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Chem. Res. Toxicol. 2000, 13, 736-741
Reactivity of Peroxynitrite versus Simultaneous Generation of •NO and O2•- toward NADH Sara Goldstein* and Gidon Czapski Department of Physical Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Received May 1, 2000
The oxidation of NADH by peroxynitrite takes place indirectly via the radical intermediates formed during the self-decomposition of peroxynitrite, i.e., •OH and •NO2, and the oxidation yield exceeds 29% at relatively high NADH concentrations. The efficiency of oxidation of NADH by peroxynitrite is hardly affected by the presence of bicarbonate at physiological pH, and is remarkably increased when authentic peroxynitrite is replaced by low and equal fluxes of •NO and O2•-. We determined the rate constants for the reactions of NADH with •OH, CO3•-, and •NO to be (2.0 ( 0.2) × 1010, (1.4 ( 0.3) × 109, and (4.0 ( 2.0) × 103 M-1 s-1, respectively. We 2 show that the reaction of NADH with •OH in aerated solution does not form O2•-, whereas the other one-electron oxidants oxidize NADH to NAD•, which in turn very efficiently reduces oxygen to O2•-. These results suggest that at physiological pH the oxidation of NADH by peroxynitrite in the absence or presence of bicarbonate occurs mainly through the reactions of NADH with •OH or CO3•-, which are formed in about equal yields. The oxidation of NADH by continuous generation of •NO and O2•- proceeds via a chain mechanism, and therefore, the oxidation yield increases upon decreasing the flux of the radicals, and is higher than that obtained with authentic peroxynitrite.
Introduction Peroxynitrite (ONOOH/ONOO-) is a powerful oxidant that can be produced in biological systems via the diffusion-limited reaction between •NO and O2•- (1, 2). The reactivity of peroxynitrite toward biological molecules (3) and its very high toxicity toward cells (4) are assumed to be the potential cause of a number of diseases (5). Peroxynitrite ion is fairly stable, but its conjugate peroxynitrous acid (ONOOH, pKa ) 6.6) decomposes rapidly to yield about 30% •OH and •NO2 in the bulk of the solution (6, 7). In the presence of bicarbonate, the reaction of peroxynitrite ion with CO2 competes efficiently with the self-decomposition of peroxynitrite (8), and about 33% •NO2 and CO3•- reach the bulk of the solution (911). Peroxynitrite has been shown to oxidize NADH in aerated solutions (13). Recently, it has been argued that peroxynitrite reacts directly with NADH under physiological conditions, and can compete efficiently with known peroxynitrite scavengers such as cysteine (14). The oxidation efficiency of NADH was found to be ≈25%, but was reduced to ≈8% in the presence of bicarbonate (14). Surprisingly, the efficiency of oxidation of NADH by SIN-1 (3-morpholinosydonimine), which generates equal fluxes of •NO and O2•-, was considerably higher than that obtained with authentic peroxynitrite, regardless of the presence or absence of bicarbonate (14). The authors found this difference hard to explain due to the lack of knowledge about the chemical characteristics of authentic and in situ generation of peroxynitrite (14). In the study presented here, we show that peroxynitrite oxidizes NADH indirectly through the radical intermedi* To whom all correspondence should be directed. Telephone: 9722-6586478. Fax: 972-2-6586925. E-mail:
[email protected].
ates formed during its self-decomposition. We also show that the efficiency of oxidation of NADH by peroxynitrite is hardly affected by the presence of bicarbonate at physiological pH, and is remarkably increased when authentic peroxynitrite is replaced by low and equal fluxes of •NO and O2•-. We suggest a model that clearly explains these results.
Experimental Procedures Chemicals. All chemicals were analytical grade and were used as received. The water was purified using a Milli-Q water purification system. Peroxynitrite was prepared by having nitrite react with acidified hydrogen peroxide in a quenchedflow system (15). The concentration of peroxynitrite was determined using an 302 of 1670 M-1 cm-1 (16). Reduced β-nicotinamide adenine dinucleotide (NADH) from grade III yeast was obtained from Sigma. The concentration of NADH was determined using an 340 of 6200 M-1 cm-1. The concentration of CO2 in bicarbonate solutions was calculated using a pK of 6.2 for the hydration of CO2 (17). Apparatus. Stopped-flow kinetic measurements were carried out using the Bio SX-17MV sequential stopped-flow system from Applied Photophysics with a 1 cm long mixing cell. Peroxynitrite solution (pH 12) was mixed in a 1:1 ratio with 0.2 M phosphate buffer in the absence and presence of bicarbonate. The buffered solutions in the absence of bicarbonate were purged with argon for at least 1 h to remove CO2. The decomposition of peroxynitrite was followed at 302 nm. The reaction of peroxynitrite with NADH in the absence and presence of bicarbonate was studied in the presence of 10 µM DTPA (diethylenetriaminepentaacetic acid) by following the bleaching of the absorbance at 340 nm. The amount of NADH consumed by peroxynitrite was determined using an 340(ONOO-) of 850 M-1 cm-1 (2) and in the presence of bicarbonate also using an 340(O2NOO-) of 400 M-1 cm-1 (18). All experiments were carried out at 22 °C, and the final pH was measured at the outlet of the flow system. Pulse radiolysis experiments were carried using a 5 MeV Varian 7715 linear accelerator (0.2-1.5 µs electron pulses, 200
10.1021/tx000099n CCC: $19.00 © 2000 American Chemical Society Published on Web 07/12/2000
NADH Oxidation by Peroxynitrite
Chem. Res. Toxicol., Vol. 13, No. 8, 2000 737
Table 1. Rates and Oxidation Efficiencies of the Reaction of NADH with Peroxynitritea [peroxynitrite] [NADH] (µM) (mM) pH 124 200 117 71 72 72 200 169 112 64 36 66 177 177 65 69 69
0.33 0.33 0.33 0.21 0.11 0.35 0.35 0.35 0.35 0.15 0.32 0.32 0.17 0.09
7.6 7.6 7.6 7.6 7.6 7.6 7.0 7.0 7.0 7.0 7.0 7.0 6.5 6.5 6.5 6.5 6.5
rate (s-1) 0.12 0.095 0.10 0.11 0.10 0.10 0.31 0.29 0.29 0.29 0.29 0.27 0.56 0.47 0.45 0.43 0.43
yieldb simulated yieldc (%) (%) 34 38 42 34 34 33 34 38 43 29 43 53 42 32
34 39 44 37 31 35 38 43 50 32 34 43 34 30
Table 2. Rates and Oxidation Efficiencies of the Reaction of NADH with Peroxynitrite in the Presence of Bicarbonate at pH 7.6a [peroxynitrite] [CO2] [NADH] kobs(1) yield(1)b kobs(2) yield(2)b (µM) (mM) (µM) (s-1) (s-1) (%) (%) 145 79 77 42 41 48 48
1.2 1.2 1.2 1.2 1.2 0.6 0.6
246 123 123 63 244 122
27.8 27.4 26.2 26.8 25.7 13.7 13.1
35 36 36 37 35 35
0.72 0.73 0.72 0.74 0.76 0.72
4 2 3 2 4 3
a Each experiment was repeated at least four times, and the experimental error was less than 3%. b Yield ) ∆[NADH]consumed/ [peroxynitrite].
a Each experiment was repeated at least four times, and the experimental error was less than 3%. b Yield ) ∆[NADH]consumed/ [peroxynitrite]. c Simulated yields were obtained using the proposed model (reactions 3-14) and assuming that k6 ) 4 × 103 M-1 s-1 and k8 ) 4.5 × 105 M-1 s-1.
mA current). All measurements were taken at room temperature in a 4 cm optical path length cell using three light passes. γ-Radiolysis experiments were carried out with a 137Cs source. Absorbed doses were determined using the Fricke dosimeter (19). Modeling of the experimental results was carried out using INTKIN, a noncommercial program developed at Brookhaven National Laboratories by H. A. Schwarz.
Results Reaction of NADH with Peroxynitrite. The reaction of peroxynitrite with NADH was studied under limiting concentrations of peroxynitrite at pH 6.5-7.6. The decay of NADH was first-order, and kobs was found to be independent of [NADH]o and [peroxynitrite]o, and within experimental error identical to that of the selfdecomposition of peroxynitrite at the same pH (Table 1). The efficiency of oxidation of NADH by peroxynitrite exceeded 29% at a relatively high [NADH]o (Table 1). Reaction of NADH with Peroxynitrite in the Presence of Bicarbonate. The reaction of peroxynitrite with CO2 in the presence of NADH was studied under limiting concentrations of peroxynitrite. The decay of the absorbance at 340 nm followed two sequential first-order reactions, where kobs of the first fast decay was linearly dependent upon [CO2], and identical to that measured for the decay of peroxynitrite in the absence of NADH at 302 nm. The rate of the second slow process was independent of [peroxynitrite], [CO2], and [NADH], and decreased upon decreasing the pH from 0.74 ( 0.02 s-1 at pH 7.6 to 0.47 ( 0.03 s-1 at pH 6.5. Typical results at pH 7.6 are given in Table 2. Simultaneous Generation of •NO and O2•- in the Presence of NADH and Bicarbonate. Radiation sources were used to generate essentially equal amounts of •NO and O2•- in solutions containing 10 mM nitrite, 1 M methanol, 0.33 mM CO2, 10 µM DTPA, 100 µM NADH, and 50 mM phosphate buffer (pH 7.5). γ
H2O 98 eaq-(2.6), •OH(2.7), H•(0.6), H2(0.45), H2O2(0.7), H3O+(2.6) (1)
Figure 1. Consumption of 100 µM NADH as a function of the dose delivered at various dose rates into aerated solutions also containing 10 mM nitrite, 1 M methanol, 0.33 mM CO2, and 50 mM phosphate buffer (pH 7.5).
(The numbers in parentheses are G values, which represent the number of molecules formed per 100 eV of energy absorbed by pure water. An additional contribution to these yields of ca. 7% was assumed due to track scavenging.) Upon irradiation of the solution described above, eaq- reacts with NO2- to form NO22-, which in turn reacts with phosphate to generate •NO. The hydroxyl radical is converted into O2•- via its reaction with methanol, and H• is partially converted into •NO via its reaction with NO2- and partially into O2•- via its reaction with methanol and oxygen. The reaction mechanism of this system was described in detail elsewhere (20). The consumption of 100 µM NADH was found to be linear with the dose at various dose rates (Figure 1). The oxidation yields were calculated from the slope of the lines assuming that G(•NO) ) G(O2•-) ) 3.15 (Table 3). Oxidation of NADH by Carbonate Radical. The carbonate radical was generated upon pulse irradiation of a N2O-saturated solution containing 0.2 M carbonate at pH 10. Under these conditions eaq- is converted to •OH via its reaction with N2O, and •OH reacts with CO32- to form CO3•- (12). The decay rate of CO3•- at 600 nm (600 ) 1860 M-1 cm-1) was enhanced in the presence of NADH, and the kinetics changed from second- to firstorder. The decay of CO3•- was accompanied by a simultaneous first-order formation of a transient species with a maximum absorption at 400 nm and an 400 of 2100 ( 170 M-1 cm-1. This transient species decayed slowly via second-order kinetics where 2k ) (2.1 ( 0.2) × 108 M-1 s-1. The spectrum and half-life of this transient are
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Chem. Res. Toxicol., Vol. 13, No. 8, 2000
Goldstein and Czapski
Table 3. Efficiency of Oxidation of NADH by Essentially Equal Generation of •NO and O2•- in the Presence of 0.33 mM CO2 at pH 7.5 dose rate
[•NO] ≈ [O2•-]
∆[NADH]consumed
yield (%)
1.04 ( 0.09 Gy/pulse 8.83 ( 0.19 Gy/min 1.37 ( 0.10 Gy/min 0.97 ( 0.09 Gy/min
3.28 ( 0.28 µM/pulse 2.78 ( 0.06 µM/min 0.43 ( 0.03 µM/min 0.31 ( 0.03 µM/min
1.18 ( 0.02 µM/pulse 1.81 ( 0.02 µM/min 0.35 ( 0.03 µM/min 0.36 ( 0.01 µM/min
36 ( 4 65 ( 2 81 ( 14 116 ( 16
Table 4. Efficiency of Oxidation of NADH by CO3•-, Br2•-, and •OH in the Presence of Oxygena ∆[NADH]consumed/[R•]
Figure 2. Observed first-order rate constant for the decay of 3.4 µM CO3•- as a function of [NADH]. All solutions were N2Osaturated and contained 0.2 M carbonate (pH 10).
identical to those reported previously for NAD• (21, 22). The observed decay rate constant of CO3•- and the observed formation rate of NAD• were linearly dependent on [NADH]o, resulting in a k2 of (1.4 ( 0.1) × 109 M-1 s-1 (Figure 2).
CO3•- + NADH f NAD• + HCO3-
(2)
The efficiency of oxidation of NADH by CO3•- in the presence of oxygen was determined upon γ-irradiation of a solution containing 150 µM NADH and 10 µM DTPA at pH 10, which was saturated with a mixture of 80% N2O and 20% O2. Under these conditions, the concentration of N2O is sufficiently high to scavenge eaq-, but 10% of the radicals formed by the radiation are converted into O2•- due to the fast reaction of H• with O2 (12). The oxidation yield in the absence of O2 was found to be 102 ( 5%, but increased to 146 ( 7% in the presence of 20% O2. The efficiency of oxidation of NADH by CO3•- could not be determined at physiological pH because the reaction of •OH with HCO3- is too slow (12). Therefore, we replaced 0.2 M carbonate with 50 mM NaBr. Under these conditions, •OH is converted to Br2•-, which very efficiently oxidizes NADH to NAD• (12). The oxidation yields obtained via the reaction of NADH with Br2•- in aerated solutions and at various pHs are summarized in Table 4. The oxidation yield in acidic solutions could not be determined because the consumption of NADH was no longer linear with respect to the dose. Oxidation of NADH by Hydroxyl Radical. The rate constant for the reaction of •OH with NADH was determined from the competition between NADH and 0.5 mM iodide for •OH in N2O-saturated solutions at pH 7.7 (1 mM phosphate buffer), assuming that k(•OH + I-) ) 1.1 × 1010 M-1 s-1 (12). The reciprocal of the yield of I2•- was found to be linear with [NADH]o, resulting in k3 ) (2.0 ( 0.2) × 1010 M-1 s-1 (results not shown).
R•
[NADH] (mM)
pH
experiment
simulatedb
CO3•Br2•Br2•Br2••OH •OH •OH •OH
0.15 0.20 0.20 0.20 0.13 0.15 0.15 0.17
10 10 7.8 6.9 10 7.9 6.9 6.2
1.42 ( 0.07 1.50 ( 0.04 1.74 ( 0.03 2.63 ( 0.12 1.02 ( 0.05 1.19 ( 0.04 1.37 ( 0.07 2.19 ( 0.26
1.35 1.49 1.71 2.70 1.00 1.15 1.44 2.05
a The consumption of NADH was linear with the dose (e150 Gy), and the yields were determined from the slopes of such lines. The dose rate was 6.84 Gy/min. b Simulated yields were obtained assuming that k8 ) 4.5 × 105 M-1 s-1, that k9 ) 25 M-1 s-1, and that the reaction of NADH-OH with O2 does not yield O2•-. The simulation took into account the fact that 10% of the radicals formed by the radiation yields O2•- due to the reaction of H• with O2 (12).
•
OH + NADH f NADH-OH
(3)
The reaction of •OH with NADH forms an OH-adduct as indicated by the difference in the decay at 400 nm between NAD• and the transient species formed via reaction 3 (21). The efficiency of oxidation of NADH by •OH in the presence of oxygen was determined upon γ-irradiation of solutions saturated with a mixture of 80% N2O and 20% O2 and containing 140-160 µM NADH and 10 µM DTPA at various pHs. The results are summarized in Table 4. Reaction of NADH with Nitrogen Dioxide. Approximately 20 µM •NO2 was generated by pulse irradiation of deaerated solutions containing 0.5 M NO3- and 0.05 M NO2- at pH 7 (5 mM phosphate buffer). Under these conditions, the reaction of •OH with NO2- forms • NO2, and that of the eaq- with NO3- forms NO32-, which in turn reacts with phosphate to generate •NO2 (12). The decay of •NO2 was followed at 400 nm ( ) 200 M-1 cm-1), and corresponded to the well-established pathway (23): •
NO2 + •NO2 h N2O4 k4 ) 4.5 × 108 M-1 s-1 and k-4 ) 6.9 × 103 s-1 (4)
N2O4 + H2O f NO3- + NO2- + 2H+ k5 ) 1 × 103 s-1 (5) The addition of up to 0.24 mM NADH had hardly any effect on the half-life of •NO2, resulting in a k6 of
