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Chem. Res. Toxicol. 1996, 9, 1382-1390
Reactions of Oxymyoglobin with NO, NO2, and NO2under Argon and in Air Ruth S. Wade and Charles E. Castro* The Environmental Toxicology Graduate Program, University of California, Riverside, California 92521, and CEC Consulting, 1090 Madison Place, Laguna Beach, California 92651 Received March 12, 1996X
Oxymyoglobin under argon reacts with NO2- and NO2 (N2O4) to produce metmyoglobin in a spectrally clean process with clear isosbestic points. In both cases, the reactant is NO2-. The second-order rate constant for NO2- or N2O4 is the same: d(Mb+)/dt ) k(MbO2)(NO2-) where k ) 0.21 ( 0.02 L mol-1 s-1. The reaction of MbO2 with NO under argon is a complex process and entails the generation of Mb+ and OONO- (peroxynitrite) in the first step. The latter (λmax, 302 nm) was poorly resolved from more intense protein absorbtion in the 300-nm region. However, at pH 9, the change in absorbance corresponded exactly to a quantitative production of the OONO- ion. Hydroxyl radicals from it were trapped with ethylene-1,2-13C. The initial step is followed in sequence by the rapid formation of MbNO+. The iron(III)-nitrosyl adduct hydrolyzes slowly to MbII and NO2- (k ) 8.0 ( 0.8 × 10-5 s-1). MbII then rapidly associates with NO, and MbNO is the final product of this reaction. Oxymyoglobin is inert to NO3-. In contrast to the results under argon, in air the reactions of MbO2 with NO2-, NO, and NO2 (N2O4) all proceed in the same autocatalytic fashion with kave (for the autocatalytic rates) = 9 ( 5 L mol-1 s-1. Nitrite is the initial reactant in all cases. Isosbestic points are not observed in the visible spectrum, and additional porphyrin iron-ligated species are intermediates. Based upon work with iron porphyrins [J. Org. Chem. (1996) 61, 6388-6395 and J. Am. Chem. Soc. (1996) 118, 3894-3895], it is proposed that ozone may be an intermediate in the autocatalysis.
Introduction In 1898, Gangee described the oxidation of oxyhemoglobin to methemoglobin by nitrite ion (1). In the ensuing century, the reaction has received considerable attention (2-7). It can be the basis of fatal methemoglobinemia (8). Children and infants are particularly susceptible to the reaction. Nitrite is also capable of generating carcinogenic nitrosamines in vivo (9). Moreover, nitrate ion is reduced in vivo to nitrite by intestinal bacteria (8). In soil, nitrite is an intermediate in the denitrification processes that result in nitrogen or ammonia (10). It is now well established that the reactions of oxyhemoglobin and oxymyoglobin with nitrite ion are complex and autocatalytic (2). However, the exact nature of the autocatalysis remains poorly defined. Nitrogen dioxide, nitric oxide, peroxide, superoxide, peroxynitrite, and peroxynitrate have all been implicated. The most recent kinetic investigations (2, 5) suggest that nitrogen dioxide is the main autocatalytic agent. Nitric oxide is also reported to react rapidly with the oxyhemeproteins to produce the met species (11, 12). The reaction has been widely used as an assay for NO in various tissues (12, 13). However, the important biomessenger molecule NO (14-17) is now known to hydrolyze in aerobic solutions to nitrite ion (18, 19). Furthermore, NO synthase (20) and its target enzyme guanylate cyclase (21) are both heme proteins. The former is a P450-like enzyme and must, at some point, entail an iron-oxygen intermediate. In addition, NO reacts very rapidly with the iron(II) form of many heme proteins, * To whom correspondence should be addressed at the Laguna Beach address. X Abstract published in Advance ACS Abstracts, November 1, 1996.
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including deoxyhemoglobin and myoglobin to form stable heme-NO adducts (21-28). NO also rapidly affiliates with the corresponding iron(III) proteins and porphyrins (29, 30). The resulting iron(III)-NO+ adducts are capable of NO transfer to a range of nucleophiles (31) including nucleic acid bases (32) and water. Nitrogen dioxide is also a highly toxic gas (33), and it has been implicated in the autocatalytic oxidation of oxyhemoglobin and myoglobin (2). Finally, the oxides of nitrogen and nitrite and nitrate ions are interrelated by simple oxygen oxidation and hydrolysis: H2O
2NO + O2 f N2O4 h 2NO2 98 2H+ + NO2- + NO3- (1) H2O
NO + NO2 h N2O3 98 2H+ + 2NO2-
(2)
These processes are rapid (18, 34). In aerobic aqueous solution, nitrite is the product of NO oxidation. The rate process is first order in oxygen and second order in nitric oxide, with k ) 6 × 106 L2 mol-2 s-1 at 22 °C (18). Rate constants for the dimerization of NO2 and the combination of NO with NO2 are 9 × 108 and 1.1 × 109 L mol-1 s-1, respectively (18). Rate constants for the hydrolysis of N2O3 and N2O4 are both approximately 1.0 × 103 s-1 (34). The equilibrium between NO2 and N2O4 is dynamic and highly temperature dependent (45). In the liquid phase, at the boiling point (21 °C), the free radical NO2 is only present to the extent of 0.1%. Throughout this paper, we use the notation NO2 and nitrogen dioxide to indicate the gaseous mixture of NO2 and N2O4 at equilibrium. At 22 °C, our operating temperature, this mixture is nearly all dinitrogen tetroxide even though the brown colored radical nitrogen dioxide is visible in it. © 1996 American Chemical Society
Oxymyoglobin with NO, NO2, and NO2- under Argon and Air
Given the complexity of N-O bonded species that may be present in any reaction and the potential reaction of iron-oxyiron(II) and iron(III) heme proteins with them, it is not surprising that the autocatalytic oxidation of oxyheme proteins is not fully characterized. We have attempted to simplify this problem by studying the reactions of each N-O entity (NO, NO2, NO2-, NO3-) with oxymyoglobin under argon and in air. We have chosen MbO2 because the protein is well defined (33) and more stable than the hemoglobin tetramer or a P450O2 adduct.
Experimental Section Horse heart myoglobin (Sigma type IV) in 0.05 M phosphate buffer (pH 7.4) and 0.1 M KCl was first oxidized with potassium ferricyanide, reduced with sodium dithionite, and passed through a G-25 Sephadex column in air. For runs in air, the oxy complex emanating from the column was diluted to appropriate concentrations. Potassium nitrate and nitrite were Fischer reagent grade. Nitrogen dioxide (dinitrogen tetroxide) (Matheson Research Grade) was stored in a 1-L three-necked flask equipped with inlet and outlet stopcocks and a serum-capped stopcock for gas removal. The flask was repeatedly evacuated and filled with argon 15 times before the final evacuated flask was filled with NO2. Samples were removed by gas-tight syringes that had been thoroughly flushed with argon and emptied before puncturing the serum cap. GC analyses of this gas on a 9 M 8-Å, 1/8-in. molecular sieve column at 30 °C (30 mL/min He) exhibited only a trace of argon (
