Romero Natalia, Radi Rafael, Linares Edlaine, Augusto Ohara, Detweiler Charles D, Mason Ronald P, Denicola Ana
Departamento de Bioquímica, Facultad de Medicina, 11800 Montevideo, Uruguay.
J Biol Chem. 2003 Nov 7;278(45):44049-57. doi: 10.1074/jbc.M305895200. Epub 2003 Aug 13.
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.
过氧亚硝酸根是一种在体内血管内形成的强氧化剂，它可以扩散到红细胞上，并通过与氧合血红蛋白的快速反应（2×10⁴ m⁻¹ s⁻¹）被大量消耗。过氧亚硝酸根与氧合血红蛋白反应生成高铁血红蛋白的反应机制仍有待阐明。在这项工作中，我们在生物学相关条件下，使用毫摩尔浓度的氧合血红蛋白以及氧合血红蛋白相对于过氧亚硝酸根化学计量过量的情况下研究了该反应。结果支持一种反应机制，该机制涉及亚铁血红素的净单电子氧化、过氧亚硝酸根异构化为硝酸根、超氧自由基和过氧化氢的产生。过氧亚硝酸根在血红素铁内部的均裂使得大约10%产率的高铁血红蛋白得以形成，其可以以珠蛋白部分的还原当量为代价衰变为高铁血红蛋白。事实上，使用2-甲基-2-亚硝基丙烷和5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）的自旋捕获研究证明了酪氨酸基和半胱氨酸基衍生自由基的形成。DMPO还抑制了共价连接的二聚体产物，并导致了DMPO-血红蛋白加合物的形成。除非使用过量的过氧亚硝酸根相对于氧合血红蛋白，否则未观察到血红蛋白硝化，这与二氧化氮的少量形成一致。所获得的结果支持氧合血红蛋白作为过氧亚硝酸根相关的血管内清除剂的作用。
