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

血红素铜氧化酶和一氧化氮还原酶的生物合成蛋白质模型

• 批准号: 7144246
• 负责人: Yi Lu
• 金额: $ 28.91万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7144246
• 关键词: Raman spectrometry; X ray crystallography; binding sites; calcium binding protein; chemical models; cytochrome c peroxidase; electron spin resonance spectroscopy; enzyme activity; hemoprotein; infrared spectrometry; iron; manganese; metal complex; metalloenzyme; model design /development; myoglobin; nitric oxide synthase; nuclear magnetic resonance spectroscopy; peroxidases; protein structure function; site directed mutagenesis

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.

描述（由申请人提供）：该项目的一般目标是更深入地了解血红素 - 波波氧化酶（HCO）（HCO）中血红素 - 铜点的结构和功能，以及一氧化氮还原酶（NOR）中的血红素铁位点。为了实现我们的目标，我们使用稳定，易于生产且特征良好的血红素蛋白（Sperm Whale Myoglobin，SWMB）作为制造HCO和NOR的生物合成模型的脚手。如光谱和X射线晶体学所证明的那样，我们现在已经成功地将CUB位点设计到SWMB（称为CUBMB）中。将使用位置定向的诱变和表达的蛋白质连接（EPL），分别将天然和非天然氨基酸引入金属结合位点。模型蛋白的详细研究将使用诸如UV-VIS，EPR，FTIR，RR，EXAFS，NRVS和NMR以及X射线晶体学等光谱工具进行。动力学研究将在蛋白质模型上进行，以洞悉天然酶的机理。具体目的是1）阐明幼崽在调节血红素 - 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（即A/ O类型）在调节O2结合和还原方面的作用； 3）阐明共价连接His-Tyr在调节幼崽的结合亲密和几何形状，血红素 - 铜中心的还原电位以及HCO的反应性中的作用； 4）描述金属在非血红金属位点确定活性中的作用。 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氏病，利综合症和衰老有关。无机氮循环中也不是关键酶之一。精心研究的反硝化酶可以为哺乳动物酶提供潜在的结构和光谱模型，这些酶在各种信号转导途径中生产和利用NO。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个酶提供分子基础。