Structure Function Relationship in Hemeproteins

血红素蛋白的结构功能关系

• 批准号: 6433792
• 负责人: Syun-Ru Yeh
• 金额: $ 28.44万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6433792
• 关键词: Escherichia coli; Mycobacterium bovis; Mycobacterium tuberculosis; Raman spectrometry; bacterial proteins; enzyme activity; enzyme complex; enzyme mechanism; hemoglobin; ligands; microorganism metabolism; nitric oxide; oxygen consumption; oxygen microelectrode; prostaglandin endoperoxide synthase; protein protein interaction; protein structure function; site directed mutagenesis; ultraviolet spectrometry

The objective of this proposal is to elucidate the protein-ligand interactions and structure/function relationships in three new bacterial hemoglobins (Hb) and two mammalian prostaglandin H synthases (PGHS-1 and PGHS-2). The two bacterial hemoglobins from Mycobacterium tuberculosis (HbN and HbO) belong to a newly discovered truncated hemoglobin family, which are characterized by a novel two-over-two alpha-helical sandwich motif, the absence of the A- helix and the presence of an extended loop substituting for most of the F-helix. The physiological functions of HbN and HbO are not established but because O2 delivery in unicellular organisms is a diffusion-controlled process, functions other than oxygen transport have been put forth. The bacterial hemoglobin from E. coli (Hmp) is a flavohemoglobin consisting of a heme-containing globin-like domain and a FAD-containing reductase domain. It is believed that the function of Hmp is to detoxify NO and other reactive nitrogen species. The structural properties of the three bacterial hemoglobins will be fully characterized. Based on our preliminary resonance Raman studies, we postulate that the heme pockets of these bacterial hemoglobins are tailored to perform chemistry, such as oxygen activation, and that they may share structural and functional similarities to peroxidases. This hypothesis will be tested by studies of the reactions of these hemoglobins with NO, hydrogen peroxide and peroxynitrite. The possible role of these hemoglobins in protecting the microorganisms against attack by reactive nitrogen intermediates will be explored by monitoring NO and O2 consumption in wild-type and hemoglobin knock-out cells. Related reactions will be studied in the peroxidase sites of two PGHS isoforms, which play an essential role in the synthesis of prostaglandins. Preliminary data suggests that the proximal bond that coordinates the heme to the polypeptide is quite weak in PGHS's, which is very unusual for peroxidases. Experiments are proposed to test the functional consequences of this finding. These hemeprotein systems provide an excellent model for investigating fundamental structural properties that underlie biological reactivity.

该提案的目的是阐明三种新细菌血红蛋白 (Hb) 和两种哺乳动物前列腺素 H 合酶（PGHS-1 和 PGHS-2）中的蛋白质-配体相互作用和结构/功能关系。 来自结核分枝杆菌的两种细菌血红蛋白（HbN 和 HbO）属于新发现的截短血红蛋白家族，其特征在于新型的二二二α螺旋夹心基序、缺乏A螺旋和存在A螺旋。延长环取代了大部分 F 螺旋。 HbN 和 HbO 的生理功能尚未确定，但由于单细胞生物中 O2 的输送是一个扩散控制的过程，因此已经提出了氧运输以外的功能。 来自大肠杆菌 (Hmp) 的细菌血红蛋白是一种黄素血红蛋白，由含有血红素的球蛋白样结构域和含有 FAD 的还原酶结构域组成。 据信，Hmp 的功能是解毒 NO 和其他活性氮物质。三种细菌血红蛋白的结构特性将得到充分表征。 根据我们的初步共振拉曼研究，我们假设这些细菌血红蛋白的血红素袋是为了执行化学反应而设计的，例如氧活化，并且它们可能与过氧化物酶具有结构和功能相似性。 这一假设将通过研究这些血红蛋白与一氧化氮、过氧化氢和过氧亚硝酸盐的反应来检验。通过监测野生型和血红蛋白敲除细胞中NO和O2的消耗，将探索这些血红蛋白在保护微生物免受活性氮中间体攻击方面的可能作用。将研究两种 PGHS 亚型的过氧化物酶位点的相关反应，这两种亚型在前列腺素的合成中发挥重要作用。 初步数据表明，在 PGHS 中，协调血红素与多肽的近端键相当弱，这对于过氧化物酶来说是非常不寻常的。 建议进行实验来测试这一发现的功能后果。 这些血红素蛋白系统为研究生物反应性的基本结构特性提供了一个极好的模型。