Femtosecond Coherence Spectroscopy and Ultrafast Kinetic Investigations of Heme P

描述（由申请人提供）：该项目旨在扩展我们对血红素蛋白的结构、功能和动力学的理解，如细胞色素P450、哺乳动物过氧化物酶、一氧化氮合酶、可溶性鸟苷酸环化酶、硝基磷蛋白和细胞色素c。这些蛋白质参与广泛的催化、信号传导和电子传递过程，并且能够具有惊人的广泛功能，即使血红素轴向配体是相同的。这表明蛋白质结构及其对血红素结构的影响起着重要的功能作用。利用相干光谱学和飞秒光学“泵浦探测”技术，可以在水环境中激发和分析血红素的“软”面外（OOP）低频振动模式。这些振动模式以前没有被记录下来，因为它们很难用传统的光谱方法获得。它们落在环境热激发区（<200cm-1 ~300K），因此最有可能被蛋白质用作反应坐标。观察到的这些“软”模式的相干光谱强度取决于由蛋白质结构引起的血红素结构扭曲的大小。血红素“圆顶”模式在双原子配体结合反应中的重要性证明了这些OOP血红素运动在功能上的重要性。低频血红素运动的丰富频谱才刚刚开始被欣赏，因为更广泛的蛋白质和模型化合物被检查。本项目旨在探索血红素蛋白中静态扭曲和热激发低频振动的功能作用。畸变（如血红素“褶皱”和“鞍状”）改变了铁原子与其周围分子框架之间的电子轨道相互作用，假设会影响金属中心的氧化还原电位。沿着这些相同的、热可及的OOP坐标的振动运动是调解和控制电子转移的极好候选者。相干光谱学在水溶液中探测这些模式具有独特的定位。例如，我们将检查电子转移伙伴，如Pdx和CYP101，以监测当蛋白质复合物形成时发生的低频频谱变化。Fe-S蛋白的低频模式也将被检查。超快时间尺度上的动力学探针，时间跨度超过10年，将用于研究与生化反应相关的电子重排之后立即发生的快速时间尺度非平衡过程。例如，肌红蛋白中氧与血红素再结合的两个双相表现出非常不同的阿伦尼乌斯前因子，这表明重组的熵势垒与时间有关。这种非平衡过程将被研究，以了解它们是否允许血红素蛋白增强不同类型双原子配体之间的区分。公共卫生相关性：该项目涉及血红素和铁硫金属蛋白，具有广泛的健康相关意义。许多代谢性疾病状态涉及涉及这些蛋白质的催化、信号传导和/或电子传递过程的中断。所有涉及分子电子传递和/或利用血红素或铁硫辅助因子的生物过程都与本研究有关。对蛋白质结构如何与金属辅助因子相互作用，以及如何在分子水平上转化为振动动力学和功能的基本理解，将为那些关注任何水平代谢紊乱治疗的人带来更深入的见解。金属蛋白的基础研究对于我们对人体的综合认识是必不可少的。该项目深入研究了这些系统的基础问题。

DESCRIPTION (provided by applicant): This project aims to extend our understanding of the structure, function, and dynamics of heme proteins such as cytochrome P450, mammalian peroxidases, nitric oxide synthase, soluble guanylate cyclase, nitrophorin, and cytochrome c. These proteins are involved in broad array of catalytic, signaling, and electron transport processes and are capable of an amazingly broad range of functions, even when the heme axial ligands are identical. This indicates that the protein architecture, and its influence on the heme structure, plays an important functional role. By using coherence spectroscopy, a femtosecond optical "pump-probe" technique, the "soft" out-of-plane (OOP) low-frequency vibrational modes of the heme can be excited and analyzed even in an aqueous environment. These vibrational modes have not been documented previously because they are difficult to access using traditional spectroscopic methods. They fall in the region of ambient thermal excitations (<200cm-1 ~300K) and are therefore most likely to be utilized as reaction coordinates by proteins. The observed coherence spectral intensities of these "soft" modes depend upon the magnitude of the heme structural distortions that are induced by the protein architecture. These OOP heme motions are functionally significant, as demonstrated by the importance of the heme "doming" mode in the diatomic ligand binding reaction. The rich spectrum of the low-frequency heme motions is just beginning to be appreciated, as a wider variety of proteins and model compounds is examined. This project aims to explore the functional role of both static distortions and thermally excited low-frequency vibrations in heme proteins. Distortions (such as heme "ruffling" and "saddling") that alter the electronic orbital interactions between the iron atom and its surrounding molecular framework are hypothesized to affect the redox potential of the metal center. Vibrational motions along these same, thermally accessible, OOP coordinates are excellent candidates to mediate and control electron transfer. Coherence spectroscopy is uniquely positioned to probe these modes in aqueous solution. For example, we will examine electron transfer partners, such as Pdx and CYP101, in order to monitor changes in the low frequency spectrum that occur when the protein complex is formed. The low frequency modes of Fe-S proteins will also be examined. Kinetic probes on ultrafast timescales, stretching over 10 decades in time, will be used to study the rapid time-scale, non-equilibrium processes, that take place immediately following the electronic rearrangements associated with biochemical reactions. For example, the two geminate phases for oxygen rebinding to the heme in myoglobin exhibit very different Arrhenius prefactors, suggesting that the entropic barrier for recombination is time dependent. Such non-equilibrium processes will be studied to learn if they allow heme proteins to enhance discrimination between different classes of diatomic ligands. PUBLIC HEALTH RELEVANCE: This project has a wide range of health related implications involving heme and iron-sulfur metalloproteins. Many metabolic disease states involve disrupted catalytic, signaling, and/or electron transport processes that involve such proteins. All biological processes that involve molecular electron transport, and/or utilize the heme or iron-sulfur cofactors, are related to this research. A fundamental understanding of how the protein architecture interacts with the metal co-factors, and how this translates into vibrational dynamics and function at the molecular level, will lead to deeper insights for those concerned with the treatment of metabolic disorders at any level. Basic research on metalloproteins is essential to our composite understanding of the human body. This project is deeply involved with investigations of these systems at a fundamental level.