CO Rebinding Kinetics and Molecular Dynamics Simulations Highlight Dynamic Regulation of Internal Cavities in Human Cytoglobin

Cytoglobin (Cygb) was recently discovered in the human genome and localized in different tissues. It was suggested to play tissue-specific protective roles, spanning from scavenging of reactive oxygen species in neurons to supplying oxygen to enzymes in fibroblasts. To shed light on the functioning of such versatile machinery, we have studied the processes supporting transport of gaseous heme ligands in Cygb. Carbon monoxide rebinding shows a complex kinetic pattern with several distinct reaction intermediates, reflecting rebinding from temporary docking sites, second order recombination, and formation (and dissociation) of a bis-histidyl heme hexacoordinated reaction intermediate. Ligand exit to the solvent occurs through distinct pathways, some of which exploit temporary docking sites. The remarkable change in energetic barriers, linked to heme bis-histidyl hexacoordination by HisE7, may be responsible for active regulation of the flux of reactants and products to and from the reaction site on the distal side of the heme. A substantial change in both protein dynamics and inner cavities is observed upon transition from the CO-liganded to the pentacoordinated and bis-histidyl hexacoordinated species, which could be exploited as a signalling state. These findings are consistent with the expected versatility of the molecular activity of this protein.

细胞球蛋白（Cytoglobin, Cygb）是近年在人类基因组中发现的蛋白质，广泛分布于多种人体组织。研究表明其具备组织特异性的保护功能，涵盖神经元内活性氧的清除、成纤维细胞内酶的氧气供给等多个方面。为阐明这类多功能蛋白的作用机制，我们针对细胞球蛋白内气态血红素配体的转运过程展开了研究。一氧化碳再结合实验呈现出复杂的动力学模式，存在多个不同的反应中间体，分别对应来自临时停靠位点的再结合、二级重组反应，以及双组氨酸血红素六配位反应中间体的形成与解离。配体通过特异性通路释放进入溶剂相，其中部分通路借助临时停靠位点完成转运。由E7位组氨酸（HisE7）介导的血红素双组氨酸六配位结构，其伴随的能垒发生显著变化，这一变化或可调控血红素远端反应位点处反应物与产物的进出通量。从结合一氧化碳的状态转变为五配位及双组氨酸六配位状态时，蛋白质动力学与内部空腔均发生显著改变，这一转变可作为一种信号状态加以利用。上述研究结果与该蛋白分子活性具备多功能性的预期相符。