Reactions of Oxymyoglobin with NO, NO₂, and NO₂^- under Argon and in Air

Abstract

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 (lambda MAX, 302 nm) was poorly resolved from more intense protein absorption in the 300-nm region. However, at pH 9, the change in absorbance corresponded exactly to a quantitative production of the OONO-ion. Hydroxy radicals from it were trapped with ethylene-1, 2-(13) C. 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 x 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) approximately equal to 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.

Reaction of Human Hemoglobin with Peroxynitrite

“…A similar transient metHb-peroxynitrite complex was detected during the ⅐ NO-mediated oxidation of oxyHb, which rapidly evolves to metHb and nitrate (12). Importantly, oxidation of oxymyoglobin by ⅐ NO also yields an intermediate complex, which promotes the oxidation of ethylene-1,2-13 C as detected by NMR (13); this result indicates that a hydroxyl radical-like species arises at low yields from the iron-catalyzed homolysis of complexed peroxynitrite and that it can escape the solvent cage to initiate free radical chemistry (13). In addition, superoxide anion released in the first step of the reaction (Equation 8) would spontaneously dismutate to O 2 and H 2 O 2 (Equation 12), which would initiate secondary oxidation reactions in oxy-or metHb to ferrylHb (10,17,18) and promote consumption of pre-existing nitrite in peroxynitrite preparations (Table I), which is facilitated by autocatalytic mechanisms (16) The oxidation of oxyHb by peroxynitrite yields a transient ferrylHb intermediate (8,9).…”

“…The alternative scenarios for the peroxynitrite/oxyHb reaction are further complicated by the fact that the reactions of nitrite (NO 2 Ϫ ) and nitrogen dioxide ( ⅐ NO 2 ) with oxyHb could promote the formation of hydrogen peroxide (13,16) which in turn can secondarily react with oxy-and metHb to yield ferrylHb (Equation 3) or ferrylHb with a second oxidation equivalent centered in the globin moiety, respectively (10,17,18).…”

Diffusion of peroxynitrite across erythrocyte membranes

“…2, Table 1) which indicates that the predominant species reacting with hemoglobin is the protonated form of peroxynitrite, peroxynitrous acid. The oxidation of hemoglobin is not specific for peroxynitrite, since the same oxidation product can be obtained by reactions of oxyhemoglobin with peroxynitrite decomposition products such as nitrite (NO 2 Ϫ ) and nitrogen dioxide ( ⅐ NO 2 ) (45,46). However, the peroxynitrite-mediated oxidation of hemoglobin is significantly faster than the reaction with nitrite, which is kinetically characterized by a lag period followed by an autocatalytic phase (45).…”

Nitrogen monoxide and oxidative stress: composition and intensity of cellular oxidative bursts cocktail. A study through artificial electrochemical synapses on single human fibroblasts

“…Finally, the NO-dependent oxidation of hemoproteins such as oxy-hemoglobin [32] and oxy-myoglobin [33] can also lead to peroxynitrite formation (eq. 3).…”