Dynamics of Lysine as a Heme Axial Ligand: NMR Analysis of the Chlamydomonas reinhardtii Hemoglobin THB1
Dynamics of Lysine as a Heme Axial Ligand: NMR Analysis of the Chlamydomonas reinhardtii Hemoglobin THB1
复制标题
赖氨酸作为血红素轴向配体的动力学：莱茵衣藻血红蛋白 THB1 的 NMR 分析
DOI：
10.1021/acs.biochem.6b00926
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Lecomte, Juliette T.
中科院分区：
文献类型：
--
作者：
Preimesberger, Matthew R.;Majumdar, Ananya;Lecomte, Juliette T.
莱茵衣原体的硝酸盐代谢涉及THB 1，THB 1是一种被认为具有一氧化氮双加氧酶（NOD）功能的单体血红蛋白。NOD活性需要分子氧和一氧化氮结合，然后是血红素铁的单电子氧化和硝酸盐释放。不同于五配位的黄血红蛋白，这是有效的NOD，THB 1使用两个铁轴配体：保守的近端组氨酸和远端赖氨酸（赖氨酸53）。作为氧化态（铁）和还原态（亚铁）的配体，Lys 53预期会降低与电子转移相关的重组能，从而促进铁酶的还原。然而，在亚铁THB 1中，Lys 53必须被置换以用于底物结合。为了表征Lys 53动力学，通过NMR光谱在各种pH、温度和压力下研究了THB 1。结构信息表明，蛋白质折叠和Lys 53环境是独立的氧化态。高压核磁共振实验提供的证据表明，通过快速平衡（在1 bar，298 K下为103 -4 × 103 s-1），Lys 53发生位移，其中Lys 53是中性的，并且是配位的。一旦脱配位，Lys 53能够朝向溶剂取向并被质子化。15 Nz交换光谱测量的赖氨酸的整体解配位/重取向/质子化过程在化学位移时间标度上是缓慢的（101- 102 s-1，pH = 6.5，298 K），在两种铁氧化还原状态下。因此，在亚铁THB 1的重取向/质子化步骤似乎呈现出一个显着的障碍，为双氧结合，因此，NOD营业额。结果说明远端配体动力学在调节多步血红素氧化还原反应动力学中的作用。
Nitrate metabolism inChlamydomonas reinhardtiiinvolves THB1, a monomeric hemoglobin thought to function as a nitric oxide dioxygenase (NOD). NOD activity requires dioxygen and nitric oxide binding followed by a one-electron oxidation of the heme iron and nitrate release. Unlike pentacoordinate flavohemoglobins, which are efficient NODs, THB1 uses two iron axial ligands: the conserved proximal histidine and a distal lysine (Lys53). As a ligand in both the oxidized (ferric) and reduced (ferrous) states, Lys53 is expected to lower the reorganization energy associated with electron transfer and therefore facilitate reduction of the ferric enzyme. In ferrous THB1, however, Lys53 must be displaced for substrate binding. To characterize Lys53 dynamics, THB1 was studied at various pH, temperatures, and pressures by NMR spectroscopy. Structural information indicates that the protein fold and Lys53 environment are independent of the oxidation state. High-pressure NMR experiments provided evidence that displacement of Lys53 occurs through fast equilibrium (∼3–4 × 103s–1at 1 bar, 298 K) with a low-population intermediate in which Lys53 is neutral and decoordinated. Once decoordinated, Lys53 is able to orient toward solvent and become protonated. The global lysine decoordination/reorientation/protonation processes measured by15Nz-exchange spectroscopy are slow on the chemical shift time scale (101–102s–1at pH ≈ 6.5, 298 K) in both iron redox states. Thus, reorientation/protonation steps in ferrous THB1 appear to present a significant barrier for dioxygen binding, and consequently, NOD turnover. The results illustrate the role of distal ligand dynamics in regulating the kinetics of multistep heme redox reactions.