Mechanism of NO-induced oxidation of myoglobin and hemoglobin
Mechanism of NO-induced oxidation of myoglobin and hemoglobin
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DOI：
10.1021/bi960442g
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发表时间：
1996-06-04
期刊：
影响因子：
2.9
通讯作者：
Olson, JS
中科院分区：
文献类型：
--
作者：
Eich, RF;Li, TS;Olson, JS
一氧化氮(NO)参与多种生理功能，包括神经传递、血小板聚集、巨噬细胞功能和血管扩张。细胞外血红蛋白消耗NO和随后的血管收缩被认为是在基于血红蛋白的O-2携带者的体内试验中报告的轻度高血压事件的原因。内皮细胞中NO的耗竭很可能是由于NO与微动脉及周围组织中的氧合血红蛋白的氧化反应所致，为了确定这一关键反应的机制，我们测量了各种不同的重组抹香鲸肌红蛋白(Mb)和人血红蛋白(Hb)在NO诱导下的氧化动力学。观察到的速率与[NO]呈线性关系，但与[O-2]无关。NO诱导氧化MBO(2)和HBO(2)的双分子速率常数很大(野生型蛋白质的k‘(ox，NO)=30-50muM(-1)S(-1))，与简单的一氧化氮与脱氧Mb和Hb结合的速率常数相似，可逆的NO结合和NO诱导的氧化都发生在两个步骤：(1)一氧化氮进入血红素袋的远端；(2)非共价结合的一氧化氮与铁原子快速反应生成Fe2+-N=O或与Fe2+-O-O-Delta反应生成Fe3+-OH2和硝酸盐。当His(E7)被脂肪残基取代时，抹香鲸Mb的氧化和结合速率常数都增加了。这些突变体在远端口袋中缺乏极性相互作用，这通常阻止不进入蛋白质。用芳香族氨基酸取代亮氨酸(B10)和Val(E11)，减少远端神经袋的体积，可显著抑制NO诱导的MBO(2)氧化。后者的结果为减少基于细胞外血红蛋白的O-2载体引起的高血压事件提供了一种蛋白质工程策略。通过检测PHC(B10)和Phe(E11)替换对NO诱导的重组人血红蛋白α和β亚基氧化速率的影响，探索了这一方法。
Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. The consumption of NO by extracellular hemoglobin and subsequent vasoconstriction have been suggested to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O-2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue, In order to determine the mechanism of this key reaction, we have measured the kinetics of NO-induced oxidation of a variety of different recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rates depend linearly on [NO] but show no dependence on [O-2]. The bimolecular rate constants for NO-induced oxidation of MbO(2) and HbO(2) are large (k'(ox,NO)=30-50 mu M(-1) s(-1) for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb and Hb, Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitric oxide with the iron atom to produce Fe2+-N=O or with Fe2+-O-O-delta- to produce Fe3+-OH2 and nitrate, Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the distal pocket which normally hinder NO entry into the protein. Decreasing the volume of the distal pocket by replacing Leu(B10) and Val(E11) with aromatic amino acids markedly inhibits NO-induced oxidation of MbO(2). The latter results provide a protein engineering strategy for reducing hypertensive events caused by extracellular hemoglobin-based O-2 carriers, This approach has been explored by examining the effects of Phc(B10) and Phe(E11) substitutions on the rates of NO-induced oxidation of the alpha and beta subunits in recombinant human hemoglobin.