Protein dynamics govern the oxyferrous state lifetime of an artificial oxygen transport protein
Protein dynamics govern the oxyferrous state lifetime of an artificial oxygen transport protein
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DOI：
10.1016/j.bpj.2023.10.022
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发表时间：
2023-11-21
影响因子：
3.4
通讯作者：
Koder，Ronald L.
中科院分区：
文献类型：
--
作者：
Zhang，Lei;Brown，Mia C.;Koder，Ronald L.
人们早就知道，改变蛋白质侧链，使血红素辅因子封闭或暴露于水，可以极大地影响氧化亚铁血红素状态的稳定性。在这里，我们证明了动态驱动的水渗透到人工氧转运蛋白的核心的速率也与oxyferrous状态寿命通过减少全球动态，而不改变活性位点的结构，通过简单的连接的两个单体在一个同型二聚体的人工氧转运蛋白使用富含甘氨酸的循环。这两个螺旋的拴系不显著影响活性位点结构、五配位血红素结合亲和力、还原电位或气态配体亲和力。然而，它确实显著降低了蛋白质核心的水合作用，如通过共振拉曼光谱、骨架酰胺氢交换和掩埋组氨酸侧链中的pKa位移所证明的。这进一步破坏了电荷掩埋的能态，并使氧铁态的寿命增加了近三倍。这些数据是第一个直接的证据，动态驱动的水渗透是一个限速步骤，这些复合物的氧化。它还表明，限制水渗透的结构刚性是金属酶结构中的一个关键设计特征，并为早期创建氧结合蛋白的尝试的失败和成功提供了解释。
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.