PDF(Pubmed)
Abstract：
It has long been known that the alteration of protein side chains that occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here, we demonstrate that the rate of dynamically driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme-binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core, as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity that limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins.
摘要：
早就知道,闭塞血红素辅因子或使血红素辅因子暴露于水的蛋白质侧链的改变可极大地影响氧合血红素态的稳定性。在这里，我们证明了动态驱动的水渗透到人工氧转运蛋白核心的速率也通过减少全球动力学与氧铁状态寿命相关。而不改变活性位点的结构，通过使用富含甘氨酸的环将同二聚体人工氧转运蛋白中的两个单体简单连接。这两个螺旋的束缚不会显着影响活性位点的结构，五配位血红素结合亲和力，还原电位,或气态配体亲和力。确实如此，然而,如共振拉曼光谱所示，显着减少蛋白质核心的水合作用，主链酰胺氢交换，和pKa在埋藏的组氨酸侧链中移位。这进一步使电荷掩埋的纠缠态不稳定，并使氧亚铁态的寿命几乎增加了三倍。这些数据是动态驱动的水渗透是这些络合物氧化中的限速步骤的第一个直接证据。它进一步证明了限制水渗透的结构刚性是金属酶构建中的关键设计特征，并为早期尝试创建氧结合蛋白的失败和成功提供了解释。
