Mechanism of Dioxygen Reduction by Heme-Copper Oxidases

本研究的主要目的是阐明血红素-铜氧化酶将二氧还原成水过程中电子和质子转移的机理。我们的具体目标将集中在四个问题上：1。采用CO流动闪蒸法研究细菌血红素-铜氧化酶将二氧还原为水的机理。时间分辨多通道光学吸收光谱，结合奇异值分解（SVD）和全局指数拟合分析，将用于跟踪电子和质子转移动力学，并推断瞬态中间体的紫外-可见光谱。这些研究将为血红素-铜氧化酶双氧还原反应的机理提供新的认识。2. 我们将研究在不同氧化状态下，利用由光解合成的双氧载体原位产生的双氧与牛心脏和细菌氧化酶的反应。我们还将把这种方法扩展到核糖核苷酸还原酶（RNR）的快速双氧结合和激活，在这种情况下，反应发生得太快，无法通过传统的停流方法进行监测。3. 细菌氧化酶的分子内电子转移，bo3来自E. coil， aa3来自球形红杆菌和ba3来自嗜热热菌将使用光活化染料硫脲-三磺酸酯（up）进行研究，该染料与氧化酶上的单活性半胱氨酸残基共价连接。时间分辨光学吸收光谱，结合SVD和全局指数拟合，将用于确定存在的中间产物的光谱和单个电子转移步骤的速率常数。通过改变标记半胱氨酸和初始电子受体之间的距离，并通过位点定向诱变在假定的电子转移途径中引入断裂，将获得关于血红素-铜氧化酶分子内电子转移途径的详细信息。4. 我们建议制造细胞色素氧化酶活性位点的化学类似物，包括His-Tyr交联二肽和环五肽（His-Pro-Glu-Val-Tyr），并结合或不结合cu配体。这些类似物将使用多光谱方法进行研究，包括稳态和时间分辨紫外-可见光谱、FTIR和ESR。

The primary objective of this research is to elucidate the mechanism of electron and proton transfer during the reduction of dioxygen to water by heme-copper oxidases. Our specific aims will focus on four problems: 1. The mechanism of the reduction of dioxygen to water by bacterial heme-copper oxidases will be studied by the CO flow-flash method. Time-resolved multichannel optical absorption spectroscopy, in conjunction with singular value decomposition (SVD) and global exponential fitting analysis, will be used to follow the kinetics of electron and proton transfer and to deduce the UV-Vis spectra of the transient intermediates. These studies should provide new insight into the mechanism of the dioxygen reduction reaction by heme-copper oxidases. 2. We will investigate the reaction of dioxygen with bovine heart and bacterial oxidases in different oxidation states using dioxygen which is produced in situ by photodissociating synthetic dioxygen carriers. We will also extend this approach to rapid dioxygen binding and activation in ribonucleotide reductase (RNR), in which the reactions occur too rapidly to be monitored by conventional stopped-flow methods. 3. The intramolecular electron transfer in the bacterial oxidases, bo3 from E. coil, aa3 from Rhodobacter sphaeroides and ba3 from Thermus thermophilus will be investigated using a photoactivatable dye, thiouredopyrene-trisulfonate (TUPS), covalently linked to single reactive cysteine residues on the oxidases. Time-resolved optical absorption spectroscopy, in conjunction with SVD and global exponential fitting, will be used to determine the spectra of the intermediates present and the rate constants of individual electron transfer steps. By varying the distance between the labeled cysteine and the initial electron acceptor and by introducing breaks into presumed electron transfer pathways by site-directed mutagenesis, detailed information regarding intramolecular electron transfer pathways in heme-copper oxidases will be obtained. 4. We propose to make chemical analogs of the active site of cytochrome oxidase, including the His-Tyr cross-linked dipeptide and the cyclic pentapeptide (His-Pro-Glu-Val-Tyr) with and without Cu-ligands incorporated. The analogs will be studied using a multispectroscopic approach, including steady-state and time-resolved UV-Vis spectroscopy, FTIR and ESR.