Structure Function Relationship in Hemeproteins

本研究的目的是阐明三种新的细菌血红蛋白（Hb）和两种哺乳动物前列腺素H-天冬氨酸酶（PGHS-1和PGHS-2）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，其特征在于新的两个在两个α-螺旋上的三明治基序，A-螺旋的缺失和取代大部分F-螺旋的延长环的存在。 HbN和HbO的生理功能尚未确定，但由于单细胞生物体中的O2输送是一个扩散控制的过程，因此已经提出了除氧运输以外的功能。 从E.大肠杆菌（Hmp）是由含血红素的球蛋白样结构域和含FAD的还原酶结构域组成的黄素血红蛋白。 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。 基于我们初步的共振拉曼研究，我们假设这些细菌血红蛋白的血红素口袋是定制的，以执行化学，如氧活化，并且它们可能与过氧化物酶具有结构和功能上的相似性。 这一假设将通过研究这些血红蛋白与NO、过氧化氢和过氧亚硝酸盐的反应来检验。这些血红蛋白在保护微生物免受活性氮中间体攻击的可能作用将通过监测野生型和血红蛋白敲除细胞中的NO和O2消耗来探索。相关的反应将在两个PGHS异构体的过氧化物酶位点进行研究，这两个异构体在三尖杉酯碱的合成中起着至关重要的作用。 初步的数据表明，在PGHS中，血红素与多肽的近端结合非常弱，这对于过氧化物酶来说是非常不寻常的。 提出实验来测试这一发现的功能后果。 这些血红素蛋白系统提供了一个很好的模型，研究基础的生物反应性的基本结构特性。

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.