Mechanism of Dioxygen Reduction by Heme-Copper Oxidases

描述(申请人提供)：这项研究的主要目的是阐明血红素铜氧化酶将氧气还原为水过程中电子和质子转移的机制。我们的具体目标将集中在四个问题上：1.用CO流动闪蒸的方法研究野生型和突变型细菌血红素铜氧化酶还原水中氧气的机理。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的动力学，并推导出过渡中间体的UV-Vis光谱。这些研究将为研究血红素-铜氧化物酶的氧还原反应机理提供新的思路。2.利用光活化染料硫代吡喃三磺酸(TUPS)研究了细菌氧化酶--大肠杆菌的bo3、球形红杆菌的aa3和嗜热杆菌的ba3的分子内电子转移。将使用时间分辨光学吸收光谱来确定中间体的光谱。通过改变标记的半胱氨酸和初始电子受体之间的距离，以及通过定点突变在假定的电子转移途径中引入断裂，将获得关于血红素-铜氧化物酶分子内电子转移途径的详细信息。3.我们将合成细胞色素氧化酶活性中心的化学类似物，包括掺入和不掺入铜配体的环状五肽(His-Pro-Glu-Val-Tyr)。这些类似物将使用稳态和时间分辨的UV-Vis光谱、FTIR和EPR进行研究，这将有助于深入了解交联剂在细胞色素氧化酶功能中的作用。4.一氧化氮(NO)是一种重要的生物调节剂。我们研究的一个新方向是了解NO如何与血红素铜氧化酶相互作用。用时间分辨多通道光学吸收光谱研究了Ru亚硝酰配合物的光解动力学，以及光生NO与血红素-铜氧化物酶及其转化中间体的反应。这些研究将绕过停流技术施加的速率限制，并提供关于NO调节细胞色素氧化酶活性的信息。

DESCRIPTION (provided by applicant): 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 4 problems: 1. The mechanism of the reduction of dioxygen to water by wild-type and mutant 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. The intramolecular electron transfer in the bacterial oxidases, bo3 from E. coli, aa3 from Rhodobacter sphaeroides and ba3 from Thermus thermophilus, will be investigated using a photoactivatable dye, thiouredopyrenetrisulfonate (TUPS), covalently linked to single reactive cysteine residues on the oxidases. Time-resolved optical absorption spectroscopy will be used to determine the spectra of the intermediates. 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. 3. We will synthesize chemical analogs of the active site of cytochrome oxidase, including the cyclic pentapeptide (His-Pro-Glu-Val-Tyr) with and without Cu-ligands incorporated. The analogs will be studied using steady-state and time-resolved UV-Vis spectroscopy, FTIR and EPR, which will provide insight into the role of the cross-link in cytochrome oxidase function. 4. Nitric oxide (NO) has emerged as an important biological regulatory agent. A new direction in our research is to understand how NO interacts with heme-copper oxidases. The photodissociation dynamics of ruthenium nitrosyl complexes and the reaction of the photoproduced NO with heme-copper oxidases and their turnover intermediates will be investigated using time-resolved multichannel optical absorption spectroscopy. These studies will circumvent rate limitation imposed by stopped-flow techniques and provide information regarding NO regulation of cytochrome oxidase activity.