CAREER: Vibrational Spectroscopic Studies of the Dynamics and Mechanism of Amphitrite Ornata Dehaloperoxidase

职业：Amphitrite Ornata 脱卤过氧化物酶动力学和机制的振动光谱研究

• 批准号: 9874895
• 负责人: Stefan Franzen
• 金额: $ 44.5万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9874895&HistoricalAwards=false
• 关键词: CAREER; Vibrational; Spectroscopic; Studies; Dynamics

FranzenMCB 9874895This is a CAREER award. The research component addresses biophysical and biochemical aspects of the function and, the marine worm dynamics of the novel enzyme dehaloperoxidase (DHP) from the marine worm, A. ornata. From a biochemical point of view there is intense interest in understanding the ability of the DHP enzyme to dehalogenate bromo- and fluorophenol by an oxidative mechanism to produce quinone. From a biophysical point of view there is a strong structural analogy with myoglobin. The structural analogy will be exploited to study the significance of conformational substates and structural relaxations of proteins as has been discussed in hundreds of biophysical studies of myoglobin. Time-resolved vibrational and transient absorption spectroscopy will be applied to the study of structural relaxations of the DHP enzyme including both the motions of the peptide backbone and the motions sensed by the heme. Novel rapid-mixing and stopped-flow experiments will be combined with these spectroscopies. The biophysical studies will focus on a test of hypotheses concerning structure/function relations that have been applied to myoglobin. Biochemical studies will contribute spectroscopic information on the mechanism of oxygen activation and substrate oxidation. The educational component focuses on modernizing the physical laboratory to include flash photolysis and resonance Raman spectroscopy experiments. Classical biophysical experiments on electron transfer and ligand dynamics will be included in the curriculum. Computational chemistry will be developed as part of the undergraduate curriculum in statistical mechanics and spectroscopy. The research will serve as an example for much of this effort since there is now an extensive literature on both experiment and computation of myoglobin that serves as model for future studies of heme proteins. The impact of these studies will be felt by more than one hundred chemistry majors who graduate annually. The focus on DHP will provide an example of the utility of these studies for enzyme function. This is both an exciting research area for graduate students and an interesting forum for interaction between the research program and undergraduate education.The study applies vibrational spectroscopy to the structure and dynamics of a recently discovered heme enzyme, dehaloperoxidase of Amphitrite ornata. The enzyme is capable of replacing bromine and fluorine with hydrogen or oxygen in a phenolic substrate. The mechanism involves binding of peroxide to the heme, but the details are not yet understood. One aspect of the research is to study mechanistic intermediates in collaboration with Dr. Dawson and Dr. Lebioda at the University of South Carolina. A second aspect of the studies addresses central issues in the interpretation of protein biophysics. The study addresses the importance of protein conformations for enzyme function. The goals are to understand how protein fluctuations affect catalysis and to understand specific structure changes that accompany binding of peroxide or the substrate and control by biological feedback mechanisms. Since dehaloperoxidase is structurally similar to myoglobin it provides a very interesting test of the generality of conclusions for protein biophysics based on studies of myoglobin. The educational component focuses on modernizing the physical laboratory using laser kinetics and light scattering experiments. Classical biophysical experiments on electron transfer and ligand dynamics will be included in the curriculum. The focus on DHP will provide an example of the utility of these studies for enzyme function. This is both an exciting research area for graduate students and an interesting forum for interaction between the research program and undergraduate education.

FranzenMCB 9874895这是一个职业奖。 研究内容涉及海洋蠕虫中新型酶脱卤过氧化物酶（DHP）的功能和海洋蠕虫动力学的生物物理和生化方面。装饰。从生物化学的角度来看，有强烈的兴趣，了解的能力，DHP酶脱卤溴和氟苯酚的氧化机制，以产生醌。从生物物理学的观点来看，肌红蛋白在结构上有很强的相似性。结构类比将被用来研究蛋白质的构象亚态和结构松弛的意义，这在肌红蛋白的数百项生物物理研究中已经讨论过。时间分辨的振动和瞬态吸收光谱将被应用到DHP酶的结构弛豫的研究，包括肽骨架的运动和血红素感测的运动。新的快速混合和停流实验将与这些光谱相结合。生物物理学研究将集中在已应用于肌红蛋白的结构/功能关系的假设的测试。生物化学研究将有助于氧活化和底物氧化机制的光谱信息。教育部分侧重于物理实验室的现代化，包括闪光光解和共振拉曼光谱实验。电子转移和配体动力学的经典生物物理实验将包括在课程中。计算化学将作为统计力学和光谱学本科课程的一部分。这项研究将作为这方面努力的一个例子，因为现在有大量关于肌红蛋白的实验和计算的文献，可以作为未来血红素蛋白研究的模型。这些研究的影响将被每年毕业的100多名化学专业学生所感受到。对DHP的关注将为这些研究对酶功能的效用提供一个例子。这是一个令人兴奋的研究领域的研究生和研究计划和本科教育之间的互动一个有趣的论坛。这项研究应用振动光谱的结构和动力学最近发现的血红素酶，脱卤过氧化物酶的装饰双壁藻。该酶能够用酚底物中的氢或氧取代溴和氟。其机制涉及过氧化物与血红素的结合，但细节尚不清楚。这项研究的一个方面是与南卡罗来纳州大学的道森博士和莱比奥达博士合作研究机械中间体。研究的第二个方面解决了蛋白质生物物理学解释中的核心问题。这项研究解决了蛋白质构象对酶功能的重要性。目标是了解蛋白质波动如何影响催化作用，并了解伴随过氧化物或底物结合的特定结构变化以及生物反馈机制的控制。由于脱卤过氧化物酶在结构上与肌红蛋白相似，它为基于肌红蛋白研究的蛋白质生物物理学结论的普遍性提供了一个非常有趣的测试。教育部分侧重于使用激光动力学和光散射实验使物理实验室现代化。电子转移和配体动力学的经典生物物理实验将包括在课程中。对DHP的关注将为这些研究对酶功能的效用提供一个例子。这既是研究生的一个令人兴奋的研究领域，也是研究计划和本科教育之间互动的一个有趣论坛。