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

描述（由申请人提供）：该项目的总体目标是更深入地了解血红素-铜氧化酶（HCO）中血红素-铜位点和一氧化氮还原酶（NOR）中血红素-铁位点的结构和功能。为了实现我们的目标，我们使用一种稳定的，易于生产的，和良好的特点血红素蛋白（抹香鲸肌红蛋白，swMb）作为支架，使HCO和NOR的生物合成模型。我们现在已经成功地将CuB位点设计成swMb（称为CuBMb），如光谱学和X射线晶体学所示。将采用定点诱变和表达蛋白连接（EPL）将天然和非天然氨基酸分别引入金属结合位点。将使用光谱工具如UV-vis、EPR、FTIR、RR、EXAFS、NRVS和NMR以及X射线晶体学对模型蛋白进行详细研究。将对蛋白质模型进行动力学研究，以深入了解天然酶的机制。具体目标是1）阐明CuB在调节血红素-铜中心的还原电位、O2结合亲和力和HCO的反应机制中的作用; 2）定义血红素类型的作用（即，a/ o或B型）在调节O2结合和还原中的作用; 3）阐明共价连接的His-Tyr在调节Cu B的结合亲和力和几何结构、血红素-铜中心的还原电位和HCO的反应性中的作用;和4）描绘非血红素金属位点中的金属在确定NOR活性中的作用。HCO催化生物圈中90%的分子氧还原。HCO缺陷或自然发生的突变与阿尔茨海默病，Leigh综合征和衰老有关。NOR是无机氮循环的关键酶之一。研究反硝化酶可能提供潜在的结构和光谱模型的哺乳动物酶，产生和利用NO在各种信号转导途径。这项工作将为医疗保健做出重要贡献，因为它将为理解对人类健康重要的两种酶提供分子基础。

DESCRIPTION (provided by applicant): The general goal of the project is to provide deeper insight into the structure and function of the heme-copper site in heme-copper oxidases (HCO) and the heme-iron site in nitric oxide reductase (NOR). To achieve our goals, we use a stable, easy-to-produce, and well-characterized heme protein (sperm whale myoglobin, swMb) as the scaffold for making biosynthetic models of HCO and NOR. We have now successfully engineered a CuB site into swMb (called CuBMb), as demonstrated by both spectroscopy and X-ray crystallography. Site-directed mutagenesis and expressed protein ligation (EPL) will be employed to introduce natural and unnatural amino acids, respectively, into the metal-binding sites. Detailed studies of the model proteins will be carried out using spectroscopic tools such as UV-vis, EPR, FTIR, RR, EXAFS, NRVS, and NMR, as well as X-ray crystallography. Kinetic studies will be performed on the protein models to provide insight into the mechanism of the native enzymes. Specific aims are to 1) elucidate the role of CuB in modulating the reduction potentials of the heme-copper center, O2 binding affinity, and reaction mechanism of HCO; 2) define the role of heme types (i.e., a/ o or b types) in regulating O2 binding and reduction; 3) clarify the role of the covalently linked His-Tyr in modulating the binding affinity and geometry of CuB, the reduction potential of the heme-copper center, and reactivity of HCO; and 4) delineate the role of the metal in the non-heme metal site in determining NOR activity. HCOs catalyze 90% of molecular oxygen reduction in the biosphere. HCO deficiencies or naturally occurring mutations have been linked to Alzheimer's disease, Leigh syndrome, and aging. NOR is one of the key enzymes in the inorganic nitrogen cycle. Well-studied denitrification enzymes may provide potential structural and spectroscopic models for mammalian enzymes that produce and utilize NO in a variety of signal transduction pathways. The work will make important contributions to healthcare, as it will provide a molecular basis for understanding two enzymes important to human health.