BIOENERGETIC MECHANISMS IN MULTICENTER ENZYMES

三种金属酶，细胞色素氧化酶，甲烷单加氧酶（MMO）和可溶性鸟苷酸（sGC）的运作机制将进行研究。 细胞色素氧化酶通过催化分子氧还原为水来维持线粒体中的电子转移活性。 它还通过以化学计量泵送质子与转运至O2的电子直接促进跨膜质子梯度，如下所示：4cyt c2+ +O2+8H+in产生4cyt c3+ +4H+ out +2H2O。双氧化学发生在血红素α 3/CuB双核中心。 我们和其他人提出，CuB直接参与质子泵。 我们最近的数据还表明，一个独特的，组氨酸交联酪氨酰自由基发生在O2还原，并参与驱动质子易位。 通过使用时间分辨的拉曼光谱，我们打算测量振动光谱的部分代谢的中间体在O2还原和评估的机制，蛋白质施加质子控制这些反应的速率。 定点突变体的sphaeroides氧化酶是可用的，我们将表征特定氨基酸的功能作用。 我们打算使用FTIR光谱，磁共振，计算方法，化学建模，和化学标记，以评估是否氧化还原活性酪氨酰残基密切参与O-O键裂解和测试模型的配体交换在CuB网站，可能是相关的质子泵中的拟议作用。 对于MMO，我们计划表征化合物Q中间体。 我们的拉曼数据表明，这种物种，进行氧键插入化学，具有Fe-2+ O2+金刚石锥结构。 对于sGC，我们将研究允许有效NO/O2区分的分子机制，并开始旨在了解NO结合如何触发环化酶活性的工作。

The mechanisms by which three metalloenzymes, cytochrome oxidase, methane monooxygenase (MMO), and soluble guanylate (sGC) operate will be studied. Cytochrome oxidase maintains electron-transfer activity in the mitochrondrion by catalyzing the reduction of dioxygen to water. It also contributes directly to the transmembrane proton gradient by pumping protons stoichiometrically with electrons transported to O2, as follows: 4 cyt c2+ +O2+8H+in yields 4cyt c3+ +4H+ out +2H2O. Dioxygen chemistry occurs at the heme alpha3/CuB binuclear center. We and others have proposed that CuB is directly involved in the proton pump. Our recent data also suggests that a unique, histidine cross-linked tyrosyl radical occurs during O2 reduction and is involved in driving proton translocation. By using time-resolved Raman spectroscopy, we intend to measure vibrational spectra for partially metabolized intermediates in O2 reduction and to assess mechanisms by which the protein imposes proton control on the rates of these reactions. Site-directed mutants of sphaeroides oxidase are available, and we will characterize the functional roles of specific amino acids. We intend to use FTIR spectroscopy, magnetic-resonance, computational methods, chemical modeling, and chemical labeling to assess whether redox-active tyrosyl residue is intimately involved in O-O bond cleavage and to test models for ligand exchange at the CuB site that may be relevant to its proposed role in the proton pump. For MMO, we plan to characterize the Compound Q intermediate. Our Raman data indicate that this species, which carries out oxygen-bond insertion chemistry, has an Fe-2+ O2+ diamond cone structure. For sGC, we will study the molecular mechanisms that allow efficient NO/O2 discrimination and begin work aimed at understanding how NO binding triggers cyclase activity.