Mielcarek Andreas, Blauenburg Bastian, Miethke Marcus, Marahiel Mohamed A
Philipps-University Marburg, Department of Chemistry/Biochemistry, Marburg, Germany.
Philipps-University Marburg, Department of Chemistry/Biochemistry, Marburg, Germany; Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, United States of America.
PLoS One. 2015 Mar 31;10(3):e0122538. doi: 10.1371/journal.pone.0122538. eCollection 2015.
Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen-deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis.
铁作为一种元素，是所有真核生物和大多数细菌维持生命所必需的。尽管已经对几种细菌获取铁的策略进行了充分研究，但对于铁在细胞内的运输途径及其进入辅因子生物合成系统的过程却知之甚少。在本研究中，我们调查了枯草芽孢杆菌中血红素成熟的铁依赖性过程，并首次提供了结构证据，证明一种frataxin同源物（Fra）与一种亚铁螯合酶（HemH）之间存在物理相互作用。Fra被认为在不同细胞途径中既作为调节成分又作为铁伴侣发挥作用，而HemH催化血红素b生物合成的最后一步。从粗细胞裂解物中共纯化时观察到Fra和HemH之间存在特异性相互作用，此外，通过使用重组蛋白进行分析型尺寸排阻色谱也观察到了这种相互作用。氢-氘交换实验确定了Fra/HemH相互作用界面的情况，并揭示Fra是亚铁螯合酶HemH的特异性亚铁供体。通过使用枯草芽孢杆菌一氧化氮合酶bsNos作为代谢靶标酶，证实了Fra介导的铁转移后体外生成的血红素b辅因子的功能利用。互补的突变分析证实，Fra是血红素b辅因子成熟及后续靶向定位的必需成分，因此是枯草芽孢杆菌铁依赖性生理学中的关键参与者。
