Study of copper oxidoreductases through metalloprotein engineering

描述（由申请人提供）：我们将使用金属蛋白设计和重新设计来解决许多铜金属酶共同的重要功能特征。铜蛋白是一类重要的金属生物分子，人体中至少有十几种酶利用铜进行结构和功能。大多数人类铜酶被归类为氧化还原酶，并参与一系列过程，如电子转移和底物的氧化或还原。铜在这些蛋白质中的共同作用是电子转移，如在血浆铜蓝蛋白（CP）或细胞色素c氧化酶（CcO）中，以及与O2的反应性，如在肽基？羟基化单加氧酶（PHM）、酪氨酸酶或胺氧化酶。因此，Cu酶中的共同主题包括1）Cu介导的电子转移和2）Cu催化的具有底物氧化和还原的氧化学。本提案的目标是使用蛋白质设计和重新设计来探索和阐明铜金属酶的这些共同主题。我们将通过模拟非偶联双核铜蛋白家族来实现这一点，该蛋白家族包含电子传递中心和催化中心。因此，我们将模拟PHM，多巴胺？单加氧酶（DBM）和使用蛋白天青蛋白作为配体或支架的亚硝酸盐还原酶（NiR）。使用蛋白质设计方法，我们将模拟这些酶的结构和功能。我们将检查和调整我们的模型中的结构元素的不同层，这些结构元素被假设为对本机系统中的活动至关重要。这些包括1）调节两个Cu位点的铜氧化还原电位，2）调整Cu位点之间的电子转移途径，以及3）在我们的模型中进行突变以促进底物结合。所有这些组件放在一起是试图耦合在一起，并测试不同的因素，被认为是重要的催化铜酶的适当功能：一个结构上足够的铜网站的设计，氧化还原电位的调整，电子转移路径的修改，并结合底物，如氧。本研究的长期目标是了解铜金属酶的这些性质，并阐明天然系统的机制。这些项目同样重要的一个目标是指导和培训本科生和硕士。学生在多学科生物化学科学的成功事业。 公共卫生相关性：人类有十几种铜酶，其中许多利用铜促进电子转移反应并用分子氧氧化底物。我们将使用蛋白质金属位点设计来整合和调整不同层的结构元素到天然系统的模型中，这些模型测试假设对功能至关重要的因素。确定导致这些酶中铜活性的特征有助于了解各种疾病的潜在因素。

DESCRIPTION (provided by applicant): We will use metalloprotein design and redesign to address important functional features common to many copper metalloenzymes. Copper proteins are an important class of metallobiomolecules with at least a dozen enzymes in the human body utilizing copper for structure and function. Most human copper enzymes are classified as oxidoreductases and are involved in an array of processes, such as electron transfer, and oxidation or reduction of substrates. Common roles of copper in these proteins are electron transfer, such as in ceruloplasmin (CP) or cytochrome c oxidase (CcO), and reactivity with O2, such as in peptidyl ?-hydroxylating monooxygenase (PHM), tyrosinase, or amine oxidases. Therefore, themes common among Cu enzymes include 1) Cu mediated electron transfer and 2) Cu catalyzed oxygen chemistry with both oxidation and reduction of substrate. The goal of this proposal is to use protein design and redesign to explore and elucidate these common themes of Cu metalloenzymes. We will accomplish this by modeling the family of noncoupled dinuclear copper proteins, which contain both an electron transfer center and a catalytic center. We therefore will model the di-copper centers of PHM, dopamine ?-monooxygenase (DBM), and nitrite reductase (NiR) using the protein azurin as the ligand or scaffold. Using the protein design methodology, we will model these enzymes structurally as well as functionally. We will examine and adjust different layers of structural elements in our models that are hypothesized to be essential for activity in the native systems. These include 1) modulating the copper redox potentials of the two Cu sites, 2) tuning the electron transfer pathway between the Cu sites, and 3) making mutations to encourage substrate binding in our models. All of these components taken together are attempts to couple together and test different factors that are deemed important for the proper function of a catalytic Cu enzyme: the design of a structurally adequate copper site, the tuning of redox potential, the modification of electron transfer paths, and the binding of substrates such as oxygen. The long term goal of the research is to understand these properties of Cu metalloenzymes and shed light on the mechanisms of the native systems. Equally as important a goal for these projects is the mentoring and training of undergraduate and M.S. students for successful careers in multidisciplinary biochemical sciences. PUBLIC HEALTH RELEVANCE: There are over a dozen human copper enzymes, many of which utilize copper to facilitate electron transfer reactions and oxidize substrate with molecular oxygen. We will use protein metal site design to incorporate and adjust different layers of structural elements into models of native systems that test factors hypothesized to be essential for function. Defining the features that lead to activity of copper in these enzymes can contribute to an understanding of factors underlying various diseases.