Reaction-pathway selection in the structural dynamics of a heme protein.
Reaction-pathway selection in the structural dynamics of a heme protein.
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血红素蛋白结构动力学中的反应途径选择
作者：
Nienhaus;Meuwly;Nienhaus
在任何给定的时间点，无数的化学反应都在活细胞中发生。最普遍的是蛋白质与其他蛋白质和DNA/RNA、脂质、糖部分和小配体分子的相互作用。所有这些反应形成了一个微调和相互调节的复杂网络，维持细胞的重要功能。在分子水平上探索这些反应的机理细节是一项具有挑战性的奋进，但对于推进医学，生物学和生物技术来说是必不可少的。解决蛋白质结构是这一追求的先决条件，蛋白质能量学和动力学是进一步研究以了解生物分子反应的关键成分。近年来，强大的计算方法来检查蛋白质的能量学和动力学与生物分子反应在硅片上已经开发出来。在此，我们结合时间分辨光谱与分子动力学（MD）模拟阐明一个有趣的反应途径选择机制，在脑红蛋白（Ngb），一个小血红素蛋白从珠蛋白家族。[1]Ngb主要在神经组织中表达，已知在缺氧条件下提供神经保护;其确切的生理功能仍在争论中。[2]血红蛋白长期以来一直作为探索蛋白质动力学和蛋白质-配体相互作用的模型系统。[3-9]它们在由血红素辅基的亚铁（Fe II）铁提供的内部活性位点处结合双氧和其他小配体。各种各样的光谱和结构研究揭示了这种生物学上“简单”的反应的惊人复杂性。[10-17]与血红蛋白不同，血红蛋白在不存在外源配体的情况下具有空缺的第六个Fe II配位，Ngb结合内源配体，即远端组氨酸的咪唑侧链His 64。对CO连接的鼠Ngb（双突变体C55 S-C120 S，本文中称为野生型（wt）NgbCO）进行闪光光解实验，并进行光吸收监测，得到图1a中所示的反应方案。[18用短激光脉冲光解CO产生亚稳的五配位脱氧物质NgbP，其沿着沿着不同的路径弛豫回到NgbCO。从蛋白质内解离的CO分子的直接（成对）再结合在生理温度下以亚微秒时间尺度发生，并且在本文中不考虑。大部分CO分子在光解后逃逸到溶剂中，并在毫秒时间尺度上与血红素铁结合（图1a，过程Ia）。它们与内源性His 64配体竞争血红素铁上的第六个空配位位点（图1a，过程Ib），这从动力学中存在的两个步骤中可以看出（图1b）。在第一步中，CO或His 64在第六血红素铁配位结合;第二，较慢的步骤表明，六配位脱氧Ngb（NgbH）物种持续存在，直到结合的His 64配体热解离，并被更紧密结合的CO分子取代（图1a，过程II）。
At any given point in time, a myriad of chemical reactions are taking place in a living cell. Most prevalent are protein interactions with other proteins and DNA/RNA, lipids, sugar moieties, and small ligand molecules. All these reactions form a finely tuned and mutually regulated complex network that sustains the vital functions of the cell. Exploring the mechanistic details of such reactions at the molecular level is a challenging yet essential endeavor for advancing medicine, biology, and biotechnology. Solving protein structures is a prerequisite in this pursuit, and protein energetics and dynamics are further crucial ingredients that need to be studied to understand biomolecular reactions. In recent years, powerful computational methods to examine protein energetics and dynamics associated with biomolecular reactions in silico have been developed. Herein, we have combined time-resolved spectroscopy with molecular-dynamics (MD) simulations to elucidate an intriguing reaction-pathway selection mechanism in neuroglobin (Ngb), a small heme protein from the globin family.[1] Ngb is primarily expressed in neuronal tissue and is known to provide neuroprotection under hypoxic conditions; its precise physiological function is still under debate.[2] Hemoglobins have long served as model systems for exploring protein dynamics and protein–ligand interactions.[3–9] They bind dioxygen and other small ligands at an interior active site provided by a ferrous (FeII) iron of a heme prosthetic group. A wide variety of spectroscopic and structural studies have revealed a stunning complexity of this biologically “simple” reaction.[10–17]Unlike hemoglobin, which has a vacant sixth FeII coordination in the absence of an exogenous ligand, Ngb binds an endogenous ligand, that is, the imidazole side chain of the distal histidine, His64. Flash-photolysis experiments on CO ligated murine Ngb (double mutant C55S-C120S, herein referred to as wild-type (wt) NgbCO) with optical-absorption monitoring gave the reaction Scheme depicted in Figure 1 a.[18, 19] Photodissociation of CO with a short laser pulse generates metastable pentacoordinate deoxy species, NgbP, which relaxes back to NgbCO along different pathways. Direct (geminate) rebinding of dissociated CO molecules from within the protein occurs on sub-microsecond time scales at physiological temperatures and is not considered herein. A large fraction of CO molecules escape into the solvent after photodissociation and bind to the heme iron on the millisecond time scale (Figure 1a, process Ia). They compete with the endogenous His64 ligand for the vacant sixth coordination site at the heme iron (Figure 1a, process Ib), which is evident from the presence of two steps in the kinetics (Figure 1b). In the first step, CO or His64 bind at the sixth heme iron coordination; the second, slower step indicates that the hexacoordinate deoxy Ngb (NgbH) species persists until the bound His64 ligand thermally dissociates and is replaced by the more tightly bound CO molecule (Figure 1a, process II).