Electron Transfer in Iron and Copper Proteins

描述（由申请人提供）：许多生物能量转导途径依赖于电子或空穴通过蛋白质长距离（>30？）的快速运动。许多氧化还原酶，特别是参与氧活化和产生的那些，需要在高电位下转移空穴，其中氧化还原活性氨基酸的侧链可以参与其中。必须满足严格的设计要求，以沿着特定路径快速有效地传输电荷，并防止氧化还原等价物的偏离路径扩散和能量流的中断。在这项研究计划中，控制多步电子隧穿通过色氨酸（目标1）和酪氨酸（目标2）自由基的分子参数将被检查。将制备几种天青蛋白突变体，其中色氨酸残基位于铜活性位点和表面附着的光敏剂之间。动力学测量将用于阐明氧化还原位点分离距离，驱动力和色氨酸环境对远程电荷传输动力学的作用。酪氨酸被假定为介导在几种氧化还原酶中的远程空穴传输。天青蛋白突变体中的酪氨酸残基被放置作为介质的远程电子转移将被制备和研究。还将制备其中质子接受残基在中间酪氨酸残基的氢键距离内的突变蛋白。预期这些近端质子受体通过酪氨酸自由基促进长程跳跃。氨基酸侧链自由基通常存在于参与氧活化和形成化学的酶中。这类酶的重要成员是血红素-硫醇盐单加氧酶，例如细胞色素P450和一氧化氮合酶（NOS）。这些酶利用分子氧来实现涉及有机底物的广泛的羟基化反应。尽管在许多实验室中进行了广泛的研究，但关于这些酶中活性羟基化试剂的性质仍然存在问题：铁基物质（即，Fe-氧代化合物I和II）经常被调用。蛋白质标记和激光猝灭光化学将用于开发新的见解血红素硫醇单加氧酶的活性中间体。而不是处理酶与活性氧化合物，光化学产生的单电子，外层氧化剂共价结合到蛋白质的外围将被用来消除电子从休息酶和生产铁化合物。努力将集中在细胞色素P450（目标3）和表现出NOS样反应性的细菌酶（目标4）中化合物I和II的氧化生成。公共卫生相关性：还原和氧化（氧化还原）反应是一系列代谢过程中的重要转化。无数的疾病都与氧化还原途径的失败或中断有关。阐明控制生物氧化还原过程的基本化学因素将导致对疾病机制的更深入理解，并指导新疗法的开发。

DESCRIPTION (provided by applicant): A great many biological energy transduction pathways depend upon the rapid movement of electrons or holes over long distances (>30 ¿) through proteins. Many redox enzymes, particularly those involved in oxygen activation and production, require the transfer of holes at high potentials where the sidechains of redox-active amino acids can become involved. Stringent design requirements must be met to transport charges rapidly and efficiently along specific pathways and prevent the off- path diffusion of redox equivalents and the disruption of energy flow. In this research program, the molecular parameters that control multistep electron tunneling through tryptophan (Aim 1) and tyrosine (Aim 2) radicals will be examined. Several azurin mutants in which tryptophan residues lie between the Cu active site and a surface-attached photosensitizer will be prepared. Kinetics measurements will be used to elucidate the roles of redox-site separation distance, driving force, and tryptophan environment on the dynamics of long-range charge transport. Tyrosine is postulated to mediate long-range hole transport in several redox enzymes. Azurin mutants in which tyrosine residues are placed to serve as mediators of long-range electron transfer will be prepared and studied. Mutant proteins in which proton accepting residues are within hydrogen-bonding distance of the intermediate tyrosine residue also will be prepared. These proximal proton acceptors are expected to facilitate long-range hopping via tyrosine radicals. Amino-acid sidechain radicals often are found in enzymes involved in oxygen activation and formation chemistry. Important members of this class of enzymes are the heme-thiolate mono- oxygenases such as cytochrome P450 and nitric oxide synthases (NOS). These enzymes utilize molecular oxygen to effect a wide range of hydroxylation reactions involving organic substrates. Despite extensive investigations in many laboratories, questions remain regarding the nature of the active hydroxylating reagent in these enzymes: ferryl species (i.e., Fe-oxo Compounds I and II) are often invoked. Protein labeling and laser flash-quench photochemistry will be used to develop new insights into the reactive intermediates in heme-thiolate mono-oxygenases. Rather than treating the enzymes with reactive oxygen compounds, photochemically generated one-electron, outer-sphere oxidants covalently bound to the protein periphery will be used to remove electrons from the resting enzyme and produce ferryl compounds. Efforts will focus on oxidative generation of Compounds I and II in cytochromes P450 (Aim 3) and a bacterial enzyme that exhibits NOS-like reactivity (Aim 4). PUBLIC HEALTH RELEVANCE: Reduction and oxidation (redox) reactions are vital transformations in a wide array of metabolic processes. Countless diseases are associated with failures or disruptions of redox pathways. Elucidation of the fundamental chemical factors that control biological redox processes will lead to deeper understanding of disease mechanisms and guide the development of new therapies.