Eakanunkul Suntara, Lukat-Rodgers Gudrun S, Sumithran Suganya, Ghosh Arundhati, Rodgers Kenton R, Dawson John H, Wilks Angela
Department of Pharmaceutical Sciences, School of Pharmacy, 20 Penn Street, University of Maryland, Baltimore, Maryland 21201, USA.
Biochemistry. 2005 Oct 4;44(39):13179-91. doi: 10.1021/bi050422r.
The heme uptake systems by which bacterial pathogens acquire and utilize heme have recently been described. Such systems may utilize heme directly from the host's hemeproteins or via a hemophore that sequesters and transports heme to an outer membrane receptor and subsequently to the translocating proteins by which heme is further transported into the cell. However, little is known of the heme binding and release mechanisms that facilitate the uptake of heme into the pathogenic organism. As a first step toward elucidating the molecular level events that drive heme binding and release, we have undertaken a spectroscopic and mutational study of the first purified periplasmic heme-binding protein (PBP), ShuT from Shigella dysenteriae. On the basis of sequence identity, the ShuT protein is most closely related to the class of PBPs typified by the vitamin B(12) (BtuF) and iron-hydroxamate (FhuD) PBPs and is a monomeric protein having a molecular mass of 28.5 kDa following proteolytic processing of the periplasmic signaling peptide. ShuT binds one b-type heme per monomer with high affinity and bears no significant homology with other known heme proteins. The resonance Raman, MCD, and UV-visible spectra of WT heme-ShuT are consistent with a five-coordinate high spin heme having an anionic O-bound proximal ligand. Site-directed ShuT mutants of the absolutely conserved Tyr residues, Tyr-94 (Y94A) and Tyr-228 (Y228F), which are found in all putative periplasmic heme-binding proteins, were subjected to UV-visible, resonance Raman, and MCD spectroscopic investigations of heme coordination environment and rates of heme release. The results of these experiments confirmed Tyr-94 as the only axial heme ligand and Tyr-228 as making a significant contribution to the stability of heme-loaded ShuT, albeit without directly interacting with the heme iron.
最近已描述了细菌病原体获取和利用血红素的血红素摄取系统。此类系统可直接从宿主的血红素蛋白获取血红素，或通过一种血红素载体来获取，该载体螯合血红素并将其转运至外膜受体，随后转运至转运蛋白，血红素通过该转运蛋白进一步转运至细胞内。然而，对于促进血红素摄取到致病生物体中的血红素结合和释放机制知之甚少。作为阐明驱动血红素结合和释放的分子水平事件的第一步，我们对痢疾志贺氏菌中首个纯化的周质血红素结合蛋白（PBP）ShuT进行了光谱和突变研究。基于序列同一性，ShuT蛋白与以维生素B12（BtuF）和铁异羟肟酸酯（FhuD）PBP为代表的PBP类别关系最为密切，并且是一种单体蛋白，在周质信号肽进行蛋白水解处理后分子量为28.5 kDa。ShuT每个单体以高亲和力结合一个b型血红素，并且与其他已知的血红素蛋白没有明显的同源性。野生型血红素 - ShuT的共振拉曼光谱、磁圆二色光谱和紫外可见光谱与具有阴离子O结合近端配体的五配位高自旋血红素一致。对所有假定的周质血红素结合蛋白中都存在的绝对保守的Tyr残基Tyr - 94（Y94A）和Tyr - 228（Y228F）进行定点ShuT突变体，对其进行血红素配位环境和血红素释放速率的紫外可见光谱、共振拉曼光谱和磁圆二色光谱研究。这些实验结果证实Tyr - 94是唯一的轴向血红素配体，Tyr - 228对负载血红素的ShuT的稳定性有重大贡献，尽管它不直接与血红素铁相互作用。
