Electron Transfer in Iron and Copper Proteins

描述(申请人提供)：许多生物能量转导途径依赖于电子或空穴在蛋白质中的长距离快速移动(&gt；30)。许多氧化还原酶，特别是那些参与氧气活化和产生的酶，需要在高电位下转移空穴，在那里可以参与氧化还原活性氨基酸的侧链。必须满足严格的设计要求，才能沿着特定的路径快速有效地传输电荷，并防止氧化还原当量的偏离路径扩散和能量流中断。在这项研究计划中，将研究控制色氨酸(目标1)和酪氨酸(目标2)自由基的多步电子隧穿的分子参数。将制备几个色氨酸残基位于铜活性中心和表面附着光敏剂之间的天青突变体。动力学测量将被用来阐明氧化还原位分离距离、驱动力和色氨酸环境对远程电荷传输动力学的作用。酪氨酸被认为在几种氧化还原酶中介导长距离空穴运输。将制备和研究酪氨酸残基作为长程电子转移介体的天青突变体。还将制备质子接受残基在中间酪氨酸残基氢键距离内的突变蛋白质。这些近端的质子受体有望通过酪氨酸自由基促进远距离跳跃。氨基酸侧链自由基经常存在于参与氧气活化和形成化学的酶中。这类酶的重要成员是血红素硫酸盐单加氧酶，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。尽管在许多实验室进行了广泛的调查，但关于这些酶中活性羟化试剂的性质仍然存在疑问：经常引用铁基物种(即铁氧化合物I和II)。蛋白质标记和激光闪光猝灭光化学将被用来开发对血红素硫酸盐单加氧酶中的活性中间体的新见解。与用活性氧化合物处理酶不同，光化学产生的单电子、共价结合到蛋白质外围的外球氧化剂将被用来从休眠的酶中去除电子并产生铁基化合物。工作重点将集中在细胞色素P450中化合物I和II的氧化生成(目标3)和表现出一氧化氮合酶样反应的细菌酶(目标4)。与公共卫生相关：还原和氧化(氧化还原)反应在广泛的代谢过程中是至关重要的转变。无数的疾病都与氧化还原途径的失效或中断有关。阐明控制生物氧化还原过程的基本化学因素将有助于加深对疾病机制的理解，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.