Vanadyl(IV) binding to mammalian ferritins. An EPR study aided by site-directed mutagenesis
Vanadyl(IV) binding to mammalian ferritins. An EPR study aided by site-directed mutagenesis
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DOI：
10.1016/s0162-0134(00)00046-5
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发表时间：
2000-05-30
影响因子：
3.9
通讯作者：
Chasteen, ND
中科院分区：
文献类型：
--
作者：
Grady, JK;Shao, JL;Chasteen, ND
在其代谢过程中，钒被认为与铁储存蛋白铁蛋白相关。为了阐明可能的钒结合位点的蛋白质，VO 2+结合哺乳动物铁蛋白进行了研究，使用定点突变和EPR光谱。VO 2+去铁蛋白EPR谱的人H-链（100%H），L-链（100%L），马脾（84%L，16%H）和羊脾（45%L，55%H）铁蛋白揭示了α和β VO 2+物种的存在下，在所有的蛋白质，这意味着这些物种的配体之间的H-和L-链是保守的。α物种比β物种稳定性差，并随pH值的增加而降低，表明这两种物种与pH值无关，这一结果与早期的建议相反。在H-和L-链铁蛋白中保守的几个残基（Asp-131、Glu-134、His-118和His-128）的定点HuHF变体的EPR光谱表明，在三重通道的外部开口附近的His-118可能是VO 2+的配体，并且负责EPR光谱中的β信号。数据表明，VO 2+不结合三重通道内的Asp-131和Glu-134残基，也不结合亚铁氧化酶位点残基Glu-62或His-65或推定的成核位点残基Glu-61、64、67。虽然铁氧化酶位点不是VO 2+结合的位点，但该位点的残基Glu-62和His-65突变为Ala会影响位于约17埃处的His-118处的VO 2+结合。因此，VO 2+自旋探针的研究提供了一个窗口铁蛋白的结构变化中没有看到在大多数以前的工作，并表明，点突变引起的长期影响时，必须仔细考虑从蛋白质的诱变研究得出的结论。(C)2000 Elsevier Science Inc. All rights reserved.
During its metabolism, vanadium is known to become associated with the iron storage protein, ferritin. To elucidate probable vanadium binding sites on the protein, VO2+ binding to mammalian ferritins was studied using site-directed mutagenesis and EPR spectroscopy. VO2+ apoferritin EPR spectra of human H-chain (100% H), L-chain (100% L), horse spleen (84% L, 16% H) and sheep spleen (45% L, 55% H) ferritins revealed the presence of alpha and beta VO2+ species in all the proteins, implying that the ligands for these species are conserved between the H- and L-chains. The alpha species is less stable than the beta species and decreases with increasing pH, demonstrating that the two species are not pH-related, a result contrary to earlier proposals. EPR spectra of site-directed HuHF variants of several residues conserved in H- and L-chain ferritins (Asp-131, Glu-134, His-118 and His-128) suggest that His-118 near the outer opening of the three-fold channel is probably a ligand for VO2+ and is responsible for the beta signals in the EPR spectrum. The data indicate that VO2+ does not bind to the Asp-131 and Glu-134 residues within the three-fold channels nor does it bind at the ferroxidase site residues Glu-62 or His-65 or at the putative nucleation site residues Glu-61,64,67. While the ferroxidase site is not a site for VO2+ binding, mutation of residues Glu-62 and His-65 of this site to Ala affects VO2+ binding at His-118, located some 17 Angstrom away. Thus, VO2+ spin probe studies provide a window on structural changes in ferritin not seen in most previous work and indicate that long-range effects caused by point mutations must be carefully considered when drawing conclusions from mutagenesis studies of the protein. (C) 2000 Elsevier Science Inc. All rights reserved.