Reaction of Human Hemoglobin with Peroxynitrite

Abstract

Peroxynitrite, a strong oxidant formed intravascularly in vivo, can diffuse onto erythrocytes and be largely consumed via a fast reaction (2 ؋ 10 4 M ؊1 s ؊1 ) with oxyhemoglobin. The reaction mechanism of peroxynitrite with oxyhemoglobin that results in the formation of methemoglobin remains to be elucidated. In this work, we studied the reaction under biologically relevant conditions using millimolar oxyhemoglobin concentrations and a stoichiometric excess of oxyhemoglobin over peroxynitrite. The results support a reaction mechanism that involves the net one-electron oxidation of the ferrous heme, isomerization of peroxynitrite to nitrate, and production of superoxide radical and hydrogen peroxide. Homolytic cleavage of peroxynitrite within the heme iron allows the formation of ferrylhemoglobin in ϳ10% yields, which can decay to methemoglobin at the expense of reducing equivalents of the globin moiety. Indeed, spin-trapping studies using 2-methyl-2-nitroso propane and 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated the formation of tyrosyl-and cysteinyl-derived radicals. DMPO also inhibited covalently linked dimerization products and led to the formation of DMPO-hemoglobin adducts. Hemoglobin nitration was not observed unless an excess of peroxynitrite over oxyhemoglobin was used, in agreement with a marginal formation of nitrogen dioxide. The results obtained support a role of oxyhemoglobin as a relevant intravascular sink of peroxynitrite.Peroxynitrite, 1 a strong oxidant formed intravascularly in vivo by the diffusion-limited reaction between nitric oxide ( ⅐ NO) and superoxide (O 2 . ) radicals (1-5), rapidly oxidizes human oxyhemoglobin (oxyHb) 2 (k 2 ϭ 2 ϫ 10 4 M Ϫ1 s Ϫ1 at 37°C and pH 7.4 (6,7)) to yield methemoglobin (metHb) as the final product. Peroxynitrite can behave as either a one-or two-electron oxidant during its direct reaction with transition metal centers; therefore, it is not apparent how metHb is formed. Recently, an initial two-electron oxidation process has been proposed leading to the formation of a high oxidation state intermediate, ferrylhemoglobin (ferrylHb) (8, 9). However, the ferrylHb intermediate detected during reaction of oxyHb with peroxynitrite has a very short half-life (i.e. milliseconds), and it can only be clearly observed when trapped with sodium sulfide (8, 9). Even under excess of sodium sulfide, the yields of sulfo-hemoglobin obtained were low (ϳ 10 -15% of metHb yield). This contrasts with the two-electron oxidation product obtained with oxyHb under excess of hydrogen peroxide (H 2 O 2 ), which yields a relatively stable ferrylHb intermediate (i.e. minutes) that can be observed directly by spectrophotometric techniques (10). If peroxynitrite oxidizes oxyHb by a two-electron oxidation process, nitrite and molecular oxygen should be produced in addition to ferrylHb (Equation 1):Neither nitrite nor molecular oxygen yields have been assessed yet, so the mechanism proposed in Equation 1 cannot be confirmed with the available data. Other authors have previously prop...

Nitric oxide, oxidants, and protein tyrosine nitration

“…In this context, oxyhemoglobin (oxyHb), present at 5 mM in red blood cells, serves as a sink to capture intravascularly formed nitrating species. Indeed, a fraction of intravascularly formed peroxynitrite could diffuse into red blood cells before enacting target molecule reactions with plasma components (83) and could undergo a fast reaction with oxyHb, which results in its isomerization to NO 3 Ϫ , without significant formation of nitrating species (82 (60,85). In cases where SOD is not close enough to O 2 Ϫ sources, it can enhance peroxynitritedependent nitration (7,8).…”

Intraphagosomal Peroxynitrite as a Macrophage-derived Cytotoxin against Internalized Trypanosoma cruzi

“…The reaction between ⅐ NO and O 2 . yields peroxynitrite (19,20), a strong one-and twoelectron oxidant and a precursor of secondary species including hydroxyl ( ⅐ OH), nitrogen dioxide ( ⅐ NO 2 ), and carbonate (CO (22), tyrosine nitration (23)) transition metal-containing centers (24), lipids (25), and nucleic acids (26), reactions that can ultimately promote cell death by either apoptotic or necrotic pathways (27). The role of various oxidative mechanisms in the microbicidal activity of macrophages depends on a number of factors including, in the case of the oxidative mechanisms, a dynamic balance between the activation state of macrophages and the sensitivity of targets cells to oxidants (12,28).…”

A Non‐damaging Method to Analyze the Configuration and Dynamics of Nitrotyrosines in Proteins

“…In fact, reduction of nitro groups to amine groups has already been reported for haem proteins. [12] Still, we were unable to finding any correlation signal. Even though MALDI-TOF analysis (Figure 3 B, left panel) showed a mass difference of (47 AE 3) Da between h-Y67 and h-Y67 N , which is consistent with a replacement of a hydrogen atom by a nitro group.…”

“…[9] Thus, recent reports on the effects of protein nitration rely on the treatment of isolated proteins with this chemical. [10][11][12][13][14][15][16] In general, phenol rings are preferentially nitrated at ortho positions (e carbons in Tyr residues) relative to the hydroxyl group. Still, nitration could also take place at meta carbons, although the yield in this case is lower.…”