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

这是一个职业奖项。研究部分涉及海洋蠕虫脱氢过氧化物酶(DHP)的功能和海洋蠕虫动力学的生物物理和生化方面的研究。从生物化学的角度来看，人们对了解DHP酶通过氧化机制使溴和氟苯酚脱卤化以产生苯二酚的能力非常感兴趣。从生物物理学的角度来看，肌红蛋白在结构上与肌红蛋白有很强的相似之处。结构类比将被用来研究蛋白质构象亚态和结构松弛的意义，就像在成百上千的肌红蛋白生物物理研究中所讨论的那样。时间分辨振动和瞬时吸收光谱将被用于研究DHP酶的结构松弛，包括肽骨架的运动和被血红素感觉到的运动。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于对已应用于肌红蛋白的有关结构/功能关系的假说进行测试。生化研究将提供氧活化和底物氧化机制的光谱信息。教育部分的重点是使物理实验室现代化，以包括闪光光解和共振拉曼光谱实验。电子转移和配基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工作的一个例子，因为现在有大量关于肌红蛋白的实验和计算的文献，作为未来对血红素蛋白的研究的模型。每年毕业的100多名化学专业学生将感受到这些研究的影响。对DHP的关注将为这些研究在酶功能方面的应用提供一个例子。这对研究生来说既是一个令人兴奋的研究领域，也是研究项目和本科教育之间互动的一个有趣的论坛。这项研究将振动光谱应用于最近发现的一种血红素酶--脱卤素过氧化物酶的结构和动力学。该酶能够在酚类底物中用氢或氧取代溴和氟。其机制涉及过氧化氢与血红素的结合，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中心问题。这项研究阐述了蛋白质构象对酶功能的重要性。目标是了解蛋白质波动是如何影响催化作用的，并了解伴随着过氧化氢或底物结合以及生物反馈机制控制的特定结构变化。由于脱氢过氧化物酶在结构上类似于肌红蛋白，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检验。教育部分的重点是利用激光动力学和光散射实验使物理实验室现代化。电子转移和配基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在酶功能方面的应用提供一个例子。这对研究生来说既是一个令人兴奋的研究领域，也是研究项目和本科教育之间互动的一个有趣的论坛。

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.