Mechanism of Dioxygen Reduction by Heme-Copper Oxidases

描述（由申请人提供）：本研究的主要目的是阐明血红素-铜氧化酶将二氧还原为水的电子和质子转移机制。我们的具体目标将集中在四个问题上：1。采用CO流动闪蒸法研究了野生型和突变型细菌血红素-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. 细菌氧化酶中的bo3来自大肠杆菌，aa3来自球形红杆菌和ba3来自嗜热热菌，将使用一种可光激活的染料硫脲聚氰脲三磺酸盐（up）来研究分子内电子转移，这种染料与氧化酶上的单活性半胱氨酸残基共价连接。时间分辨光吸收光谱法将用于测定中间体的光谱。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。3. 我们将合成细胞色素氧化酶活性位点的化学类似物，包括含和不含cu配体的环五肽（His-Pro-Glu-Val-Tyr）。这些类似物将使用稳态和时间分辨紫外-可见光谱，FTIR和EPR进行研究，这将深入了解交联在细胞色素氧化酶功能中的作用。4. 一氧化氮（NO）已成为一种重要的生物调节剂。了解NO与血红素-铜氧化酶的相互作用是我们研究的一个新方向。利用时间分辨多通道光学吸收光谱技术研究钌亚硝基配合物的光解动力学以及光解产物NO与血红素-铜氧化酶及其转化中间体的反应。这些研究将规避由止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.