CAREER: Correlating Metalloenzyme Structure with Reactivity By Tunneling Electrons in Crystals

归根结底，生命的本质是控制电荷的运动。多肽链具有大量的构象和化学状态，这使得大自然可以调节金属中心的反应性，并指导蛋白质内部和蛋白质之间的电子流动。Crane博士的研究目标是开发和应用新的光化学方法来研究生物学中氧化还原化学和远程电子转移（ET）的结构基础。光敏剂将用于引发单蛋白晶体中的远程ET，其中结构可以通过x射线晶体学精确定义。设计的晶体系统可以固定供体和受体的取向，通过诱变和同位素取代来探测蛋白质和水结构对电子隧穿的影响。ET速率将在金属修饰的azurins晶体和细胞色素c与细胞色素c过氧化物酶之间的配合物晶体中直接确定。蛋白质金属中心的氧化还原反应将在晶体中由光诱导的ET驱动，因此对催化重要的激活态结构可以通过冷冻晶体学或时间分辨衍射技术来确定。将确定过氧化物氧化酶和过氧化物氧化铁的结构，它们是生物氧活化的重要中间体，以及色氨酸和酪氨酸自由基，它们是各种高电位生物氧化还原化学中的新兴参与者。原子结构将与电子结构相关联，用磁学和光谱学探测，以了解金属辅助因子与多肽链之间的反应性相互调节。本研究结果将为详细定义的结构状态提供一套远程ET速率，为迄今未观察到的金属蛋白催化物种提供新结构，并为通过x射线衍射研究蛋白质动力学提供新技术。与这项研究相关的教育活动旨在将结构、动力学、热力学和化学反应性等基本概念与复杂的生物过程（如能量转导、信号转导、环境敏感性和反应）联系起来。主要分为两大类：1)指导高中生、本科生和研究生进行研究；2)康奈尔大学新本科和研究生课程的开发和教学。K-12阶段的发现式学习也将得到推广。上述研究旨在提供适合所有水平的学生的项目。目前正在教授一门新的研究生课程“蛋白质的物理化学”，第二门课程“生物无机化学”将于2003年提出。其他课程活动包括为化学专业大一新生开设化学结构和键合的入门课程，以及酶动力学的跨系短期课程。在所有活动中都强调以一种有趣的方式来解决问题。

Ultimately, the essence of life is the controlled movement of charge. The vast number of conformational and chemical states available to the polypeptide chain allows nature to tune the reactivity of metal centers and direct electron flow within and between proteins. The goal of Dr. Crane's research is to develop and apply new photochemical methods for studying the structural basis of redox chemistry and long-range electron transfer (ET) in biology. Photosensitizers will be used to initiate long-range ET in single protein crystals, where structure can be precisely defined by X-ray crystallography. Designed crystal systems that fix donor and acceptor orientations allow effects of intervening protein and water structure on electron tunneling to be probed by mutagenesis and isotopic substitution. ET rates will be determined directly in crystals of metal-modified azurins and crystals of complexes between cytochrome c and cytochrome c peroxidase. Redox reactions at protein metal centers will be driven in crystals by photoinduced ET so that structures of activated states important for catalysis can be determined by cryo-crystallography or time-resolved diffraction techniques. Structures will be determined for peroxidase peroxo-iron and oxo-iron species, important intermediates in biological oxygen activation, and for tryptophan and tyrosine radicals, emerging players in a wide variety of high-potential, biological redox chemistries. Atomic structure will be correlated with electronic structure probed with magnetic and optical spectroscopies to understand the reciprocal tuning of reactivity between metallocofactors and the polypeptide chain. The results of this research will provide sets of long-range ET rates for structural states defined in detail, new structures of hitherto unobserved metalloprotein catalytic species, and new techniques for studying protein dynamics by X-ray diffraction.Educational activities connected with this research aim to relate fundamental concepts in structure, kinetics, thermodynamics and chemical reactivity to complex biological processes such as energy transduction, signal transduction, environmental sensitivity and response. Effort falls into two general categories: 1) mentoring high-school, undergraduate, and graduate students in research; and 2) the development and teaching of new undergraduate and graduate courses at Cornell University. Discovery-based learning at the K-12 level will also be promoted. The research described above is designed to provide projects appropriate for students of all levels. A new graduate course, " the physical chemistry of proteins" is now being taught and a second "Bio-inorganic chemistry" will be presented in 2003. Other course activities include development of an introductory course for freshman chemistry majors in chemical structure and bonding, and an interdepartmental short course in enzyme kinetics. A playful approach to addressing problems will be emphasized in all activities.