Mechanism of peroxynitrite interaction with cytochrome c

Kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate was studied. It was found that the heme iron in ferric cytochrome c does not react directly with peroxynitrite. The rates of the absorbance changes in the Soret region of cytochrome c spectrum caused by peroxynitrite or peroxynitrite/bicarbonate were the same as the rate of spontaneous isomerization of peroxynitrite or as the rate of the reaction of peroxynitrite with bicarbonate, respectively. This means that intermediate products of peroxynitrite decomposition, OH/ NO2 or, in the presence of bicarbonate, CO3 – / NO2, are the species responsible for the absorbance changes in the Soret band of cytochrome c. Modifications of the heme center of cytochrome c by radiolytically produced radicals, OH, NO2 or CO3 – , were also studied. The absorbance changes in the Soret band caused by radiolytically produced OH or CO3 – were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals. NO2 produced radiolytically did not interact with the heme center of cytochrome c. Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced OH, NO2 or CO3 – radicals. This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H2O2 with the heme iron of cytochrome c, followed by reaction with the second substrate.

for the absorbance changes in the Soret band of cytochrome c.Modifications of the heme center of cytochrome c by radiolytically produced radicals, × OH, × NO 2 or CO 3 -× , were also studied.The absorbance changes in the Soret band caused by radiolytically produced × OH or CO 3 -× were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals.
× NO 2 produced radiolytically did not interact with the heme center of cytochrome c.Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced × OH, × NO 2 or CO 3 -× radicals.This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H 2 O 2 with the heme iron of cytochrome c, followed by reaction with the second substrate.
Cytochrome c is a heme protein which participates in electron transfer processes in biomembranes.The heme group, located in the center of the protein is covalently bound to the polypeptide backbone by Cys-14 and Cys-17.The heme iron is coordinated by His-18 and Met-80.The methionine ligand is dissociated from the heme iron in all nonnative states (Oellerich et al., 2002).Cytochrome c, in the native form, displays a very weak peroxidase-like activity (Radi et al., 1991).This activity is enhanced when cytochrome c is partially unfolded or oxidized (Hamachi et al., 1994;Gêbicka, 2001;Diederix et al., 2002;Chen et al., 2002).
Recently Cassina et al. (2000) reported nitration of the heme-vicinal Tyr-67 of ferric cytochrome c treated by an excess of peroxynitrite.Peroxynitrite-treated cytochrome c displayed an increased peroxidatic activity (Cassina et al., 2000) and was less active in the electron transfer reaction in mitochondria (Cassina et al., 2000;Nakagawa et al., 2001).It seems, however, that in contrast to the ferrous heme of cytochrome c (Thomson et al., 1995) the ferric heme of cytochrome c is not an important target for peroxynitrite (Cassina et al., 2000;Nakagawa et al., 2001;Pietraforte et al., 2001).
In this study we investigated kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate (HCO 3 -/CO 2 ).As we found that, in contrast to many hemoproteins, absorbance changes of the Soret band of cytochrome c were induced by intermediate radical products of peroxynitrite decomposition, reactions of cytochrome c with radiolytically produced hydroxyl, nitrogen dioxide and carbon trioxide radicals were also studied.

MATERIALS AND METHODS
Peroxynitrite was prepared by the method described by Pryor et al., (1995).To reduce contamination with unreacted azide, ozona-tion was continued for 15 min after obtaining the maximum concentration of peroxynitrite (Uppu et al., 1996).The final concentration of peroxynitrite was about 30 mM as measured spectrophotometrically at 302 nm (e = 1670 M -1 s -1 (Hudges & Nicklin, 1968)).Stock solutions were stored at -25°C and used within 3-4 weeks after synthesis.Nitrite concentration in the stock peroxynitrite solution, measured with Griess reagent (Grisham et al., 1996), was about 2 mM and did not change significantly during storage.Ferric cytochrome c from horse heart was obtained from Sigma.An extinction coefficient of 1.06 ´10 5 M -1 cm -1 at 410 nm (Goto et al., 1993) was used for spectrophotometric determination of cytochrome c concentration.All other chemicals were also from Sigma.
The reactions of peroxynitrite with cytochrome c, with bicarbonate or with cytochrome c in the presence of bicarbonate were studied using an Applied Photophysics SX-17 stopped-flow spectrophotometer with a 1-cm cell and mixing time <1 ms.The stock solution of peroxynitrite was diluted to the appropriate concentration with 0.01 M NaOH, cytochrome c and/or bicarbonate were dissolved in 0.2 M phosphate buffer, pH 7.0.Equal volumes of peroxynitrite and cytochrome c and/or bicarbonate were mixed in the cell and the reaction rate was monitored at 302 and 410 nm.
Autodecomposition of peroxynitrite was monitored at 302 nm after mixing peroxynitrite solution with 0.2 M phosphate buffer, pH 7.0.Replicate mixing was performed outside the stopped-flow apparatus to measure the pH of the reaction mixture.The process of mixing increased the pH by 0.1 unit.
The reactions of cytochrome c with hydroxyl, nitrogen dioxide or carbon trioxide radicals were initiated by g-irradiation of N 2 O-satu-rated cytochrome c solution without additives, with 0.1 M NaNO 2 or 0.1 M Na 2 CO 3 , respectively, in 1 mM phosphate buffer (pH was adjusted to 7.0) with a dose rate of 0.025 J kg -1 per second.Radiolysis of water saturated with N 2 O produces 0.54 mol J -1 of hydroxyl radicals (Buxton et al., 1988).Under our experimental conditions the rate of hydroxyl radical formation was 13.5 nM per second.When nitrite is present in N 2 O-saturated water, nitrogen dioxide is formed in the following reaction: Similarly, reaction of × OH with carbonate leads to the formation of CO 3 -: Under conditions where concentrations of these salts are in excess of the concentration of × OH, the yield of formation of × NO 2 and CO 3 -× should be the same as the yield of × OH Absorption spectra and kinetic measurements in long-time regime were taken on a Hewlett-Packard 8452A diode-array spectrophotometer.
All kinetic measurements were carried out at 24±0.5°C.Water from MilliQ Plus was used throughout.

RESULTS AND DISCUSSION
We measured the time-course of peroxynitrite decomposition in the absence and in the presence of cytochrome c at pH 7.1 under conditions when peroxynitrite was in excess.In both cases peroxynitrite decomposed with a rate constant of 0.4 ± 0.05 s -1 (not shown).This means that cytochrome c does not catalyze decomposition of peroxynitrite.We checked that addition of an up to 70-fold excess of peroxynitrite to cytochrome c did not result in measurable changes of absorbance in the Soret band of cytochrome c, whereas larger excess of the oxidant led to a decrease of the intensity of the Soret peak.We measured the kinetics of the absorbance changes in the Soret region of cytochrome c (410 nm) induced by different amounts of peroxynitrite and found that the rate of the absorbance changes at 410 nm was the same as the rate of spontaneous peroxynitrite decay, but the yield of bleaching increased with the increase of peroxynitrite concentration (Fig. 1).When peroxynitrite was in a 1000-fold excess over cytochrome c, the decrease of absorbance at 410 nm was about 25%.These results suggest that intermediate products of ONOOH decomposition ( × OH and/or × NO 2 ) are the species responsible for the absorbance changes of the Soret band of cytochrome c.
Similar experiments were done in the presence of an excess of bicarbonate.From the slope of the plot describing the dependence of the pseudo-first order rate constant of peroxynitrite decomposition on the concentration of bicarbonate at pH 7.1, k obs was calculated to be 885 M -1 s -1 (not shown).The absorbance changes in the Soret band of cytochrome c caused by peroxynitrite/bicarbonate were significantly faster than those caused by peroxynitrite alone and depended on the concentration of bicarbonate.From the slope of the plot of the pseudo-first order rate constant of the Soret band bleaching of cytochrome c versus bicarbonate concentration, the second order rate constant equal to 900 M -1 s -1 was obtained (Fig. 2).This value is close to the rate constant of the reaction of peroxynitrite with bicarbonate at pH 7.1.This means that the rate-determining step in the reaction(s) leading to the decrease of intensity of the Soret band of cytochrome c by peroxynitrite/bicarbonate is the formation of the radical pair × NO 2 /CO 3 -× .The yield of bleaching of the Soret band by peroxynitrite/bicarbonate was comparable to that caused by the same amount of peroxynitrite alone.We found, in accordance with Cassina et al. (2000), that the peroxidatic activity of cytochrome c treated with peroxynitrite increased with the increase of the concentration of peroxynitrite.When peroxynitrite was in an almost 1000-fold excess, we observed an about threefold increase in the peroxidase-like activity of cytochrome c (Table 1).Cytochrome c incubated with decomposed peroxynitrite (added to the 0.2 M buffer 5 min.prior to reaction with cytochrome c) displayed as low peroxidatic activity as the native protein.The increase of the peroxidatic activity of cytochrome c treated, prior to activity measurements, with peroxynitrite/bicarbonate (bicarbonate was in excess relative to peroxynitrite) was lower than that observed for cytochrome c modified with peroxynitrite alone (Table 1).We checked that bicarbonate itself, when present in reaction medium, did not influence the peroxidatic activity of native cytochrome c.
It has been suggested that the increase of peroxidatic activity of peroxynitrite-treated cytochrome c is related to nitration of Tyr-67, because cytochrome c pretreated with tetranitromethane (a nitrating agent) becomes more sensitive to H 2 O 2 -mediated inactivation (Cassina et al., 2000).On the other hand, it was shown that only a small fraction of nitrogen dioxide formed during peroxynitrite decomposition was able to nitrate tyrosine residues in cytochrome c (Cassina et al., 2000;Nakagawa et al., 2001).It is known that the presence of bicarbonate in the system en-hances the yield of peroxynitrite-induced nitration (Gow et al., 1996;Yiermilov et al., 1996;Lemercier et al., 1997).Our results showed that peroxynitrite in the absence and in the presence of bicarbonate caused similar absorbance changes in the Soret band of cytochrome c, whereas a higher increase of the peroxidatic activity of cytochrome c was observed after its reaction with peroxynitrite alone (Table 1).This means that the increase of the peroxidatic activity of cytochrome c after peroxynitrite treatment is not only due to tyrosine nitration but also to oxidation of other amino-acid residues.This leads to such a modification of protein structure which facilitates the reaction of H 2 O 2 with heme iron of cytochrome c, followed by reaction with the assay substrate (ABTS).
In order to check what is the role of each of the radical intermediates in bleaching of the Soret band of cytochrome c and in the increase of the peroxidatic activity of this protein, we analyzed the absorption spectra and Table 1.Peroxidatic activity of cytochrome c, modified by several reactants, measured as the rate of ABTS oxidation at 700 nm.
[cytochrome c] = 2.7 mM; [ABTS] = 9 mM; [H 2 O 2 ] = 5 mM.peroxidatic activity of cytochrome c after reaction with radiolytically generated hydroxyl, nitrogen dioxide or carbon trioxide radicals.We found that under conditions when × OH or CO 3 -× radicals were the reactive species, the intensity of the Soret band of cytochrome c decreased with the dose (Fig. 3).The faster decrease of cytochrome c absorbance at 410 nm caused by CO 3 -× may be explained by the more selective action of CO 3 -× than × OH radicals.Hydroxyl radicals react with all amino acids with rate constants higher than 10 7 M -1 s -1 (Buxton et al., 1988) and hence the aminoacid residues located on the surface of the protein are primary targets for × OH attack.Hydroxyl radicals add to aromatic, or abstract H-atom from aliphatic amino-acid residues.Then, transfer of the radical site to the inner part of the protein molecule may take place.In the case of some hemoproteins (among others, cytochrome c) redox transformation of the heme center was observed as a result of × OH-induced radical transfer processes (Simic & Taub, 1977;Whitburn et al., 1982;Gêbicka & Gêbicki, 1992).Oxidation of proteins by CO 3 -× is expected to mainly concern tryptophan, cysteine, tyrosine and methionine residues (Neta et al., 1988), i.e. the residues located in the vicinity of the heme group of cytochrome c (Yeh et al., 1998;Cassina et al., 2000).On the other hand, × NO 2 radicals formed under our experimental conditions did not cause any absorption changes in the Soret region of cytochrome c.
The peroxidatic activity of cytochrome c irradiated under conditions when × OH or CO 3 -× were formed increased significantly.For example, the peroxidase-like activity of cytochrome c treated with 73 mM × OH or CO 3 -× increased about 21-and 19-fold, respectively.After the same time of irradiation, the peroxidatic activity of cytochrome c exposed to × NO 2 radicals increased only 1.5-fold.Nitrogen dioxide is known to oxidize cysteine, tryptophan and tyrosine.In the case of tryptophan and tyrosine, the rate constant of the reaction with × NO 2 is about two to three orders of magnitude lower than with CO 3 -× and × OH (Buxton et al., 1988;Neta et al., 1988).On the other hand, the rate of dimerization of × NO 2 is of the order of 10 8 M -1 s -1 (Neta et al.,1988) and, as a consequence, the protein damage caused by × NO 2 should be significantly lower than that caused by the same concentration of CO 3 -× or × OH.It has been shown (Prütz et al., 1985) that tyrosine is nitrated in the presence of × NO 2 .In the first step phenoxyl radicals are formed, which then react further with × NO 2 to form nitrotyrosine.It should be stressed that the nitrating power of nitrogen dioxide is significantly enhanced in the presence of × OH and CO 3 -× .This is due to the fact that × OH and CO 3 -× very quickly oxidize tyrosine to phenoxyl radical, which then recombines with × NO 2 with a diffusion-controlled rate constant (Prütz et al., 1985;Augusto et al., 2002).
The increase of the peroxidatic activity of cytochrome c caused by radiolytically produced × OH or CO 3 -× is much more significant than that observed after peroxynitrite treatment (more than 300 mM × OH should be formed during decomposition of 1 mM peroxynitrite -see above).This means that modification of the cytochrome c molecule by × OH and CO 3 -× radicals produced homoge- neously during water radiolysis is more pronounced than that caused by radicals formed during decomposition of peroxynitrite under our experimental conditions.In the latter case, × OH/ × NO 2 or CO 3 -× / × NO 2 radicals are formed in a cage, and even after escape, some of them may recombine.Additionally, a fraction of × OH or CO 3 -× radicals produced during peroxynitrite decomposition may react with nitrite (a contaminant presents in peroxynitrite solution).Thus the concentration of × OH and CO 3 -× radicals which attack the cytochrome c molecule is probably much smaller than that estimated from peroxynitrite concentration.Summing up, our results show that, in contrast to many other hemoproteins, the heme iron in cytochrome c does not react directly with peroxynitrite.Intermediate radical prodcts of peroxynitrite decomposition, × OH/ × NO 2 , or in the presence of bicarbonate, CO 3 -× / × NO 2 , are the species responsible for the absorbance changes in the Soret region of cytochrome c.These changes probably reflect structural modifications in the vicinity of the heme, although damage of the heme center by × OH or CO 3 -× is also possible.It seems that nitrogen dioxide does not react with the heme iron of cytochrome c.Peroxynitrite treated cytochrome c exhibits an increased peroxidase-like activity.This means that modification of protein structure (oxidation of amino acids and/or tyrosine nitration) facilitates reaction of H 2 O 2 with the heme iron of cytochrome c.