Distal lysine (de)coordination in the algal hemoglobin THB1: A combined computer simulation and experimental study
Distal lysine (de)coordination in the algal hemoglobin THB1: A combined computer simulation and experimental study
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DOI：
10.1016/j.jinorgbio.2021.111455
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发表时间：
2021-04-18
影响因子：
3.9
通讯作者：
Capece，Luciana
中科院分区：
文献类型：
--
作者：
Julio Plana，Laia;Martinez Grundman，Jaime E.;Capece，Luciana
THB 1是来自绿色藻类莱茵衣藻的单体截短血红蛋白。在不存在外源配体和中性pH下，THB 1的血红素基团由两个蛋白残基Lys 53和His 77协调。THB 1被认为是一氧化氮双加氧酶，O2的远端结合需要Fe-Lys 53键的断裂，伴随着质子化和赖氨酸从血红素腔排出到溶剂中。核磁共振光谱和晶体学数据提供了动态和结构的见解的过程，但机制的细节尚未完全阐明。我们应用计算机模拟和定点诱变实验相结合，阐明这个问题。分子动力学模拟和混合量子力学/分子力学约束优化进行了探索的性质之间的过渡的脱配位和赖氨酸结合态的亚铁血红素在THB 1。Lys 49和Arg 52与赖氨酸结合的THB 1的X射线结构中的血红素丙酸盐形成离子相互作用，被观察到有助于将Lys 53保持在蛋白质腔内部，并在过渡中发挥关键作用。制备了Lys 49 Ala、Arg 52 Ala和Lys 49 Ala/Arg 52 Ala THB 1变体，并通过实验表征了置换对Lys（去）配位平衡的影响，以与计算预测进行比较。结果加强了蛋白质-丙酸相互作用的动态作用，并强烈建议，分裂的Fe-Lys 53键和随后的构象重排是由远端腔体内的氨基质子化促进。
THB1 is a monomeric truncated hemoglobin from the green algaChlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2requires the cleavage of the Fe–Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein–propionate interactions and strongly suggested that cleavage of the Fe–Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.