Role of PheE15 Gate in Ligand Entry and Nitric Oxide Detoxification Function of Mycobacterium tuberculosis Truncated Hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O2 and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O2/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.结核分枝杆菌（Mycobacterium tuberculosis）的截短型血红蛋白N（truncated hemoglobin N，HbN）具备强效的一氧化氮双加氧酶（nitric oxide dioxygenase，NOD）活性，可帮助宿主抵御亚硝化应激并提升其体内存活能力。尽管分子量较小，HbN的蛋白质基质却拥有一条双分支隧道，由正交分布的短通道与长通道组成，可连接血红素活性位点（heme active site）与蛋白质表面。研究人员提出了一种全新的双路径机制，用以驱动O₂和NO向远端血红素空腔（distal heme cavity）的迁移：氧气主要通过短通道完成迁移，而配体诱导的构象变化则通过充当门控的苯丙氨酸（PheE15）残基，调控长隧道分支的开放以实现NO的转运。研究人员通过定点诱变（site-directed mutagenesis）与分子模拟技术，探究了PheE15门控残基对HbN NOD功能的调控作用。将PheE15分别突变为丙氨酸、异亮氨酸、酪氨酸与色氨酸的突变体，其O₂/CO结合动力学特征无显著差异，但NOD活性却出现明显下降。分子模拟结果证实，PheE15位点的突变会对长隧道结构造成显著改变，进而可能影响配体的迁移过程。上述结果表明，PheE15门控在调控氧合状态的HbN中通过长隧道分支的NO扩散过程中发挥关键作用，进而影响HbN的NOD功能。