Femtosecond Coherence Spectroscopy & Ultrafast Kinetic Study of Heme Proteins

这个项目旨在扩大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、可溶性鸟苷环化酶 这些蛋白质参与了广泛的催化、信号转导和细胞色素c的合成。 电子传输过程，并能够具有令人惊讶的广泛功能，即使当 血红素轴向配体是相同的。这表明蛋白质的结构，以及它对 血红素的结构，起着重要的功能作用。通过使用相干光谱，一飞秒 光学“泵浦-探测”技术，“软”的离面(OOP)低频振动模 即使在水环境中，血红素也可以被激发和分析。这些振动模式并没有 以前记录过，因为它们很难使用传统光谱进行访问 方法：研究方法。它们落在环境热激发区(&lt；200 cm-1~300K)，因此最 很可能被蛋白质用作反应的配位体。观测到的相干谱强度 这些“软”模式取决于血红素结构扭曲的程度 蛋白质结构。这些面向对象的血红素运动在功能上意义重大，如 血红素“穹顶”模式在双原子配基结合反应中的重要性。丰富的光谱， 作为一种更广泛的蛋白质和模型，低频率的血红素运动才刚刚开始被人们所认识 对化合物进行了研究。该项目旨在探索静态扭曲和 血红素蛋白中的热激发低频振动。扭曲(如亚铁血红素“褶皱”和 改变铁原子与其周围环境之间的电子轨道相互作用 假设分子骨架影响金属中心的氧化还原电位。振动的 沿着这些相同的、可热访问的Oop坐标的运动是进行调解的极佳候选 并控制电子转移。相干光谱学在探测这些模式方面具有独特的地位 水溶液。例如，我们将检查电子转移伙伴，如PDX和CYP101，在 以监测当蛋白质复合体形成时发生的低频频谱的变化。 还将研究铁-S蛋白的低频模式。超快时间尺度上的动力学探测器， 超过10年的时间，将被用来研究快速的时间尺度的非平衡过程， 这发生在与生化相关的电子重排之后 反应。例如，肌红蛋白中氧与血红素重新结合的两个双相。 非常不同的Arrhenius前置因素，表明重组的熵障碍是时间 依附的。将对这种非平衡过程进行研究，以了解它们是否允许血红素蛋白 加强对不同类别的双原子配体的区分。

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.