Malone Simon A, Lewin Allison, Kilic Mehmet A, Svistunenko Dimitri A, Cooper Chris E, Wilson Michael T, Le Brun Nick E, Spiro Stephen, Moore Geoffrey R
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK.
J Am Chem Soc. 2004 Jan 21;126(2):496-504. doi: 10.1021/ja036483z.
Ferritins are iron-storage proteins capable of holding up to 4500 Fe(3+) ions within a single water-soluble protein shell made from 24 polypeptide chains. The Glu128Arg/Glu135Arg mutants of Escherichia coli and Rhodobacter capsulatus bacterioferritins are unable to associate into 24-meric structures, with dimers of polypeptide chains being their stable forms. The aerobic addition to these of up to 8-10 or 14-20 Fe(2+) ions per dimer, respectively, results in the oxidation of the added Fe(2+) to Fe(3+). Gel permeation chromatography and sedimentation equilibrium studies confirm that the Fe(3+) ions are associated with the polypeptide dimer, and the lack of intense EPR signals from magnetically isolated Fe(3+) ions confirms the formation of one or more antiferromagnetically coupled clusters of Fe(3+) ions. The effect of Fe(3+) chelators on iron-loaded subunit dimers is to remove the Fe(3+) from the protein, but to do so slowly, consistent with it not being merely adventitiously associated with protein. These data provide experimental support for the presence of nucleation centers for the mineral cores in bacterioferritins and indicate that these proteins are not simply acting as vessels in which hydrolysis of Fe(3+) occurs independent from the protein surface. From analyses of X-ray structures and amino acid sequence comparisons, possible nucleation sites are identified.
铁蛋白是铁储存蛋白，能够在由24条多肽链构成的单个水溶性蛋白壳内储存多达4500个Fe(3+)离子。大肠杆菌和荚膜红细菌细菌铁蛋白的Glu128Arg/Glu135Arg突变体无法组装成24聚体结构，多肽链二聚体是其稳定形式。向这些二聚体中分别有氧添加多达8 - 10个或14 - 20个Fe(2+)离子，会导致添加的Fe(2+)氧化为Fe(3+)。凝胶渗透色谱和沉降平衡研究证实Fe(3+)离子与多肽二聚体相关联，并且从磁性分离的Fe(3+)离子缺乏强烈的电子顺磁共振信号，证实形成了一个或多个反铁磁耦合的Fe(3+)离子簇。Fe(3+)螯合剂对负载铁的亚基二聚体的作用是从蛋白质中去除Fe(3+)，但速度缓慢，这与它不仅仅是偶然与蛋白质结合一致。这些数据为细菌铁蛋白中矿质核心的成核中心的存在提供了实验支持，并表明这些蛋白质不仅仅是充当Fe(3+)水解独立于蛋白质表面发生的容器。通过对X射线结构的分析和氨基酸序列比较，确定了可能的成核位点。
