseradish peroxidase and H(2)O(2). Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Ala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAAAC). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H(2)O(2) and MPO/H(2)O(2)/NO(2)(-). The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH(3)) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H(2)O(2)/NO(2)(-). However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H(2)O(2)/NO(2)(-)/YCSSCH(3) system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H(2)O(2)/(*)NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed.     

Tyrosine iminoxyl radical formation from tyrosyl radical/nitric oxide and nitrosotyrosine

The quenching of the Y(D)(.) tyrosyl radical in photosystem II by nitric oxide was reported to result from the formation of a weak tyrosyl radical-nitric oxide complex (Petrouleas, V., and Diner, B. A. (1990) Biochim. Biophys. Acta 1015, 131-140). This radical/radical reaction is expected to generate an electron spin resonance (ESR)-silent 3-nitrosocyclohexadienone species that can reversibly regenerate the tyrosyl radical and nitric oxide or undergo rearrangement to form 3-nitrosotyrosine. It has been proposed that 3-nitrosotyrosine can be oxidized by one electron to form the tyrosine iminoxyl radical (>C=N-O*). This proposal was put forth as a result of ESR detection of the iminoxyl radical intermediate when photosystem II was exposed to nitric oxide (Sanakis, Y., Goussias, C., Mason, R. P., and Petrouleas, V. (1997) Biochemistry 36, 1411-1417). A similar iminoxyl radical was detected in prostaglandin H synthase-2 (Gunther, M. R., Hsi, L. C., Curtis, J. F., Gierse, J. K., Marnett, L. J., Eling, T. E., and Mason, R. P. (1997) J. Biol. Chem., 272, 17086-17090). Although the iminoxyl radicals detected in the photosystem II and prostaglandin H synthase-2 systems strongly suggest a mechanism involving 3-nitrosotyrosine, the iminoxyl radical ESR spectrum was not unequivocally identified as originating from tyrosine. We report here the detection of the non-protein L-tyrosine iminoxyl radical generated by two methods: 1) peroxidase oxidation of synthetic 3-nitroso-N-acetyl-L-tyrosine and 2) peroxidase oxidation of free L-tyrosine in the presence of nitric oxide. A newly developed ESR technique that uses immobilized enzyme was used to perform the ESR experiments. Analysis of the high resolution ESR spectrum of the tyrosine iminoxyl radical generated from free tyrosine and nitric oxide reveals a 28.4-G isotropic nitrogen hyperfine coupling and a 2.2-G proton hyperfine coupling assigned to the proton originally ortho to the phenoxyl oxygen.     

Nitric oxide inhibits peroxidase activity of cytochrome c.cardiolipin complex and blocks cardiolipin oxidation

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c.CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO(-), respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c.CL complex, NO chemically reacted with H(2)O(2)-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H(2)O(2)-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H(2)O(2)-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c.CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3'-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1'2,2'-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1'2,2'-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c.CL complexes.     

Excess no predisposes mitochondrial succinate-cytochrome c reductase to produce hydroxyl radical

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O(2)(?-) generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate-cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/(?)OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron-H(2)O(2)-dependent Fenton reaction or O(2)(?-)-dependent Haber-Weiss mechanism. Direct EPR measurement of SCR at 77K indicated the formation of a nonheme iron-NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce (?)OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.     

Tyrosyl free radical formation in the small subunit of mouse ribonucleotide reductase

Each R2 subunit of mammalian ribonucleotide reductase contains a pair of high spin ferric ions and a tyrosyl free radical essential for activity. To study the mechanism of tyrosyl radical formation, substoichiometric amounts of Fe(II) were added to recombinant mouse R2 apoprotein under strictly anaerobic conditions and then the solution was exposed to air. Low temperature EPR spectroscopy showed that the signal from the generated tyrosyl free radical correlated well with the quantity of the Fe(II) added with a stoichiometry of 3 Fe(II) needed to produce 1 tyrosyl radical: 3 Fe(II) + P + O2 + Tyr-OH + H+----Fe(III)O2-Fe(III)-P + H2O. + Tyr-O. + Fe(III), where P is an iron-binding site of protein R2 and Tyr-OH is the active tyrosyl residue. The O-O bond of a postulated intermediate O2(2-)-Fe(III)2-P state is cleaved by the extra electron provided by Fe(II) leading to formation of OH., which in turn reacts with Tyr-OH to give Tyr-O.. In the presence of ascorbate, added to reduce the monomeric Fe(III) formed, 80% of the Fe(II) added produced a radical. The results strongly indicate that each dimeric Fe(III) center during its formation can generate a tyrosyl-free radical and that iron binding to R2 apoprotein is highly cooperative.     

A long-lived o-semiquinone radical anion is formed from N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial substance

N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial peptide, generates hydrogen peroxide (H(2)O(2)) that exerts antitumour activity. We have investigated the precise mechanism of H(2)O(2) production from 5-S-GAD by autoxidation aiming to understand its action toward tumour cells. Using the electron spin resonance (ESR) technique, we detected a strong signal due to radical formation from 5-S-GAD. Surprisingly, the ESR signal of the radical derived from 5-S-GAD appeared after incubation for 30 min at 37 degrees C in the buffer at pH 7.4; the signal was persistently detected for 10 h in the absence of catalytic metal ions. The computer simulation of the observed ESR spectrum together with the theoretical calculation of the spin density of the radical species indicates that an o-semiquinone radical anion was formed from 5-S-GAD. We demonstrated that H(2)O(2) is produced via the formation of superoxide anion O2(.-) by the electron-transfer reduction of molecular oxygen by the 5-S-GAD anion, which is in equilibrium with 5-S-GAD in the aqueous solution. The radical formation and the subsequent H(2)O(2) production were inhibited by superoxide dismutase (SOD), when the antitumour activity of 5-S-GAD was inhibited by SOD. Thus, the formation of the o-semiquinone radical anion would be necessary for the antitumour activity of 5-S-GAD as an intermediate in the production of cytotoxic H(2)O(2).     

Vanadyl-induced Fenton-like reaction in RNA. An ESR and spin trapping study.

Vanadyl (VO2+) complexed to RNA reacts with hydrogen peroxide in a Fenton-like manner producing hydroxyl radicals (.OH). The hydroxyl radicals can be spin trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) forming the DMPO-OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO-ETOH) confirms the production of hydroxyl radicals by the RNA/VO2+ complex. When the reaction between the RNA/VO2+ complex and H2O2 is carried out in the presence of the spin trap 2-methyl-2-nitrosopropane (MNP), radicals produced in the reaction of .OH with RNA are trapped. Base hydrolysis of the MNP-RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well-resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (aN = 1.48 mT, a beta N = 0.25 mT and a beta H = 0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a beta-nitrogen and beta-hydrogen. The results suggest that the .OH generated in the RNA/VO2+ reaction with H2O2 add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.     

Transmembrane nitration of hydrophobic tyrosyl peptides. Localization,characterization, mechanism of nitration,and biological implications

We have shown previously that peroxynitrite-induced nitration of a hydrophobic tyrosyl probe is greater than that of tyrosine in the aqueous phase (Zhang, H., Joseph, J., Feix, J., Hogg, N., and Kalyanaraman, B. (2001) Biochemistry 40, 7675-7686). In this study, we have tested the hypothesis that the extent of tyrosine nitration depends on the intramembrane location of tyrosyl probes and on the nitrating species. To this end, we have synthesized membrane spanning 23-mer containing a single tyrosyl residue at positions 4, 8, and 12. The location of the tyrosine residues in the phospholipid membrane was determined by fluorescence and electron spin resonance techniques. Nitration was initiated by slow infusion of peroxynitrite, co-generated superoxide and nitric oxide ((.)NO), or a myeloperoxidase/hydrogen peroxide/nitrite anion (MPO/H(2)O(2)/NO(2)(-)) system. Results indicate that with slow infusion of peroxynitrite, nitration of transmembrane tyrosyl peptides was much higher (10-fold or more) than tyrosine nitration in aqueous phase. Peroxynitrite-dependent nitration of tyrosyl-containing peptides increased with increasing depth of the tyrosyl residue in the bilayer. In contrast, MPO/H(2)O(2)/ NO(2)(-)-induced tyrosyl nitration decreased with increasing depth of tyrosyl residues in the membrane. Transmembrane nitrations of tyrosyl-containing peptides induced by both peroxynitrite and MPO/H(2)O(2)/NO(2)(-) were totally inhibited by (.)NO that was slowly released from spermine NONOate. Nitration of peptides in both systems was concentration-dependently inhibited by unsaturated fatty acid. Concomitantly, an increase in lipid oxidation was detected. A mechanism involving (.)NO(2) radical is proposed for peroxynitrite and MPO/H(2)O(2)/NO(2)(-)-dependent transmembrane nitration reactions.     

Biochemical properties of cytochrome c nitrated by peroxynitrite

Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.     

Histidinyl radical formation in the self-peroxidation reaction of bovine copper-zinc superoxide dismutase.

In the absence of suitable oxidizable substrates, the peroxidase reaction of copper-zinc superoxide dismutase (SOD) oxidizes SOD itself, ultimately resulting in its inactivation. A SOD-centered free radical adduct of 2-methyl-2-nitrosopropane (MNP) was detected upon incubation of SOD with the spin trap and a hydroperoxide (either H(2)O(2) or peracetic acid). Proteolysis by Pronase converted the anisotropic electron paramagnetic resonance (EPR) spectrum of MNP/(center dot)SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine + HO(center dot)) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/(center dot)SOD, suggesting that the latter was centered on a histidine residue. An additional hyperfine coupling was detected when histidine specifically (13)C-labeled at C-2 of the imidazole ring was used, providing evidence for trapping at that atom. All of the experimental spectra were convincingly simulated assuming hyperfine couplings to 2 nearly equivalent nitrogen atoms and 2 different protons, also consistent with trapping at C-2 of the imidazole ring. Free histidinyl radical consumed oxygen, implying peroxyl radical formation. MNP-inhibitable oxygen consumption was also observed when cuprous SOD but not cupric SOD was added to a H(2)O(2) solution. Formation of 2-oxohistidine, the stable product of the SOD-hydroperoxide reaction, required oxygen and was inhibited by MNP. These results support formation of a transient SOD-peroxyl radical.     

Immunochemical detection of nitric oxide and nitrogen dioxide trapping of the tyrosyl radical and the resulting nitrotyrosine in sperm whale myoglobin

We demonstrate herein that nitric oxide (*NO) and nitrogen dioxide (*NO2) both react with the tyrosyl radical formed in sperm whale myoglobin (swMb) by reaction with hydrogen peroxide. The tyrosyl radical was detected by Western blotting using a novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts. In the presence of DMPO, hydrogen peroxide reacts with swMb to form the DMPO tyrosyl radical as is known from both electron spin resonance and immuno-spin trapping investigations. Both *NO and NO2- significantly suppressed DMPO-Mb formation under the physiological oxygen tension of 30 mm Hg. If this inhibition of DMPO trapping of the tyrosyl radical is due, at least in part, to the reaction of the tyrosyl radical with *NO and *NO2, then nitrotyrosine should be formed. In line with this expectation, swMb treated with low concentrations of *NO or NO2- formed nitrotyrosine when hydrogen peroxide was added under 30 mm Hg oxygen tension as detected by Western blotting. The amount of nitrotyrosine generated with *NO was higher than with NO2-, implying that there are two different peroxynitrite-independent nitrotyrosine formation mechanisms and that *NO is not just a source of *NO2.     

Cytochrome c: a catalyst and target of nitrite-hydrogen peroxide-dependent protein nitration

Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide (*NO), with peroxynitrite (ONOO-) and leukocyte peroxidase-derived nitrogen dioxide (*NO2) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO2-) and hydrogen peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and *NO2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and NO2-, with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.     

Peroxidation and externalization of phosphatidylserine associated with release of cytochrome c from mitochondria

Production of reactive oxygen species (ROS) during apoptosis is associated with peroxidation of phospholipids particularly of phosphatidylserine (PS). The mechanism(s) underlying preferential PS oxidation are not well understood. We hypothesized that cytochrome c (cyt c) released from mitochondria into cytosol acts as a catalyst that utilizes ROS generated by disrupted mitochondrial electron transport for PS oxidation. Selectivity of PS oxidation is achieved via specific interactions of positively charged cyt c with negatively charged PS. To test the hypothesis we employed temporary transfection of Jurkat cells with a pro-apoptotic peptide, DP1, a conjugate consisting of a protein transduction domain, PTD-5, and an antimicrobial domain, KLA [(KLAKLAK)2], known to selectively disrupt mitochondria. We report that treatment of Jurkat cells with DP1 yielded rapid and effective release of cyt c from mitochondria and its accumulation in cytosol accompanied by production of H2O2. Remarkably, this resulted in selective peroxidation of PS while more abundant phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) remained nonoxidized. Neither PTD-5 alone nor KLA alone exerted any effect on PS peroxidation. Redox catalytic involvement of cyt c in PS oxidation was further supported by our data demonstrating that: (i) specific interactions of cyt c with PS resulted in the formation of EPR-detectable protein-centered tyrosyl radicals of cyt c upon its interaction with H2O2 in the presence of PS-containing liposomes, and (ii) integration of cyt c into cytochrome c null (Cyt c -/-) cells or HL-60 cells specifically stimulates PS oxidation in the presence of H2O2 or t-BuOOH, respectively. We further demonstrated that DP1 elicited externalization of PS on the surface of Jurkat cells and enhanced their recognition and phagocytosis by J774A.1 macrophages. Our results are compatible with the hypothesis that catalysis of selective PS oxidation during apoptosis by cytosolic cyt c is important for PS-dependent signaling pathways such as PS externalization and recognition by macrophage receptors.     

Mass spectral analysis of protein-based radicals using DBNBS. Nonradical adduct formation versus spin trapping

Protein-based radicals generated in the reaction of ferricytochrome c (cyt c) with H(2)O(2) were investigated by electrospray mass spectrometry (ESI-MS) using 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS). Up to four DBNBS-cyt c adducts were observed in the mass spectra. However, by varying the reaction conditions (0-5 molar equivalents of H(2)O(2) and substituting cyt c with its cyanide adduct which is resistant to peroxidation), noncovalent DBNBS adduct formation was inferred. Nonetheless, optical difference spectra revealed the presence of a small fraction of covalently trapped DBNBS. To probe the nature of the noncovalent DBNBS adducts, the less basic proteins, metmyoglobin (Mb) and alpha-lactalbumin, were substituted for cyt c in the cyt c/H(2)O(2)/DBNBS reaction. A maximum of two DBNBS adducts were observed in the mass spectra of the products of the Mb/H(2)O(2)/DBNBS reactions, whereas no adducts were detected following alpha-lactalbumin/H(2)O(2)/DBNBS incubation, which is consistent with adduct formation via spin trapping only. Titration with DBNBS at pH 2.0 yielded noncovalent DBNBS-cyt c adducts and induced folding of acid-denatured cyt c, as monitored by ESI-MS and optical spectroscopy, respectively. Thus, the noncovalent DBNBS-cyt c mass adducts observed are assigned to ion pair formation occurring between the negatively charged sulfonate group on DBNBS and positively charged surface residues on cyt c. The results reveal the pitfalls inherent in using mass spectral data with negatively charged spin traps such as DBNBS to identify sites of radical formation on basic proteins such as cyt c.