Scavenging of Peroxynitrite by Oxyhemoglobin and Identification of Modified Globin Residues

Abstract

Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO2 or hemoproteins, and these reactions affect its fate and toxicity. CO2 promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO2 on oxyhemoglobin were determined by mass spectrometry, and we found that αTyr42, βTyr130, and, to a lesser extent, αTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with •NO2. Dityrosine (α24−α42) and disulfides (β93−β93 and α104−α104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species •NO2 mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala−Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala−Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala−Tyr nitration was reduced by 65%. This “sink” function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.

Nitration and Inactivation of Tyrosine Hydroxylase by Peroxynitrite

“…These data suggest that the contribution of the cysteine residues in the reaction of peroxynitrite with TH is relatively small, and the second order rate constants are typical of the reactions of peroxynitrite with metal centers (15)(16)(17)30).…”

“…In addition, no loss of TH enzymatic activity was detected after peroxynitrite treatment of the Tyr 423 3 Phe mutant TH. Stopped flow experiments revealed reactivity with the ferrous iron in TH typical of metalloproteins reacting with peroxynitrite (15)(16)(17). The absence of other amino acid modifications at low peroxynitrite concentrations suggests that nitration of tyrosine 423 is responsible for the inactivation of TH by peroxynitrite.…”

Reaction of Human Hemoglobin with Peroxynitrite

“…6, lanes 2-5)), in agreement with the formation of intermolecular disulfide bridges and dityrosine, respectively. Covalent linkage between ␤-93 cysteines and ␣-tyrosines 42 and 24 has been reported after reactions of peroxynitrite and oxyHb in the presence of CO 2 (21). No differences were observed when 100 M ascorbate was added as a reductant to the electrophoresis loading buffer, ruling out cross-linking caused by oxidation during the denaturalization process (data not shown) (21).…”

“…The reaction of peroxynitrite with oxyHb is relevant in the context of peroxynitrite reactions and diffusion in the intravascular compartment, because red blood cells and oxyHb may constitute a "sink" for intravascularly formed peroxynitrite (7,20,21). Indeed, peroxynitrite can readily cross erythrocyte membranes via anion channel-dependent and simple diffusion mechanisms, and we have previously established that significant amounts of peroxynitrite can reach inside red blood cells, even in the presence of physiological concentrations of carbon dioxide and other fast reacting biotargets of peroxynitrite present in plasma and the extracellular milieu (6,7,20).…”

Reaction Intermediates and Molecular Mechanism of Peroxynitrite Activation by NO Synthases

“…This hypothesis of multiple PN decomposition routes has been already envisioned for metallo-porphyrins (38) and metallo-proteins (1). Cross reactions of PN with the Cpd-I or Cpd-II intermediates (39-43) but also iron-oxo (44)(45)(46)(47) and iron-nitrosyl complexes (42,48,49) have been commonly proposed.…”