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合成酶（PGHS-1和PGHS-2）的蛋白质-配体相互作用和结构/功能关系。结核分枝杆菌的两种细菌血红蛋白（HbN和HbO）属于一个新发现的截断血红蛋白家族，其特征是一个新的2 - 2 - 2 α -螺旋三明治基序，缺乏a-螺旋，存在一个取代大部分f -螺旋的延伸环。HbN和HbO的生理功能尚未确定，但由于单细胞生物的氧传递是一个扩散控制的过程，因此提出了除氧运输外的其他功能。大肠杆菌的细菌血红蛋白（Hmp）是一种黄血红蛋白，由含血红素的珠蛋白样结构域和含fad的还原酶结构域组成。认为Hmp的功能是解毒NO和其他活性氮。这三种细菌血红蛋白的结构特性将被充分表征。基于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口袋是专门为进行化学反应而设计的，比如氧激活，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验证。这些血红蛋白在保护微生物免受活性氮中间体攻击中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。我们将研究两种PGHS亚型的过氧化物酶位点的相关反应，这两种PGHS亚型在前列腺素的合成中起着至关重要的作用。初步数据表明，在PGHS中，协调血红素与多肽的近端键非常弱，这在过氧化物酶中是非常不寻常的。提出了实验来测试这一发现的功能后果。这些血红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。

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.