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: 6983544
• 负责人: OLOF EINARSDOTTIR
• 金额: $ 27.55万
• 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/6983544
• 关键词: Escherichia coli; bacterial proteins; circular magnetic dichroism; crosslink; cytochrome oxidase; electron spin resonance spectroscopy; electron transport; enzyme activity; enzyme mechanism; nitric oxide; oxidation reduction reaction; oxygen; peptide analog; peptide chemical synthesis; photoactivation; site directed mutagenesis; water

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）：这项研究的主要目的是阐明在将双氧氧氧化酶二氧化物还原为水中的电子和质子转移的机理。我们的具体目的将重点放在4个问题上：1。将通过CO流闪闪式方法研究野生型和突变细菌血红素氧化酶将二氧化物还原为水的机理。时间分辨的多通道光吸收光谱与奇异值分解（SVD）和全局指数拟合分析结合使用，将用于遵循电子和质子传递的动力学，并推导瞬态中间体的UV-VIS光谱。这些研究应提供有关血红素氧化酶二氧化量还原反应机理的新见解。 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 。时间分辨的光吸收光谱将用于确定中间体的光谱。通过改变标记的半胱氨酸和初始电子受体之间的距离，并通过位置定向的诱变将断裂引入假定的电子传递途径，可以获得有关分子内电子转移途径的详细信息，以详细信息获得血红素氧化酶中的分子内电子转移途径。 3。我们将合成细胞色素氧化酶的活性位点的化学类似物，包括具有和不含Cu-ligands的循环五肽（His-Pro-Glu-Val-Tyr）。将使用稳态和时间分辨的UV-VIS光谱，FTIR和EPR来研究类似物，这将提供有关交联在细胞色素氧化酶功能中的作用的洞察力。 4。一氧化氮（NO）已成为重要的生物学调节剂。我们研究中的一个新方向是了解NO如何与血红素氧化酶相互作用。将使用时间分辨的多机通道光吸收光谱研究研究，将研究硝基糖基络合物的光解离动力学和光早期NO与血红素氧化酶及其周转中间体的反应。这些研究将通过停止流量技术施加的速率限制，并提供有关细胞色素氧化酶活性的不调节的信息。