Mechanism of Dioxygen Reduction by Heme-Copper Oxidases

本研究的主要目的是阐明血红素-铜氧化酶还原分子氧生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。将通过CO流动闪蒸法研究细菌血红素铜氧化酶将氧还原成水的机理。时间分辨的多通道光学吸收光谱，结合奇异值分解（SVD）和全球指数拟合分析，将被用来跟踪电子和质子转移的动力学，并推导出瞬态中间体的紫外-可见光谱。这些研究将为血红素铜氧化酶的双氧还原反应机理提供新的见解。2.我们将研究在不同的氧化状态下，使用由光解合成的双氧载体原位产生的双氧与牛心和细菌氧化酶的反应。我们还将这种方法扩展到核糖核苷酸还原酶（RNR）中的快速双氧结合和激活，其中反应发生得太快，无法通过传统的停流方法进行监测。3.研究了大肠杆菌（E. coil，来自Rhodobacter sphaeroides的aa 3和来自Thermus thermophilus的ba 3将使用共价连接到氧化酶上的单个反应性半胱氨酸残基的可光活化染料硫脲芘三磺酸盐（TUPS）来研究。时间分辨光学吸收光谱，结合SVD和全球指数拟合，将用于确定存在的中间体的光谱和单个电子转移步骤的速率常数。通过改变标记的半胱氨酸和初始电子受体之间的距离，并通过定点诱变引入中断到假定的电子转移途径，详细的信息，在血红素铜氧化酶的分子内电子转移途径将获得。4.我们建议制作细胞色素氧化酶的活性位点的化学类似物，包括His-Tyr交联的二肽和环状五肽（His-Pro-Glu-Val-Tyr），其具有和不具有Cu-配体掺入。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.