BIOENERGETIC MECHANISMS IN MULTICENTER ENZYMES

研究了细胞色素氧化酶、甲烷单加氧酶（MMO）和可溶性鸟苷酸（sGC）三种金属酶的作用机制。细胞色素氧化酶通过催化二氧还原为水来维持线粒体中的电子转移活性。它还通过将质子以化学计量的方式泵送到O2的电子，直接促进了跨膜质子梯度，如下所示：4cyt c2+ +O2+8H+in生成4cyt c3+ +4H+ out +2H2O。二氧化学发生在血红素α 3/CuB双核中心。我们和其他人已经提出，CuB直接参与质子泵。我们最近的数据还表明，一种独特的组氨酸交联酪氨酸自由基在O2还原过程中发生，并参与驱动质子易位。通过使用时间分辨拉曼光谱，我们打算测量O2还原过程中部分代谢中间体的振动光谱，并评估蛋白质对这些反应速率施加质子控制的机制。球形氧化酶的定点突变体是可用的，我们将描述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来评估氧化还原活性酪氨酸残基是否与O-O键切割密切相关，并测试CuB位点的配体交换模型，这可能与它在质子泵中的作用有关。对于MMO，我们计划对化合物Q中间体进行表征。我们的拉曼数据表明，该物质具有Fe-2+ O2+金刚石锥结构，进行氧键插入化学反应。对于sGC，我们将研究允许有效的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.