Herold S, Exner M, Nauser T
Laboratorium für Anorganische Chemie, Eidgenössische Technische Hochschule, Universitätsstrasse 6, CH-8092 Zürich, Switzerland.
Biochemistry. 2001 Mar 20;40(11):3385-95. doi: 10.1021/bi002407m.
The second-order rate constants for the reactions between nitrogen monoxide and oxymyoglobin or oxyhemoglobin, determined by stopped-flow spectroscopy, increase with increasing pH. At pH 7.0 the rates are (43.6 +/- 0.5) x 10(6) M(-1) x s(-1) for oxymyoglobin and (89 +/- 3) x 10(6) M(-1) x s(-1) for oxyhemoglobin (per heme), whereas at pH 9.5 they are (97 +/- 3) x 10(6) M(-1) x s(-1) and (144 +/- 3) x 10(6) M(-1) x s(-1), respectively. The rate constants for the reaction between oxyhemoglobin and NO* depend neither on the association grade of the protein (dimer/tetramer) nor on the concentration of the phosphate buffer (100-1 mM). The nitrogen monoxide-mediated oxidations of oxymyoglobin and oxyhemoglobin proceed via intermediate peroxynitrito complexes which were characterized by rapid scan UV/vis spectroscopy. The two complexes MbFe(III)OONO and HbFe(III)OONO display very similar spectra with absorption maxima around 500 and 635 nm. These species can be observed at alkaline pH but rapidly decay to the met-form of the proteins under neutral or acidic conditions. The rate of decay of MbFe(III)OONO increases with decreasing pH and is significantly larger than those of the analogous complexes of the two subunits of hemoglobin. No free peroxynitrite is formed during these reactions, and nitrate is formed quantitatively, at both pH 7.0 and 9.0. This result indicates that, as confirmed from protein analysis after reacting the proteins with NO* for 10 times, when peroxynitrite is coordinated to the heme of myoglobin or hemoglobin it rapidly isomerizes to nitrate without nitrating the globins in physiologically significant amounts.
通过停流光谱法测定，一氧化氮与氧合肌红蛋白或氧合血红蛋白反应的二级速率常数随pH值升高而增大。在pH 7.0时，氧合肌红蛋白的反应速率为(43.6±0.5)×10⁶ M⁻¹·s⁻¹，氧合血红蛋白（每个血红素）的反应速率为(89±3)×10⁶ M⁻¹·s⁻¹；而在pH 9.5时，它们分别为(97±3)×10⁶ M⁻¹·s⁻¹和(144±3)×10⁶ M⁻¹·s⁻¹。氧合血红蛋白与NO反应的速率常数既不取决于蛋白质的缔合程度（二聚体/四聚体），也不取决于磷酸盐缓冲液的浓度（100 - 1 mM）。一氧化氮介导的氧合肌红蛋白和氧合血红蛋白的氧化反应通过中间过氧亚硝酸根络合物进行，这些络合物通过快速扫描紫外/可见光谱进行表征。两种络合物MbFe(III)OONO和HbFe(III)OONO具有非常相似的光谱，吸收最大值在500和635 nm左右。这些物种在碱性pH下可以观察到，但在中性或酸性条件下会迅速衰变为蛋白质的高铁形式。MbFe(III)OONO的衰变速率随pH值降低而增加，且明显大于血红蛋白两个亚基类似络合物的衰变速率。在这些反应过程中没有形成游离的过氧亚硝酸根，并且在pH 7.0和9.0时都定量地形成了硝酸盐。该结果表明，正如在蛋白质与NO反应10次后的蛋白质分析中所证实的，当过氧亚硝酸根与肌红蛋白或血红蛋白的血红素配位时，它会迅速异构化为硝酸盐，而不会以生理上显著的量硝化球蛋白。
