Characterization of the Periplasmic Heme-Binding Protein ShuT from the Heme Uptake System of Shigella dysenteriae
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- 作者：Suntara Eakanunkul ; Gudrun S. Lukat-Rodgers ; Suganya Sumithran ; Arundhati Ghosh ; Kenton R. Rodgers ; John H. Dawson ; and Angela Wilks
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：October 4, 2005
- 年：2005
- 卷：44
- 期：39
- 页码：13179 - 13191
- 全文大小：405K
- 年卷期：v.44,no.39(October 4, 2005)
- ISSN：1520-4995
文摘
The heme uptake systems by which bacterial pathogens acquire and utilize heme have recentlybeen described. Such systems may utilize heme directly from the host's hemeproteins or via a hemophorethat sequesters and transports heme to an outer membrane receptor and subsequently to the translocatingproteins by which heme is further transported into the cell. However, little is known of the heme bindingand release mechanisms that facilitate the uptake of heme into the pathogenic organism. As a first steptoward elucidating the molecular level events that drive heme binding and release, we have undertakena spectroscopic and mutational study of the first purified periplasmic heme-binding protein (PBP), ShuTfrom Shigella dysenteriae. On the basis of sequence identity, the ShuT protein is most closely related tothe class of PBPs typified by the vitamin B12 (BtuF) and iron-hydroxamate (FhuD) PBPs and is a monomericprotein having a molecular mass of 28.5 kDa following proteolytic processing of the periplasmic signalingpeptide. ShuT binds one b-type heme per monomer with high affinity and bears no significant homologywith 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, resonanceRaman, 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 makinga significant contribution to the stability of heme-loaded ShuT, albeit without directly interacting withthe heme iron.