Modulation of Homocysteine Toxicity by S ... - ACS Publications

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J. Phys. Chem. B 2010, 114, 9894–9904

Modulation of Homocysteine Toxicity by S-Nitrosothiol Formation: A Mechanistic Approach Moshood K. Morakinyo, Robert M. Strongin, and Reuben H. Simoyi* Department of Chemistry, Portland State UniVersity, Portland, Oregon 97207-0751 ReceiVed: April 23, 2010

The metabolic conversion of homocysteine (HCYSH) to homocysteine thiolactone (HTL) has been reported as the major cause of HCYSH pathogenesis. It was hypothesized that inhibition of the thiol group of HCYSH by S-nitrosation will prevent its metabolic conversion to HTL. The kinetics, reaction dynamics, and mechanism of reaction of HCYSH and nitrous acid to produce S-nitrosohomocysteine (HCYSNO) was studied in mildly to highly acidic pHs. Transnitrosation of this non-protein-forming amino acid by S-nitrosoglutathione (GSNO) was also studied at physiological pH 7.4 in phosphate buffer. In both cases, HCYSNO formed quantitatively. Copper ions were found to play dual roles, catalyzing the rate of formation of HCYSNO as well as its rate of decomposition. In the presence of a transition-metal ions chelator, HCYSNO was very stable with a halflife of 198 h at pH 7.4. Nitrosation by nitrous acid occurred via the formation of more powerful nitrosating agents, nitrosonium cation (NO+) and dinitrogen trioxide (N2O3). In highly acidic environments, NO+ was found to be the most effective nitrosating agent with a first-order dependence on nitrous acid. N2O3 was the most relevant nitrosating agent in a mildly acidic environment with a second-order dependence on nitrous acid. The bimolecular rate constants for the direct reactions of HCYSH and nitrous acid, N2O3, and NO+ were 9.0 × 10-2, 9.50 × 103, and 6.57 × 1010 M-1 s-1, respectively. These rate constant values agreed with the electrophilic order of these nitrosating agents: HNO2 < N2O3 < NO+. Transnitrosation of HCYSH by GSNO produced HCYSNO and other products including glutathione (reduced and oxidized) and homocysteineglutathione mixed disulfide. A computer modeling involving eight reactions gave a good fit to the observed formation kinetics of HCYSNO. This study has shown that it is possible to modulate homocysteine toxicity by preventing its conversion to a more toxic HTL by S-nitrosation. Introduction Homocysteine (HCYSH) is a non-protein-forming biologically active organosulfur amino acid. It is primarily produced by the demethylation of the essential, protein-forming amino acid methionine. Under normal conditions, the total plasma concentration of HCYSH usually ranges from 5 to 15 µM. A higher fasting level is described as hyperhomocysteinemia, which is further categorized into moderate and severe hyperhomocysteinemia for the HCYSH levels in the range 16 to 100 µM and >100 µM, respectively.1-3 Research over the decades has established that hyperhomocysteinemia is a risk factor in a variety of disease states including cardiovascular, neurodegenerative, and neural defects.4-7 Hyperhomocysteinemia is believed to arise from abnormal metabolism of methionine particularly in diet with deficiency of B-vitamins such as folate, vitamin B-12, and vitamin B-6.8 In the human body, methionine is used for the synthesis of a universal methyl donor Sadenosylmethionine (AdoMet).9 Upon donation of its methyl group, AdoMet is converted to S-adenosylhomocysteine (AdoHcy), which subsequently hydrolyzes to HCYSH and adenosine.9,10 In the physiological environment, HCYSH levels are regulated by remethylation to methionine by the enzyme methionine synthase in the presence of vitamin B-12 and 5,10methyltetrahydrofolate, and by trans-sulfuration to cystathionine by the enzyme cystathionine β-synthase and vitamin B-6 as cofactor.4,10 * Corresponding author.

It has become evident that an impairment to remethylation and trans-sulfuration metabolic pathways of HCYSH results in an alternative metabolic conversion of HCYSH to a chemically reactive homocysteine thiolactone (HTL).10 The formation of this cyclic thioester HTL is believed to be one of the major factors responsible for the pathogenesis of HCYSH.11 HTL is a highly reactive metabolite that causes N-homocysteinylation of proteins leading to the modification of their structural and physiological functions.12 This means that any process that can prevent the oxidation of HCYSH to HTL will alleviate the toxicity of HCYSH. One of such processes is the S-nitrosation of HCYSH to form S-nitrosohomocysteine (HCYSNO). Unlike HCYSH, HCYSNO does not support ring closure that may lead to the formation of HTL.13 It is therefore plausible to assume that HCYSH toxicity could occur when there is an imbalance between the available nitrosating agents such as nitric oxides (NOs) and the HCYSH levels in the body. A number of nitrosating agents are available in the physiological environment. They include NO and its metabolites such as N2O3, NO2, NO+, acidic nitrite, and S-nitrosothiols (RSNOs).14-16 Protonation of nitrite is readily occurring in the stomach, resulting in the formation of a nitrosating agent, nitrous acid (HNO2).16 NO has been found to be relatively unstable in biological systems because of its reaction with oxygen (O2), superoxide anion (O2 · -), and haem proteins.17 Nitrite (NO2-) is the major end product of its reaction with oxygen, and nitrite can also release NO on acidification.16 NO is an important signaling molecule that is produced endogenously via the oxidation of L-arginine to L-citrulline by the enzymes NO

10.1021/jp103679v  2010 American Chemical Society Published on Web 07/13/2010

Modulation of HCYSH Toxicity by RSNO Formation synthases.15,18,19 It is actively involved in the modification of sexual and aggressive behavior.20 It is also known to inhibit platelet aggregation as well as act as a protective agent against intestinal damage induced by endotoxins.18,21 Accumulated evidence has shown that NO and NO-derived metabolites readily react with thiols (RSH) to form S-nitrosothiols with the general structure RSNO (where R is mainly an organic group).22-25 Apart from performing the same physiological functions as NO,26 RSNOs have been shown to be endogenous reservoirs, transport, and delivery carriers of NO.27 They have been found in human plasma, platelet, and neutrophiles.11,25,28,29 S-nitrosohomocysteine, in particular, has been shown by Glow and coworkers to be an important mediator of vasorelaxation.30 Although some authors have suggested the inhibition of the S-center of HCYSH via the formation of S-nitrosothiols,13,30-33 the mechanism of reactions of NO and NO-derived nitrosating agents with HCYSH to form HCYSNO has, however, not been clearly elucidated. We therefore report in this manuscript on the kinetics and mechanisms of nitrosation of HCYSH by physiologically active nitrosating agents. Experimental Section Materials. DL-Homocysteine (HCYSH) 95%, N-acetyl-Dpenicillamine 99+% (Sigma-Aldrich), cysteine (99+%), sodium perchlorate (Acros), glutathione, sodium nitrite, sodium chloride, perchloric acid (72%), ferrous sulfate (Fisher), cuprous chloride, and cupric chloride (GSF chemicals) were used as purchased. Stock solutions of HCYSH and nitrite were prepared just before use. Water used for preparing reagent solutions was obtained from a Barnstead Sybron Corporation water purification unit capable of producing both distilled and deionized water (Nanopure). Inductively coupled plasma mass spectrometry (ICPMS) was used to evaluate quantitatively the concentrations of a number of metal ions in the water used for our reaction medium. ICPMS analysis showed negligible concentrations of transitionmetal ions, which were inadequate to affect the overall reaction kinetics and mechanism (0.43 ppb in Pb is the highest concentration of metal ions in the reagent water). Methods. All experiments were carried out at 25.0 ( 0.1 °C with a Neslab RTE-101 thermostat bath. Reactions were run at a constant ionic strength of 1.0 M (NaClO4). We monitored the progress of the nitrosation reaction spectrophotometrically by following the absorbance of S-nitrosohomocysteine (HCYSNO) at its experimentally determined absorption peak of 545 nm, where an absorptivity coefficient of 20.20 ( 0.2 M-1 cm-1 was evaluated. Although HCYSNO has a second absorption peak at 331 nm, with a much higher absorptivity coefficient (1040.00 ( 3.00 M-1 cm-1), its maximum absorption at 545 nm was used because of the lack of interference from the absorption peaks of nitrogen species NO2-, NO+, and HNO2 at this absorption wavelength. (See Figure 1). Product identification and verification was achieved by complementary techniques: UV/vis spectrophotometry, HPLC, quadrupole time-of-flight mass spectrometry and EPR spectrometry. Kinetics measurements for fast reactions were performed on a Hi-Tech Scientific double-mixing SF61-DX2 stopped-flow spectrophotometer. For slower reactions, absorbance readings were performed on a Perkin-Elmer Lambda 25 UV/vis spectrophotometer. HPLC Technique. The HPLC system utilized a Shimadzu Prominence (Columbia, MD) SPD-M10A VP diode array detector, dual LC 600 pumps, LPT-6B LC-PC interface controller, SIL-10AD auto injector, and Class-VP chromatography data system software. All samples were loaded on a reversed-phase

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Figure 1. UV/vis spectral scan of reactants and product. (a) 0.005 M HCYSH, (b) 0.005 M NaNO2, (c) 0.005 M HNO2, (d) 0.005 M HCYSNO, and (e) 0.0025 M HCYSNO.

Discovery 5 µm C18 column (Supelco, Bellefonite, PA). They were run isocractically at 0.15% trifluoroacetic acid (TFA)/ H2O-10% methanol/H2O, and HCYSNO was detected at 545 nm (for selective scans) with maximum spectral scan mode employed to detect all eluents. A constant flow rate of 1 mL/ min was maintained. All solutions for HPLC analysis were made with Milli-Q Millipore purified water and filtered with Whatman polypropylene 0.45 µm pore-size filter devices before injection (10 µL) into the column using the auto injector. To curb the interaction of the protonated amines on the analytes with the silanol groups on the stationary phase (which was causing tailing of peaks), the sodium salt of 1-octanesulfonic acid (0.005 M) was incorporated into the aqueous mobile phase. This was sufficient to neutralize the protonated amines and produce good resolution while eliminating peak tailing. Time-of-Flight Mass Spectrometry. Mass spectra were acquired on a Micromass QTOF-II (Waters Corporation, Millford, MA) quadrupole time-of -flight mass spectrometer (qTOF MS). Analytes were dissolved in 50/50 acetnitrile/water-1% formic acid mixture, and analyte ions were generated by positive-mode electrospray ionization (ESI) at a capillary voltage of 2.8 KV and a flow rate of 5 µL/min. The source block was maintained at 80 °C, and the nitrogen desolvation gas was maintained at 150 °C and a flow rate of 400 L/H. Data were visualized and analyzed with the Micromass MassLynx 4.0 software suite for Windows XP (Water Corporation, Millford, MA). EPR Measurements. A Bruker e-scan EPR spectrometer (Xband) was used for the detection of radicals. It was used to confirm the absence of thyl radicals and the release of NO upon the decomposition of HCYSNO. Detection of NO was through the use of the NO-specific spin trap, aci-nitroethane.34 The following settings were used for a typical run: microwave power, 19.91 mW; modulation amplitude, 1.41 G; receiver gain, 448; time constant, 10.24 ms; sweep width, 100 G; and frequency, 9.78 GHz. All measurements were taken at room temperature. Results Stoichiometry Determination. The stoichiometry of reaction between HNO2 and HCYSH was determined as

HNO2 + H2NCH(COOH)CH2CH2SH f H2NCH(COOH)CH2CH2SNO + H2O (R1)

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Figure 2. Mass spectrometry analysis of the product of nitrosation of HCYSH showing (A) pure HCYSH m/z 136.00 and (B) formation of HCYSNO m/z ) 164.87.

We performed reactions run for the deduction of this stoichiometry by varying the amount of HNO2 while keeping the amount of HCYSH constant. We generated HNO2 by mixing equimolar amounts of perchloric acid and sodium nitrite. The reaction was allowed to proceed to completion while rapidly scanning the reacting solution between 400 and 700 nm. The concentration of the product HCYSNO was deduced from its maximum absorption at 545 nm. This stoichiometry is the highest HNO2/HCYSH ratio that produced the maximum amount of HCYSNO. No nitrosation reaction at the N-center of homocysteine was observed. All nitrosation occurs at the S-center because it is more nucleophilic than the 2° N-center. Product Identification. Mass spectrometry was used to identify conclusively the products of the nitrosation reactions. In Figure 2, spectrum A is from pure DL-homocysteine. Spectrum B, from the product of the reaction, shows a HCYSNO m/z peak at 164.87, which is the expected peak from the positive mode of the electronspray ionization mass spectrometry technique used for this analysis. The production of NO from the decomposition of HCYSNO (reaction R2) was detected by EPR techniques through the use of the aci anion of nitroethane as a spin trap.34 In alkaline environments, nitroethane (CH3CH2NO2) forms aci anion (reac-

tion R3), which can effectively scavenge NO to form a longlived NO adduct (reaction R4).

HCYSNO f HCYSSHCY + NO

(R2)

CH3CH2NO2 + OH- f CH3CHdNO2- + H2O

(R3) CH3CHdNO2- + NO + OH- f [CH3C(NO)(NO2)] · 2- + H2O (R4) Direct detection of NO without a spin trap is difficult because of its broad ESR spectrum.35 Figure 3 shows a series of ESR spectra that confirm the production of NO from the decomposition of HCYSNO. The splitting constant is consistent with that expected for a typical aci-nitroethane-NO adduct. All spectra were scaled on the same X-Y coordinate range and run under the same conditions. Spectrum A shows that aci nitroethane on its own does not show any EPR peaks in the absence of NO. Spectra B and C show nitroethane with varying concentrations

Modulation of HCYSH Toxicity by RSNO Formation


Figure 3. EPR spectra of NO radical generated during the decomposition of HCYSNO using 0.5 M nitroethane (NE) in 1.0 M NaOH solution as spin trap. (A) 0.5 M NE, (B) 0.03 M HCYSNO, (C) 0.05 M HCYSNO, and (D) 0.03 M HCYSNO + 2.0 × 10-4 M Cu2+.

of HCYSNO, in which the peak height increases with an increase in HCYSNO concentration. Spectrum D has equal concentrations of HCYSNO as in spectrum B but with the addition of copper. It clearly demonstrates the catalytic effect of copper in effecting NO release from S-nitrosothiols. More NO is released in D than in B within the same reaction time, as reflected by the peak height, which is directly proportional to the amount of NO. Reaction Dynamics. The reaction essentially involved the S-nitrosation of HCYSH by nitrous acid, which is generated in situ by mixing perchloric acid and sodium nitrite using the double mixing mode of the Hi-Tech Scientific SF61-DX2 stopped-flow spectrophotometer. It is necessary to generate the nitrous acid in situ because of its instability to disproportionation reaction R5.

3HNO2 f H+ + NO3- + 2NO + H2O

(R5)

Unlike S-nitrosothiols of most of the primary aminothiols such as cysteine, HCYSNO is surprisingly stable at physiological pH of 7.4. Its half life was determined to be 198 h in the absence of reducing agents such as transition-metal ions and thiols, which could catalyze its decomposition. HCYSH Dependence. The rate of formation of HCYSNO from the reaction of HCYSH and HNO2 is significantly dependent on the initial concentration of HCYSH. As long as HCYSH is the limiting reactant, the rate of nitrosation and the amount of HCYSNO formed increases with an increase in the initial concentration of HCYSH (Figure 4a). The reaction shows a first-order dependence in HCYSH with an intercept kinetically indistinguishable from zero (Figure 4b). Nitrite and Acid Dependence. Change in the initial concentrations of both nitrite and acid (perchloric acid) has a large effect on the dynamics of the reaction. As the starting concentration of nitrite increases in excess acid and HCYSH, the final concentration of HCYSNO formed linearly increases, as seen in traces a-g of Figure 5a. Initial rate plot of these kinetics traces displayed a typical first-order dependence on nitrite for a nitrosation reaction in excess acid (Figure 5b).

Figure 4. (a) Absorbance traces showing the effect of varying HCYSH concentrations. The reaction shows first-order kinetics in HCYSH. [NO2-]0 ) 0.05 M; [H+]0 )0.05 M; [HCYSH]0 ) (a) 0.005, (b) 0.006, (c) 0.007, (d) 0.008, (e) 0.009, (f) 0.010, and (g) 0.011 M. (b) Initial rate plot of the data in Figure 4a. The plot shows strong dependence on initial rate of formation of HCYSNO on HCYSH.

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Figure 5. (a) Absorbance traces showing the effect of varying nitrite concentrations. The reaction shows first-order kinetics in nitrite. [HCYSH]0 ) 0.05 M; [H+]0 ) 0.05 M; [NO2-]0 ) (a) 0.005, (b) 0.006, (c) 0.007, (d) 0.008, (e) 0.009, (f) 0.010, and (g) 0.011 M. (b) Initial rate plot of the data in Figure 5a. The plot shows strong dependence on initial rate of formation of HCYSNO on nitrite.

Acid, as expected, exerts a most powerful effect on the rate of formation of HCYSNO. Figure 6a shows a series of kinetics experiments in which the concentration of acid is varied from 0.005 to 0.100 M. The concentrations of both nitrite and HCYSH for these experiments were constant and fixed at 0.05 M. Because the nitrosation experiments essentially involve the reaction of nitrous acid with HCYSH, a Table was created in which the final concentrations of nitrous acid were calculated (Table 1). The amount of HCYSNO formed increases as the concentration of acid increases until the concentration of the final nitrous acid is approximately equal to the concentration of HCYSH and saturation is attained, as shown in traces h-k in Figure 6a. The initial rate plot with respect to the final concentrations of HNO2 showed a first-order linear plot in the region where the initial concentrations of acid exceeded the nitrite concentration (Figure 6b) and a nonlinear quadratic second-order kinetics in the region where the final concentrations of HNO2 were less than the initial nitrite concentration (Figure 6c). We obtained confirmatory evidence of the observed secondorder kinetics in excess nitrite by plotting the initial rates against the squares of the final nitrous acid concentrations. A linear plot was obtained with an almost zero intercept (Figure 6d). To establish further this observed transition from second-order to first-order kinetics as the initial concentrations of acid increase

Morakinyo et al. from less than to more than the initial nitrite concentrations, a new series of HNO2-dependence experiments were performed at two fixed pHs 1.2 and 4.0 for two different HCYSH concentrations (Figure 7a,c). The experiments were done under the same conditions. In perfect agreement with our previous observation, plots of initial rates versus [HNO2] for data at pH 1.2 gave linear plots expected for first-order kinetics (Figure 7b). Also, plots of initial rates versus [HNO2] for data at pH 4.0 gave a nonlinear quadratic curve expected for second-order kinetics (Figure 7d). Catalytic Effect of Copper. Copper is an essential trace element that is indispensable for normal growth and development. It serves as a cofactor for the activity of a number of physiologically important enzymes such as methionine synthase, which plays a key role in the synthesis of methionine from homocysteine, and extracellular and cytosolic Cu-dependent superoxide dismutases (Cu2Zn2SOD), which play an important role in cellular response to oxidative stress by detoxifying the superoxide anion radical into the less harmful hydrogen peroxide.36,37 Depending on the redox environment, copper can cycle between two states as reduced Cu+ and oxidized Cu2+ ions, allowing it to play its numerous physiological roles. Figure 8 shows dual roles of this metal as a mediator of S-nitrosothiol (HCYSNO) formation and decomposition. Trace a represents a control experimental run without copper ions. Traces b-f have the same initial conditions as trace a but with varying amounts of Cu2+ ions. The plot shows a progressive increase in the rate of formation of HCYSNO with an increase in the initial concentrations of Cu2+ ions. High concentrations of Cu2+ ions give an early onset of the decomposition of HCYSNO, as shown in traces e and f. This observed effect of copper was virtually halted by the addition of excess EDTA, which sequesters copper ions. Transnitrosation. Transnitrosation is a physiologically important reaction between S-nitrosothiols (RSNO) and thiols (R′SH). It involves transfer of NO group from an S-nitrosothiol to a thiol to form a new S-nitrosothiol (reaction R6)

RSNO + R′SH h R′SNO + RSH

(R6)

In this study, S-nitrosoglutathione (GSNO) and S-nitroso-Nacetyl-D,L-penicillamine (SNAP) were used to transnitrosate HCYSH. They were synthesized as described in the literature.38,39 These two S-nitrosothiols have been shown to have excellent clinical applications.40 GSNO, in particular, is believed to be involved in the storage and transport of NO in mammals.41-43 Spectrophotometric studies of transnitrosation between primary S-nitrosothiols such as HCYSNO and GSNO are generally difficult because most nitrosothiols absorb at about the same wavelength (λmax(GSNO) ) 335 and 544 nm; λmax(HCYSNO) ) 331 and 545 nm), but we were able to follow the formation of HCYSNO upon transnitrosation of HCYSH by GSNO because HCYSNO has a slightly higher absorptivity coefficient (ε545 nm ) 20.2 M-1 cm-1) than GSNO (ε545 nm ) 17.2 M-1 cm-1).44 Figure 9a shows the transnitrosation of HCYSH by GSNO to form HCYSNO. Trace a is from pure GSNO without HCYSH. Traces b-f with equal amounts of GSNO show a monotonic increase in the amount and rate of formation of HCYSNO with an increase in the initial concentrations of HCYSH. Confirmatory evidence of the transnitrosation was achieved by ESI mass spectrometry (ESI-MS) technique. Figure 9b shows that the product of transnitrosation contains a mixture of components including HCYSNO, GSSG (oxidized glutathione), and mixed disulfide of homocysteine and glutathione.



Figure 6. (a) Absorbance traces showing the effect of varying acid concentrations. [HCYSH]0 ) 0.05 M; [NO2-]0 ) 0.05 M; [H+]0 ) (a) 0.005, (b) 0.01, (c) 0.02, (d) 0.03, (e) 0.04, (f) 0.05, (g) 0.06, (h) 0.07, (i) 0.08, (j) 0.09, and (k) 0.10 M. (b) Plot of initial rate versus nitrous acid concentrations of the experimental data in Figure 6a (traces h-k) and calculated in Table 1 at pH < 2. The plot shows a first-order dependence on the initial rate of formation of HCYSNO on the final nitrous acid concentrations. (c) Plot of initial rate versus nitrous acid concentrations of the experimental data in Figure 6a and calculated in Table 1 at pH 2 and above. The curve fit well into a polynomial of degree two, which is an indication of rate being dependent on the square of the final nitrous acid concentrations. (d) Plot of initial rate versus square of the final nitrous acid concentrations. This plot shows a second-order dependence in nitrous acid at pH 2 and above.

TABLE 1: Input Species Concentrations and Final Reagent Concentrations for Acid Dependence Experiments [H+]added

[NO2-]added

[HNO2]initial

[NO2-]final

[HNO2]final

[H3O+]final

(mol/L)

(mol/L)

(mol/L)

(mol/L)

(mol/L)

(mol/L)

0.005 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

0.005 0.01 0.02 0.03 0.04 0.05 0.05 0.05 0.05 0.05 0.05

-2

4.51 × 10 4.01 × 10-2 3.04 × 10-2 2.08 × 10-2 1.18 × 10-2 5.03 × 10-3 2.22 × 10-3 1.29 × 10-3 8.93 × 10-4 6.81 × 10-4 5.50 × 10-4

Transnitrosation by SNAP is simpler and can be monitored spectrophotometrically because of the fact that SNAP and HCYSNO absorb at different wavelengths. Figure 10a shows the decomposition of SNAP at 590 nm and simultaneous formation of HCYSNO at 545 nm. Figure 10b shows that the effect of HCYSH on its transnitrosation at a constant initial concentration of SNAP is linear, with a first-order dependence. The intercept (4.24 × 10-3 M) on the [HCYSH] axis corresponds to the equilibrium concentration of HCYSH

-3

4.94 × 10 9.86 × 10-3 1.97 × 10-2 2.92 × 10-2 3.82 × 10-2 4.50 × 10-2 4.78 × 10-2 4.87 × 10-2 4.91 × 10-2 4.93 × 10-2 4.95 × 10-2

∼pH -5

6.16 × 10 1.38 × 10-4 3.63 × 10-4 7.90 × 10-4 1.82 × 10-3 5.03 × 10-3 1.22 × 10-2 2.13 × 10-2 3.09 × 10-2 4.07 × 10-2 5.06 × 10-2

4.21 3.86 3.44 3.10 2.74 2.30 1.91 1.67 1.51 1.39 1.30

that reacts with SNAP to produce HCYSNO in agreement with the expected 1:1 stoichiometry ratio in reaction R6. The catalytic effect of copper on the rate of transnitrosation is shown in Figure 10c. Trace a is a reaction mixture without Cu2+ ion. The rate of formation of HCYSNO increases with increase in Cu2+ ion, as we have in traces b-e until saturation at f. Mechanism. The proposed mechanism for the formation of S-nitrosohomocysteine from the reactions of homocysteine

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Figure 7. (a) Absorbance traces showing the effect of varying nitrous acid concentrations at pH 1.2 in phosphate buffer. [HCYSH]0 ) 0.05 M; [NO2-]0 ) [H+]0 ) (a) 0.005, (b) 0.01, (c) 0.015, (d) 0.02, (e) 0.025, (f) 0.03, (g) 0.035, (h) 0.04, (i) 0.045, and (j) 0.05 M. (b) Combined HNO2 dependence plots at 0.05 M HCYSH (of the data in Figure 7a) and 0.09 M HCYSH showing first-order linear dependence of the rate of formation of HCYSNO on HNO2 at pH 1.2. These data were utilized in eq 8 for the evaluation of k6. (c) Absorbance traces showing the effect of varying nitrous acid concentrations at pH 4.0 in phosphate buffer. [HCYSH]0 ) 0.05 M; [NO2-]0 ) [H+]0 ) (a) 0.005, (b) 0.01, (c) 0.015, (d) 0.02, (e) 0.025, (f) 0.03, (g) 0.035, (h) 0.04, (i) 0.045, and (j) 0.05 M. (d) Combined HNO2 dependence plots at 0.05 M HCYSH (of the data in Figure 7c) and 0.09 M HCYSH showing nonlinear quadratic dependence of the rate of formation of HCYSNO on HNO2 at pH 4.0. These data were utilized in eq 9 for the evaluation of k4 and k5.

and acidic nitrite should accommodate the involvement of multiple nitrosating agents in the nitrosation process (Scheme 1). The first step is the formation of nitrous acid (reaction R7)

H+ + NO2- h HNO2

k1, k-1 ; Ka-1

(R7)

This is followed by the production of other nitrosating agents through the bimolecular decomposition of HNO2 to form dinitrogen trioxide, N2O3 (reaction R8), and protonation to form nitrosonium cation, NO+ (reaction R9)

2HNO2 h N2O3 + H2O

k2, k-2

HNO2 + H+ h NO+ + H2O

k3, k-3

(R8) (R9)

These nitrosating agents are electrophilic and attack the highly nucleophilic sulfur center of homocysteine to form S-nitrosohomocysteine (reactions R10, R11, and R12). The

Figure 8. Absorbance traces showing the effect of varying Cu2+ concentrations on the rate of formation of HCYSNO. There is a progressive increase in the rate of HCYSNO formation with increase in Cu2+ concentration. [HCYSH]0 ) [NO2-]0 ) [H+]0 ) 0.05 M; [Cu2+]0 ) (a) 0.00 M, (b) 1.0 mM, (c) 2.0 mM, (d) 3.0 mM, (e) 4.0 mM, and (f) 5.0 mM.



order of increasing electrophilicity of these nitrosating agents45 is HNO2 < N2O3 < NO+.

HCYSH + HNO2 f HCYSNO + H2O

(R10)

k4

HCYSH + N2O3 f HCYSNO + H+ + NO2-

HCYSH + NO+ h HCYSNO + H+

SCHEME 1: Reaction Pathways, Showing the Involvement of Multiple Nitrosating Agents in the Nitrosation of HCYSH to Form HCYSNO by Acidic Nitrite

k5 (R11)

k6, k-6

(R12) The mechanism of reaction can be explained by each of the nitrosating agents, whose degree of involvement in the

process of nitrosation significantly depends on the pH of their environment. For example, under pH conditions around 1.3, HNO2 and NO+ are the most relevant nitrosating agents.

Figure 9. (a) Absorbance traces showing the effect of varying HCYSH concentrations on its transnitrosation by GSNO. The plot shows the formation of HCYSNO at 545 nm. The increase in absorbance is due to the formation of HCYSNO, which has a higher absorptivity coefficient of 20.2 than 17.2 M-1 cm-1 of GSNO at 545 nm. [GSNO]0 ) 0.003 M; [HCYSH]0 ) (a) 0.0, (b) 0.005, (c) 0.01, (d) 0.02, (e) 0.03, and (f) 0.04 M. (b) An ESI-MS spectrum of a 1:5 ratio of GSNO to HCYSH. Final products were mixture of GSH, GSSG, HCYSNO, and mixed disulfide of GSH/HCYSH.

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Protonation of HNO2 to form NO+ is favored under these highly acidic conditions. NO+ is expected to be the more powerful nitrosating agent. At less acidic pHs around 3.8, for reactions in excess nitrite, as observed in traces a-e of Figure 6a, HNO2 and N2O3 are the main nitrosating agents with the rate of nitrosation by N2O3 being higher than that by HNO2, in accordance with their electrophilic trend. The overall rate of reaction, on the basis of rate of formation of HCYSNO, is given by

d[HCYSNO] dt ) k4[HCYSH][HNO2] + k5[HCYSH][N2O3] +

d[HCYSNO] dt ) k4[HCYSH][HNO2] + k5[HCYSH][N2O3] +

rate )

k6[HCYSH][NO+] Equation 2 can be further simplified by applying steady-state approximation on both N2O3 and NO+ because they are transient intermediates with negligible concentrations at any time during the reaction. They react as soon as they are produced.

rate )

(1)

k6[HCYSH][NO+] - k6[HCYSNO][H+]

Because, initially, there is no accumulation of HCYSNO at the beginning of the reaction, the last term in eq 1 can be ignored, giving

(2)

[N2O3] ) [NO+] )

k2[HNO2]2 + k5[HCYSH]

(3)

k3[HNO2][H+] k-3 + k6[HCYSH]

(4)

k-2

Substituting eqs 3 and 4 into eq 2 and simplifying gives the following rate equation

Figure 10. (a) Absorbance traces showing the effect of varying HCYSH concentrations on its transnitrosation by SNAP. The plot shows both formation of HCYSNO at 545 nm (solid lines) and decomposition of SNAP at 590 nm (dotted lines). [SNAP]0 ) 0.0049 M; [HCYSH]0 ) (a) 0.005, (b) 0.0075, (c) 0.01, (d) 0.02, (e) 0.03, (f) 0.04, and (g) 0.05 M. (b) Initial rate plot of the data in Figure 10a. The plot shows linear dependence on initial rate of formation of HCYSNO on HCYSH in the transnitrosation of HCYSH by SNAP. (c) Absorbance traces showing the effect of varying Cu2+ concentrations on the rate of decomposition of SNAP at 590 nm and simultaneous formation of HCYSNO at 545 nm (solid lines) during transnitrosation reactions. [HCYSH]0 ) 0.03 M; [SNAP]0 ) 0.01 M; [Cu2+]0 ) (a) 0.00 M, (b) 5 µM, (c) 10 µM, (d) 50 µM, (e) 100 µM, (f) 500 µM, (g) 1000 µM, and (h) 2000 µM.



TABLE 2: Mechanism Used for Simulating the S-Nitrosation of Homocysteinea reaction no.

reaction

M1

H+ + NO2- h HNO2

-1

-1

s ; 6.18 × 105 s-1

(M1)

1.10 × 10 M

HNO2 + H h NdO + H2O

(M2)

(Ka(HNO2) ) 5.62 × 10-4) 4.00 × 101 M-1 s-1; 3.33 × 109 s-1

M3

2HNO2 h N2O3 + H2O

(M3)

(KM3 ) 1.2 × 10-8 M-1) 1.59 ( 0.50 M-1 s-1; 5.30 × 102 s-1

M4

N2O3 h NO + NO2 RSH + HNO2 f RSNO + H2O

(M4) (M5)

+

M2

M5

+

+

+

RSH + NdO h RSNO + H RSH + N2O3 f RSNO + HNO2 2RSNO h RSSR + 2NO

M6 M7 M8 a

kf; kr

(M6) (M7) (M8)

9

(KM2 ) 3.0 × 10-3 M-1) 8.0 × 104 s-1; 1.1 × 109 M-1 s-1 9.0 × 10-2 M-1 s-1; ca. 0 6.57 × 1010 M-1 s-1; 3.6 × 103 M-1 s-1 9.49 × 103 M-1 s-1; ca. 0 5.0 × 10-2 M-1 s-1; ca. 0

RSH stands for homocysteine.

rate )

d[HCYSNO] dt

(

) [HCYSH][HNO2] k4 +

(

was observed. By ignoring the term containing [H+] in eq 5, we will obtain a rate eq 9

)

k3k6[H+] + k3 + k6[HCYSH] 2

k2k5[HCYSH][HNO2] k-2 + k5[HCYSH]

)

(5) rate )

d[HCYSNO] dt

) k4[HCYSH][HNO2] +

k2k5[HCYSH][HNO2]2 k-2 + k5[HCYSH]

(9)

where

[HNO2] )

[N(III)]T[H+]

(6)

Ka + [H+]

where [N(III)]T from mass balance equation for the total N(III) containing species is

[N(III)]T ) [NO2-] + [HNO2] + [HCYSNO] + [N2O3] + [NO+]

(7)

[HCYSH] is the sum of the protonated and unprotonated HCYSH. The proposed rate equation is valid from low to slightly acidic pH. Under low pH conditions, for reactions run in excess acid, the bimolecular decomposition of HNO2 to form N2O3 becomes irrelevant, and the last term in eq 5 can be ignored, resulting in first-order kinetics in nitrous acid with a rate equation given by

rate )

d[HCYSNO] dt

(

) [HCYSH][HNO2] k4 +

k3k6[H+] k3 + k6[HCYSH]

)

(8)

Rate eq 8 is supported by the kinetics data shown in Figures 6a,b and 7a,b. For reactions in excess nitrite, when the final perchloric acid concentrations tend to zero, a second-order kinetics, which fitted well into a polynomial of degree two,

Equation 9 is supported by the kinetics data shown in Figures 6a,c,d and 7c,d. As shown in eqs 8 and 9, there are five kinetics constants to be evaluated. Equilibrium constants K2 ) 3.0 × 10-3 M-1 and K3 ) 1.2 × 10-8 M-1 as well as the rate constant k-2 ) 5.30 × 102 s-1 have already been established.14,46,47 The data in Figure 7a-d provided rate constants for direct reaction of HCYSH and HNO2 of 9.00 × 10-2 M-1 s-1, HCYSH and N2O3 of 9.49 × 103 M-1 s-1, and HCYSH and NO+ of 6.57 × 1010 M-1 s-1. These values agree with the order of electrophilicity of these three nitrosating agents (HNO2 < N2O3 < NO+). Computer Simulations. A network of eight reactions, as shown in Table 2, can adequately model entire nitrosation reactions. Kintecus simulation software developed by James Ianni was used for the modeling.48 The rate constants for the formation of nitrosating agents (HNO2, N2O3, and NO+, reactions M1-M4) were taken from literature values.14,46,47 The forward and reverse rate constants used for reactions M2 and M3 fit well for the modeling and agreed with the literature values for the equilibrium constants 3.0 × 10-3 and 1.2 × 10-8 M-1 for reactions R8 and R9, respectively. Experimental data for reactions in highly acidic pH 1.2 (Figure 7a) and mildly acidic pH 4.0 (Figure 7c) were simulated using the bimolecular rate constants, k4, k5, and k6, determined according to our proposed mechanism. In highly acidic pH, the simulations were mostly sensitive to the values of rate constants kM1, kM2, and kM6. The rate of acid-catalyzed decomposition of HCYSNO was found to be halved as the initial concentrations of nitrous acid doubled. Reactions M1, M3, M4, M7, and M8 are the most relevant reactions in mildly acid pH. Figure 11a,b shows a comparison between the experimental results (traces a and b in Figure 7a,c) and our simulations. Reasonably good fits were obtained, and the simulations were able to reproduce the formation of HCYSNO in highly to mildly acidic pHs by the reaction of homocysteine and the nitrosating agents.

9904


Figure 11. (a) Comparison of experimental (solid lines, traces a and b from Figure 7c) and computer simulations (dotted lines) using the mechanism shown in Table 2 for the formation of HCYSNO in a highly acidic environment (pH 1.2). [HCYSH] ) 5.00 × 10-2 M; [HNO2] ) (a) 5.00 × 10-3 and (b) 1.00 × 10-2 M. (b) Comparison of experimental (solid lines, traces a and b from Figure 7a) and computer simulations (dotted lines) using the mechanism shown in Table 2 for the formation of HCYSNO in mildly acidic environment (pH 4.0). [HCYSH] ) 5.00 × 10-2 M; [HNO2] ) (a) 5.00 × 10-3 and (b) 1.00 × 10-2 M.

Conclusions This study has shown that the conversion of HCYSH to the highly toxic HTL can be prevented by the inhibition of the sulfur center of HCYSH by S-nitrosation. Nitrosation by dinitrogen trioxide and transnitrosation by other S-nitrosothiols such as GSNO are the most effective mean of S-nitrosation in mildly acid pHs to physiological pH 7.4. The high stability of HCYSNO at physiological pH, which rivals GSNO, is an advantage in the modulating of HCYSH toxicities by Snitrosation. Acknowledgment. This work was supported by grant numbers CHE 0619224 from the National Science Foundation and EB2044 from the National Institutes of Health. References and Notes (1) Ubbink, J. B. Homocysteine in Health and Disease; The Press Syndicate of the University of Cambridge: Cambridge, U.K., 2001; Chapter 40, pp 485-490. (2) Herrmann, W.; Herrmann, M.; Obeid, R. Curr. Drug Metab. 2007, 8, 17–31. (3) Krumdieck, C. L.; Prince, C. W. J. Nutr. 2000, 130, 365S–368S.

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