Biosynthetic Protein Models of Heme-Copper Oxidases and Nitric Oxide Reductases

项目描述（由申请人提供）：该项目的总体目标是回答生物能量学和氮循环领域的一个关键科学问题，即为什么蛋白质在使用血红素-铜中心（如血红素-铜氧化酶（HCO））时精通O-O键切割，而在使用血红素-非血红素铁中心（如一氧化氮还原酶（NOR））时有效形成N-N键。我们试图通过开发一种新的生物合成方法来克服该领域进展的关键方法障碍，该方法利用稳定，易于生产且具有良好特征的血红素蛋白（肌红蛋白）作为支架来制作HCO和NOR的结构和功能模型。在上一个资助期成功地将CuB和FeB位点设计成Mb（分别称为CuBMb和FeBMb）的基础上，通过光谱学和x射线晶体学证明，位点定向诱变和表达蛋白连接（EPL）将被用于将天然或非天然氨基酸引入金属结合位点。模型蛋白的研究将使用光谱学和x射线晶体学进行。动力学研究也将进行，以深入了解O2和NO还原的机制。与其他方法相比，我们的生物合成方法可以独特地解决三个具体目标：1)阐明负责HCO功能的结构特征，特别是CuB位点的作用，与CuB- his配体共价连接的酪氨酸，以及非共价相互作用，如水和相关的氢键网络；2)阐明NOR功能的结构特征，特别是FeB位点和附近的谷氨酸残基；3)研究HCO中血红素-铜中心与NOR中血红素-非血红素铁中心的结构和功能差异。一旦项目目标实现，就会对HCO和NOR的结构和功能有更深入的了解。将血红素-铜中心和血红素-铁中心放置在同一蛋白质框架中，并将不同的金属离子引入同一蛋白质中的CuB或FeB位点，是一个独特的机会，可以直接比较两类重要的金属结合中心的结构和功能。同时，该项目将从总体上推进我们对血红素蛋白结构、功能和设计的认识，因为从研究中获得的指导原则可能适用于广泛的其他金属蛋白。

DESCRIPTION (provided by applicant): The overall goal of the project is to answer a key scientific question in the fields of bioenergetics and the nitrogen cycle, i.e., why a protein is proficient at O-O bond cleavage when using a heme-copper center as in heme-copper oxidases (HCO), and is effective at N-N bond formation when using a heme-non-heme iron center, as in nitric oxide reductase (NOR). We seek to overcome a critical methodological barrier to progress in this field by developing a novel biosynthetic approach that utilizes a stable, easy-to-produce, and well-characterized heme protein (myoglobin) as a scaffold for making structural and functional models of HCO and NOR. Building on the success of the last grant period when both CuB and FeB sites were designed into Mb (called CuBMb and FeBMb, respectively), as demonstrated by both spectroscopy and X-ray crystallography, site-directed mutagenesis and expressed protein ligation (EPL) will be employed to introduce natural or unnatural amino acids into the metal-binding sites. Studies of the model proteins will be carried out using spectroscopy as well as X-ray crystallography. Kinetic studies will also be performed to provide insight into the mechanisms of O2 and NO reduction. This proposal contains three specific aims that our biosynthetic approach can uniquely address when compared to other approaches: 1) elucidate structural features responsible for HCO function, specifically the roles of CuB site, the tyrosine that is covalently linked to a CuB-His ligand, and non-covalent interactions, such as water and the associated hydrogen bonding network; 2) elucidate structural features responsible for NOR function, specifically the FeB site and nearby glutamate residues; and 3) examine structural and functional differences between the heme-copper center in HCO and the heme-non-heme iron center in NOR. Once the project aims are achieved, it will result in a deeper understanding of the structure and function of HCO and NOR. The placement of a heme-copper and a heme- iron center in the same protein framework, and the ability to introduce different metal ions into the CuB or FeB sites in the same protein is a unique opportunity that allows direct structural and functional comparisons of two important classes of metal-binding centers. At the same time, the project will advance our knowledge of heme protein structure, function and design in general, as the guiding principles obtained from the studies may be applicable to a broad range of other metalloproteins. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to health in a broad sense. HCOs are the terminal electron acceptors in the respiratory chain of all aerobic organisms and are required for aerobic life. Furthermore, deficiencies of or mutations in HCOs have been linked to Alzheimer's disease, Leigh syndrome and aging. Well-studied denitrification enzymes such as NOR may provide potential structural and spectroscopic models for mammalian enzymes that produce and utilize NO in a variety of signal transduction pathways. Therefore the work will make important contributions to healthcare, as it will provide a molecular basis for understanding two enzymes important to human health.