Mechanism of Dioxygen Reduction by Heme-Copper Oxidases

血红素铜氧化酶还原分子氧的机制

• 批准号: 7250853
• 负责人: OLOF EINARSDOTTIR
• 金额: $ 26.5万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7250853
• 关键词: Active Sites; Apoptosis; Biological; Bos taurus; Cattle; Cell Respiration; Chemicals; Complex; Cysteine; Dioxygen; Dyes; Electron Transport; Electronics; Electrons; Escherichia coli; Future; Heart; Heme; Histidine; Hydrogen; Individual; Inflammation; Kinetics; Label; Ligands; Link; Methods; Modeling; Muscle relaxation phase; Mutate; Nitric Oxide; Numbers; Object Attachment; Optics; Oxidants; Oxidases; Pathologic Processes; Pathway interactions; Phenols; Physiological; Play; Process; Protons; Rate; Reaction; Regulation; Research; Rhodobacter sphaeroides; Roentgen Rays; Role; Ruthenium; Site; Site-Directed Mutagenesis; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Structure; Thermus thermophilus; Time; Tyrosine; Water; Work; absorption; analog; circular magnetic dichroism; copper oxidase; crosslink; cytochrome c oxidase; in vivo; inhibitor/antagonist; insight; mutant; nanosecond; prolylglutamic acid; research study; stop flow technique; thiouredopyrenetrisulfonate; time use; valyltyrosine

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.

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分子氧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。将通过CO流动闪蒸法研究野生型和突变型细菌血红素铜氧化酶将分子氧还原为水的机制。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.研究了大肠杆菌（E.大肠杆菌中，aa 3从Rhodobacter sphaeroides和ba 3从Thermus thermophilus，将研究使用光活化染料，硫脲芘三磺酸盐（TUPS），共价连接到单一的活性半胱氨酸残基的氧化酶。将使用时间分辨吸收光谱法测定中间体的光谱。通过改变标记的半胱氨酸和初始电子受体之间的距离，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。3.我们将合成细胞色素氧化酶活性位点的化学类似物，包括掺入和不掺入铜配体的环状五肽（His-Pro-Glu-Val-Tyr）。类似物将使用稳态和时间分辨紫外-可见光谱，FTIR和EPR，这将提供深入了解细胞色素氧化酶功能中的交联作用进行研究。4.一氧化氮（NO）已成为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。钌亚硝酰络合物的光解动力学和光产生的NO与血红素铜氧化酶及其周转中间体的反应将使用时间分辨多通道光学吸收光谱进行研究。这些研究将规避停流技术所施加的速率限制，并提供有关NO调节细胞色素氧化酶活性的信息。