The NOmediated Reduction of FerrylMb and FerrylHb

It has been suggested that heme-mediated redox reactions contribute to the organ dysfunction and/or tissue damage which occur in some pathological states characterized by release of Hb and Mb into the extracellular environment [29]. Autoxidation of oxyHb and oxyMb to their met-forms releases superoxide which dis-mutates to dioxygen and hydrogen peroxide, which in turn can cause tissue damage [30]. In addition, oxidation of these two proteins generates potentially cy-totoxic products such as the ferryl species [31].

It has been proposed that the highly oxidizing species ferrylMb is, at least in part, responsible for the oxidative damage caused by the reperfusion of ischemic tissues. We have determined the rate constants for the reactions of ferrylMb and ferrylHb with NO' ((17.1 ± 0.3)x 106 m-1 s-1 and (24 ± 1)x 106 m-1 s-1 at pH 7.0 and 20 °C) [23, 24]. The large value of these rate constants implies that these reactions are very likely to occur in vivo and might represent a detoxifying pathway for ferrylMb and ferrylHb and, thus, an additional antioxidant function of NO'.

Interestingly, we have shown that also the reactions of ferrylMb and ferrylHb with NO' proceed via the rapid formation of an intermediate [19]. Because of the radical-like character of the oxo-ligand in the ferryl forms of the proteins [32], it is reasonable to assume that the first step of the reactions is rapid radical-radical recombination (Scheme 2.6.2), which leads to the O-nitrito intermediates MbFemONO and HbFemONO. As shown in Figure 2.6.3, the absorbance maxima in the spectrum of the Mb-intermediate are found at 504 nm (e504 = 8.7 mM-1 cm-1) and at 631 nm (e631 = 5.1 mM-1 cm-1), and are thus consistent with

MbFelv=0 -MbFe"'-0' + NO"-MbFel"-ONO

Scheme 2.6.2. Mechanism of formation of the O-nitrito intermediate by reaction of ferrylMb with NO..

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Wavelength (nm)

Fig. 2.6.3. Rapid-scan UV-visible spectra of the reactions of ferrylMb (14.7 |im) with NO. (50 |im in 0.1 M borate buffer at pH 9.5, 20 °C. Traces 1-7, recorded 0, 0.4, 0.8, 1.2, 1.6, 2.0, and 10 s after mixing, show the decay of the intermediate MbFemONO (bold trace 1) to MbFemOH (trace 7).

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Wavelength (nm)

Fig. 2.6.3. Rapid-scan UV-visible spectra of the reactions of ferrylMb (14.7 |im) with NO. (50 |im in 0.1 M borate buffer at pH 9.5, 20 °C. Traces 1-7, recorded 0, 0.4, 0.8, 1.2, 1.6, 2.0, and 10 s after mixing, show the decay of the intermediate MbFemONO (bold trace 1) to MbFemOH (trace 7).

this assumption (Table 2.6.1). The O-nitrito intermediates decay to nitrite and metMb and metHb, respectively, but are more stable than the corresponding per-oxynitrito complexes.

Local nitrite concentrations in tissues are linked to the amounts of NO' produced. Indeed, except for nitrate generated from the reaction of NO' with oxyHb, nitrite is the major end product of NO' metabolism. Increased nitrite levels are thus found under pathophysiological conditions, for example inflammation, when NO' production is elevated. We have found that the rate constants for the reactions of the ferryl forms of Mb and Hb with nitrite are significantly lower than those for the corresponding reaction with NO' (16 + 1 s_1 at pH 7.5 for Mb and (7.5 + 0.1)x 102 m^1 s^1 at pH 7.0 for Hb, at 20 °C) [19, 20]. Thus, the reaction with nitrite probably plays a role only when NO' has been consumed completely and large concentrations of nitrite are still present.

In contrast with the protecting role of NO', however, the reaction with nitrite generates nitrogen dioxide that can contribute to tyrosine nitration. Indeed, we have demonstrated that nitrite can cause nitration of added tyrosine in the presence of metMb (or metHb) and hydrogen peroxide [19]. As shown in Figure 2.6.4, the yield of nitrotyrosine increased with increasing nitrite concentration but reached a plateau (ca. 16% yield, relative to metMb) in the presence of 25-30 mM nitrite. On the basis of the reaction mechanism described in Scheme 2.6.3, the nitrite concentration dependence and, in particular, the plateau reached above 30 mM nitrite can be rationalized as follows. Reaction of metMb with hydrogen peroxide generates the one-electron oxidized form of ferrylMb which has an additional transient radical on the globin ('MbFelv=O) [33]. When nitrite is present in very high concentrations its reaction with either 'MbFeIv=O or MbFelv=O might outcompete the reaction of tyrosine with 'MbFeIv=O. Thus, Tyr' is generated in a concentration significantly lower than that of NO2'. Consequently, the recombina-

Fig. 2.6.4. Yield of nitrotyrosine, relative to metMb, generated by adding one equiv. H2O2 (relative to metMb) to a mixture of metMb (250-270 |im), 1 mM tyrosine, and different amounts of nitrite at pH 7.0.
Scheme 2.6.3. Mechanism of formation of nitrotyrosine by reaction of metMb and nitrite in the presence of H2O2 and tyrosine.

tion of Tyr' and NO2' to generate nitrotyrosine occurs to a lesser extent than the disproportionation of NO2' or its reaction with MbFelv=O to yield nitrate. This reaction mechanism might explain the observation that higher nitrite concentrations do not generate higher yields of nitrotyrosine.

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