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首页> 外文期刊>Biochemistry >Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli.
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Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli.

机译：EcDos蛋白中的可置换血红素轴向残基的性质，该蛋白是大肠杆菌的基于血红素的传感器。

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摘要

The EcDos protein belongs to a group of heme-based sensors that detect their ligands with a heme-binding PAS domain. Among these various heme-PAS proteins, EcDos is unique in having its heme iron coordinated at both axial positions to residues of the protein. To achieve its high affinities for ligands, one of the axial heme-iron residues in EcDos must be readily displaceable. Here we present evidence from mutagenesis, ligand-binding measurements, and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopies about the nature of the displaceable residue in the heme-PAS domain of EcDos, i.e., EcDosH. The magnetic circular dichroism spectra in the near-infrared region establish histidine-methionine coordination in met-EcDos. To determine whether in deoxy-EcDos coordination of the sixth axial position is also to methionine, methionine 95 was substituted with isoleucine. This substitution caused the ferrous heme iron to change from an exclusively hexacoordinate low-spin form (EcDosH) to an exclusively pentacoordinate high-spin form (M95I EcDosH). This was accompanied by a modest acceleration of the dissociation rates of ligands but a dramatic increase (60-1300-fold) in the association rate constants for binding of O(2), CO, and NO. As a result, the affinity for O(2) was enhanced 10-fold in M95I EcDosH, but the partition constant M = [K(d)(O(2))/K(d)(CO)] between CO and O(2) was raised to about 30 from the extraordinarily low EcDosH value of 1. Thus a major consequence of the increased O(2) affinity of this sensor was the loss of its unusually strong ligand discrimination.
机译：EcDos蛋白属于一组基于血红素的传感器，可检测与血红素结合的PAS结构域的配体。在这些各种血红素-PAS蛋白中，EcDos的独特之处在于其血红素铁在两个轴向位置均与该蛋白的残基配位。为了实现对配体的高亲和力，EcDos中的轴向血红素铁残基之一必须易于置换。在这里，我们从诱变，配体结合测量以及磁性圆二色性，共振拉曼光谱和电子顺磁共振光谱学中提供了有关EcDos血红素-PAS域（即EcDosH）中可置换残基性质的证据。在近红外区域中的磁性圆二色性光谱在met-EcDos中建立了组氨酸-蛋氨酸的配位关系。为了确定在第六轴向位置上的脱氧-EcDos配位是否也与蛋氨酸一致，用异亮氨酸取代了蛋氨酸95。这种取代导致亚铁血红素铁从完全六配位的低旋变形式（EcDosH）变为完全五配位的高旋变形式（M95I EcDosH）。这伴随着配体解离速率的适度加速，但结合O（2），CO和NO的缔合速率常数急剧增加（60-1300倍）。结果，在M95I EcDosH中，对O（2）的亲和力提高了10倍，但CO和O之间的分配常数M = [K（d）（O（2））/ K（d）（CO）] （2）从异常低的EcDosH值1提高到了大约30。因此，此传感器的O（2）亲和力增加的一个主要结果是失去了其异常强的配体分辨力。

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