title: Reaction of human hemoglobin with peroxynitrite. Isomerization to nitrate and secondary formation of protein radicals.
abstract: Peroxynitrite, a strong oxidant formed intravascularly in vivo, can diffuse onto erythrocytes and be largely consumed via a fast reaction (2 x 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 approximately 10% 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.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, 11800 Montevideo, Uruguay.
authors.forename: Natalia
authors.initials: N
authors.lastname: Romero
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
authors.forename: Edlaine
authors.initials: E
authors.lastname: Linares
authors.forename: Ohara
authors.initials: O
authors.lastname: Augusto
authors.forename: Charles D
authors.initials: CD
authors.lastname: Detweiler
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
authors.forename: Ana
authors.initials: A
authors.lastname: Denicola
mesh_terms: D015153:Blotting, Western; D003497:Cyclic N-Oxides / Q000737:chemistry; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005914:Globins / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007150:Immunohistochemistry; D007501:Iron / Q000737:chemistry; D007536:Isomerism; D008706:Methemoglobin / Q000737:chemistry; D009566:Nitrates / Q000032:analysis / Q000737:chemistry; D009573:Nitrites / Q000032:analysis; D010100:Oxygen / Q000032:analysis; D010108:Oxyhemoglobins / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000737:chemistry*; D013053:Spectrophotometry; D013113:Spin Labels; D013481:Superoxides / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D003497:Cyclic N-Oxides; D009566:Nitrates; D009573:Nitrites; D010108:Oxyhemoglobins; D013113:Spin Labels; D013481:Superoxides; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D005914:Globins; D008706:Methemoglobin; D006861:Hydrogen Peroxide; D007501:Iron; D010100:Oxygen