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almax [nm] (e [mM 1 cm

dependent. At pH 7.0 we obtained (43.6 + 0.5)x 106 m^1 s^1 and (89 + 3)x 106 s_1 (per heme) for the NO'-mediated oxidation of oxyMb and oxyHb, respectively. Approximately the same rate constants were obtained when these reactions were performed in the pH range 5.0-7.0, whereas under alkaline conditions the second-order rate constants increased continuously up to (97 + 3)x 106 m_1 s_1 and (144 + 3)x 106 m_1 s_1 (per heme) at pH 9.5, for oxyMb and oxyHb, respectively.

As mentioned above, the rates of decay of the peroxynitrite complex of the two hemoglobin subunits (36 s_1 and 7 s_1 at pH 9.5) are significantly slower than that measured for MbFemOONO (205 s_1 at pH 9.5). However, these rates are significantly faster than that for the decay of free peroxynitrite under the same conditions (0.11 s_1 at pH 9.5 [25]). These differences can be rationalized by considering the influence of the distal histidine. As depicted in Scheme 2.6.1, the distal histidine (His64 in myoglobin) could facilitate the cleavage of the O-O bond by interacting with one of the two oxygen atoms. Acceleration of the decay of perox-ynitrite when it is bound to the iron(iii) center might therefore be a consequence of the presence of both the iron and this hydrogen bond that pull from two sides on the O-O bond.

Further mechanistic studies showed that no free peroxynitrite is formed during the reactions of NO' with the oxy-forms of these proteins, and that nitrate is formed quantitatively, at both pH 7.0 and pH 9.0 [18]. Analysis of the proteins after ten cycles of oxidation by NO' and reduction by ascorbic acid indicated that fewer than 1% of the tyrosine residues are nitrated. These results show that when per-oxynitrite is coordinated to the heme of myoglobin or hemoglobin, it rapidly iso-merizes to nitrate, and thus cannot nitrate the tyrosine residues of the globin.

oxyMb

Scheme 2.6.1. The role of the distal histidine (His64) in the isomerization of peroxynitrite bound to the iron(III) center of myoglobin.

Interestingly, the iron(iii)peroxynitrito complex is not detected when the aquo-iron(iii) forms of Mb and Hb (metMb and metHb) are allowed to react with peroxynitrite under neutral or alkaline conditions [22]. Indeed, the reactivity of metMb towards peroxynitrite is regulated by the presence of the distal histidine (His64), which partly blocks the active site and stabilizes, via a strong hydrogen bond, the water ligand coordinated to the iron (see metMb structure in Scheme 2.6.1). In the presence of wild-type metMb the decay of peroxynitrite is only slightly accelerated (kcat = (1.4 + 0.1)x 104 m^1 s^1 at pH 7.4 and 20 °C [23]). In contrast, the myoglobin mutant in which His64 is replaced by an alanine (H64A) is an efficient catalyst for the isomerization of peroxynitrite (kcat = (6.0 + 0.1)x 106 m_1 s_1 at pH 7.4 and 20 °C [23]). Indeed, ion chromatographic analysis of the nitrogen-containing products showed that in the presence of 0.01 equivalents of H64A per-oxynitrite decays quantitatively to nitrate. Moreover, HPLC analysis revealed that 0.05 equivalents of H64A prevent nitration of free tyrosine by peroxynitrite almost completely, both in the absence and presence of physiological amounts of carbon dioxide (1.2 mM).

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