The Peroxynitritemediated Oxidation of OxyMb and OxyHb

Despite the presence of a large concentration of carbon dioxide in the blood (ca. 1 mM), it has been reported that peroxynitrite can diffuse across the red-blood-cell membrane and react with oxyHb [24]. The anionic form (ONOO~) crosses the ery-throcyte membrane by using the anion channel band 3 whereas peroxynitrous acid crosses the lipid membranes by rapid passive diffusion [24].

We have recently shown that the peroxynitrite-mediated oxidation of oxyMb and oxyHb proceeds via intermediate oxoiron(iv) (ferryl) complexes, which, in a second step, react further with peroxynitrite to yield metMb and metHb, respec tively [25]. The rate constants for the two steps of the reaction of peroxynitrite with oxyMb, at pH 7.3 and 20 °C, were determined as (5.4 + 0.2)x 104 s_1 and (2.2 + 0.1)x 104 m_1 s-1, respectively [25]. The corresponding rates for the reaction with oxyHb, at pH 7.0 and 20 °C, are (8.4 + 0.4)x 104 m_1 s_1 and 9.4 + 0.7 x 104 m_1 s-1, respectively [25]. These rate constants suggest that oxyMb and oxyHb can successfully compete with CO2 and, thus, might be involved in the detoxification of peroxynitrite under physiological conditions.

To act as efficient scavengers, however, it is important the proteins are not modified extensively during their reaction with peroxynitrite. To investigate this hypothesis, we have analyzed by HPLC peroxynitrite-treated proteins subjected either to acid hydrolysis or pronase digestion. Our data showed that only very low quantities of 3-nitrotyrosine are formed when peroxynitrite reacts with the oxy-form of these proteins. Comparable amounts of nitrated tyrosine residues are formed when metMb and metHb are treated with peroxynitrite under analogous conditions, but significantly larger yields are observed with apoMb and metMbCN (Figure 2.6.2). In apoMb the heme group is absent whereas in metMbCN the iron ion of the heme cannot interact directly with peroxynitrite because of the strong cyanide ligand. Taken together our data suggest that the heme center of Mb can act as an efficient scavenger of peroxynitrite, protecting the globin from nitration [26]. When the heme center is not available for direct reaction, peroxynitrite undergoes an unspecific reaction with the globin, which leads to significant tyrosine nitration.

Additional evidence for the role of oxyMb and oxyHb as scavengers of peroxynitrite is the observation that they effectively protect against peroxynitrite-mediated nitration of free tyrosine. Reaction of 200 ^m peroxynitrite with 100 ^m tyrosine leads to the formation of approximately 15% nitrotyrosine (at pH 7.4 and 20 °C). When the same reaction is carried out in the presence of 50 ^m oxyMb no nitrotyrosine can be detected. In addition, the same amount of metMb reduces the

Equivalents of peroxynitrite Fig. 2.6.2. Nitrotyrosine yields (% relative to the total tyrosine content of the protein, that is 2 tyrosine residues per heme), determined by HPLC after acid hydrolysis, from the reaction of apo-, met-, oxyMb, and metMbCN (100 |im) with different amounts of peroxynitrite (5, 10, and 20 equiv.) at 0 °C and pH 7.0.

nitration yield to 2.6%. However, 50 mM apoMb or metMbCN only slightly reduce the nitrotyrosine yield, to approximately 9 and 13%, respectively.

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