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首页> 外文期刊>Biochemistry >Role of Citrate and Phosphate Anions in the Mechanism of Iron(III) Sequestration by Ferric Binding Protein: Kinetic Studies of the Formation of the Holoprotein of Wild-Type FbpA and Its Engineered Mutants
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Role of Citrate and Phosphate Anions in the Mechanism of Iron(III) Sequestration by Ferric Binding Protein: Kinetic Studies of the Formation of the Holoprotein of Wild-Type FbpA and Its Engineered Mutants

机译：柠檬酸根和磷酸根阴离子在铁结合蛋白螯合铁（III）机理中的作用：野生型FbpA及其工程突变体形成全蛋白的动力学研究

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摘要

Ferric binding protein A (FbpA) plays a central role in the iron acquisition processes of pathogenic Neisseria gonorrheae, Neisseria meningitidis, and Haemophilus influenzae. FbpA functions as an iron shuttle within the periplasmic space of these Gram-negative human pathogens. Iron is picked up by FbpA at the periplasmic aspect of the outer membrane with concomitant acquisition of a synergistic anion. Here we report the kinetics and mechanisms involved with loading of iron(III) into iron-free FbpA using iron(III) citrate as an iron source in the presence of excess citrate or phosphate (physiologically available anions) at pH 6.5. In the presence of excess phosphate, iron(III) citrate loads into apo-FbpA in three kinetically distinguishable steps, while in the presence of excess citrate, only two steps are discernible. A stable intermediate containing iron(III) citrate-bound FbpA is observed in each case. The observation of an additional kinetic step and moderate increase in apparent rate constants suggests an active role for phosphate in the iron insertion process. To further elucidate a mechanism for iron loading, we report on the sequestration kinetics of iron(III) citrate in the presence of phosphate with binding site mutant apo-FbpAs, H9E, E57D, E57Q, Q58A, Y195F, and Y196H. Tyrosine mutations drastically alter the kinetics and hamper iron sequestration ability. H9E, E57D, and E57Q have near native iron sequestration behavior; however, iron binding rates are altered, enabling assignment of sequential side chain interactions. Additionally, this investigation elaborates on the function of FbpA as a carrier for iron chelates as well as "naked" or free iron as originally proposed.
机译：铁结合蛋白A（FbpA）在致病性淋病奈瑟氏球菌，脑膜炎奈瑟氏球菌和流感嗜血杆菌的铁获取过程中起着核心作用。 FbpA充当这些革兰氏阴性人类病原体周质空间中的铁梭。 FbpA在外膜的周质态吸收铁，并伴随获得协同阴离子。在这里，我们报告了在pH 6.5过量柠檬酸盐或磷酸盐（生理上可用的阴离子）存在下，使用柠檬酸铁（III）作为铁源将铁（III）加载到无铁FbpA中的动力学和机理。在存在过量磷酸盐的情况下，柠檬酸铁（III）通过三个动力学上可区分的步骤加载到apo-FbpA中，而在存在过量柠檬酸盐的情况下，仅可辨别两个步骤。在每种情况下均观察到稳定的中间体，该中间体含有结合柠檬酸铁（III）的FbpA。观察到额外的动力学步骤和表观速率常数的适度增加表明磷酸盐在铁插入过程中起了积极作用。为了进一步阐明铁负载的机制，我们报道了在磷酸盐存在下具有结合位点突变apo-FbpAs，H9E，E57D，E57Q，Q58A，Y195F和Y196H的螯合柠檬酸铁的动力学。酪氨酸突变极大地改变了动力学并阻碍了铁的螯合能力。 H9E，E57D和E57Q具有接近天然的铁螯合行为;但是，铁的结合率发生了变化，可以分配顺序的侧链相互作用。另外，该研究详细说明了FbpA作为铁螯合物以及最初提出的“裸”或游离铁的载体的功能。

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