From chlorite dismutase towards HemQ - the role of the proximal H-bonding network in haeme binding.
From chlorite dismutase towards HemQ - the role of the proximal H-bonding network in haeme binding.
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DOI：
10.1042/bsr20150330
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发表时间：
2016-02-08
影响因子：
4
通讯作者：
Obinger C
中科院分区：
文献类型：
--
作者：
Hofbauer S;Howes BD;Flego N;Pirker KF;Schaffner I;Mlynek G;Djinović-Carugo K;Furtmüller PG;Smulevich G;Obinger C
在结构和系统发育上密切相关的亚氯酸盐歧化酶(CLD)和hemQ在酶性质上有根本的不同。近端氢键网络的重建使Cld成为类似于HemQ的，后者被认为是结合辅血素和释放原血素。亚氯酸盐歧化酶(Cld)和hemQ在结构和系统发育上是紧密相关的血型酶，在酶性质上有根本的不同。Clds能够将亚氯酸盐转化为氯和氧气，而hemQ被认为参与了革兰氏阳性细菌的血b合成。这些蛋白质家族之间的一个显著差异涉及近端血腔的结构。Cld中明显的氢键网络，包括近端的配基组氨酸和完全保守的谷氨酸和赖氨酸残基，在hemQ中缺失。为了了解这一明显差异的功能后果，‘Candidatus Nitrospira defluvii’(NdCld)的Cld中的特定氢键被突变所破坏。通过光谱分析(UV-Vis、EPR和共振拉曼)、量热法和动力学方法对得到的突变体(E210A和K141E)进行了分析。研究表明，这些蛋白质家族的血腔结构对近端的修饰非常敏感。观察到的这种结构变化的后果包括：血球蛋白b与蛋白质之间的热稳定性和亲和力显著下降，远端空腔部分崩溃，伴随着E210A变体的低自旋状态百分比增加，伴随而来的酶活性降低，更容易自我失活。高自旋(HS)配体氟化物表现出稳定作用，部分恢复了野生型CLD的结构和功能。这些数据是关于已知的Clds的结构与功能的关系，以及在飞米米特和放线杆菌的血红素生物合成的最后一步中，hemQ作为辅酶脱羧酶的拟议功能。
Structurally and phylogenetically closely related chlorite dismutase (Cld) and HemQ differ fundamentally in their enzymatic properties. Reconstruction of the proximal H-bonding network renders Cld HemQ-like, the latter being proposed to bind coprohaeme and release protohaeme. Chlorite dismutase (Cld) and HemQ are structurally and phylogenetically closely related haeme enzymes differing fundamentally in their enzymatic properties. Clds are able to convert chlorite into chloride and dioxygen, whereas HemQ is proposed to be involved in the haeme b synthesis of Gram-positive bacteria. A striking difference between these protein families concerns the proximal haeme cavity architecture. The pronounced H-bonding network in Cld, which includes the proximal ligand histidine and fully conserved glutamate and lysine residues, is missing in HemQ. In order to understand the functional consequences of this clearly evident difference, specific hydrogen bonds in Cld from ‘Candidatus Nitrospira defluvii’ (NdCld) were disrupted by mutagenesis. The resulting variants (E210A and K141E) were analysed by a broad set of spectroscopic (UV–vis, EPR and resonance Raman), calorimetric and kinetic methods. It is demonstrated that the haeme cavity architecture in these protein families is very susceptible to modification at the proximal site. The observed consequences of such structural variations include a significant decrease in thermal stability and also affinity between haeme b and the protein, a partial collapse of the distal cavity accompanied by an increased percentage of low-spin state for the E210A variant, lowered enzymatic activity concomitant with higher susceptibility to self-inactivation. The high-spin (HS) ligand fluoride is shown to exhibit a stabilizing effect and partially restore wild-type Cld structure and function. The data are discussed with respect to known structure–function relationships of Clds and the proposed function of HemQ as a coprohaeme decarboxylase in the last step of haeme biosynthesis in Firmicutes and Actinobacteria.