Myoglobin as a Nitrite Reductase that Regulates Hypoxic Cardiac NO Signaling

描述（由申请人提供）：血红素蛋白如肌红蛋白和血红蛋白催化一氧化氮（NO）和亚硝酸盐的代谢，从而调节细胞和组织对缺氧的反应，这是一个新兴的认识。在过去的五年中，我们的团队发表了生化和生理学研究，表明血红蛋白作为亚硝酸盐还原酶在生理和病理性缺氧下产生NO的新功能，这表明肌红蛋白，另一种主要的血红蛋白，也可能具有以受氧张力影响的方式介导氮氧化物代谢的能力。与细菌亚硝酸盐还原酶类似，亚硝酸盐的质子和电子转移反应将阴离子还原为NO。我们还发现了一种新的亚硝酸盐酸酐酶活性，它可以将两个亚硝酸盐分子转化为高度扩散的亚硝化分子N2O3，从而在富含血红素的环境中实现高效的NO信号传导。在目前的研究计划中，NO/ N2O3信号通路将在肌红蛋白中进行探索，特别关注通过a)通过调节线粒体电子转移反应调节细胞代谢和b)在缺血再灌注损伤后激活净细胞保护细胞信号反应来调节细胞对缺氧和缺血应激的反应的能力。这些概念将通过验证肌红蛋白介导的亚硝酸盐代谢调节缺氧NO信号并促进NO在心脏中的生物利用度的假设来解决。更具体地说，利用体外生化和细胞培养系统中的突变肌红蛋白以及心肌梗死的体内模型，我们的目标是：1)确定缺氧时肌红蛋白亚硝酸盐还原酶活性的分子和酶机制；2)确定肌红蛋白衍生的NO和N2O3的关键分子靶点；3)确定肌红蛋白在缺血/再灌注后调控下游细胞保护信号（凋亡和线粒体生物发生）中的作用。该研究计划的成功完成将促进我们对肌红蛋白的生物学功能以及亚硝酸盐在心血管系统中的生理和药理潜力的理解。

DESCRIPTION (provided by applicant): There is an emerging appreciation that heme proteins such as myoglobin and hemoglobin catalyze the metabolism of nitric oxide (NO) and nitrite, and thereby modulate cell and tissue responses to hypoxia. Over the last five years our group has published biochemical and physiological studies that suggest a novel function for hemoglobin as a nitrite reductase that generates NO under physiological and pathological hypoxia, suggesting that myoglobin, the other major heme globin, may also possess a capability to mediate the metabolism of oxides of nitrogen in a manner influenced by oxygen tension. Analogous to the bacterial nitrite reductases, a concerted proton and electron transfer reaction to nitrite reduces the anion to NO. We have also discovered a novel nitrite anhydrase activity that converts two nitrite molecules into the highly diffusible, nitrosating molecule N2O3, allowing efficient NO signaling in a heme rich environment. In the current research proposal, the NO/ N2O3 signaling pathways will be explored in myoglobin, with special focus on an ability to regulate cellular responses to hypoxic and ischemic stress by a) modulating cellular metabolism via the regulation of mitochondrial electron transfer reactions and b) activating net cytoprotective cell signaling reactions after ischemia-reperfusion injury. These concepts will be addressed by testing the hypothesis that myoglobin-mediated nitrite metabolism regulates hypoxic NO signaling and promotes NO bioavailability in the heart. More specifically, using mutant myoglobin proteins in in vitro biochemical and cell culture systems as well as an in vivo model of myocardial infarction, we aim 1) to determine the molecular and enzymatic mechanisms underlying the nitrite reductase activity of myoglobin in hypoxia, 2) define critical molecular targets of myoglobin-derived NO and N2O3, and 3) to determine the role of myoglobin in regulating downstream cytoprotective signaling (apoptosis and mitochondrial biogenesis) after ischemia/reperfusion. Successful completion of the proposed research plan will advance our understanding of the biological function of myoglobin and the physiological and pharmacological potential of nitrite in the cardiovascular system.