Vanadyl(IV) binding to mammalian ferritins. An EPR study aided by site-directed mutagenesis

John K. Grady, Junlong Shao, Paolo Arosio, Paolo Santambrogio, N. Dennis Chasteen

Research output: Contribution to journalArticle

Abstract

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 α and β VO2+ species in all the proteins, implying that the ligands for these species are conserved between the H- and L-chains. The α species is less stable than the β 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 β 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 Å 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.

Original languageEnglish
Pages (from-to)107-113
Number of pages7
JournalJournal of Inorganic Biochemistry
Volume80
Issue number1-2
DOIs
Publication statusPublished - May 30 2000

Keywords

  • Electron paramagnetic resonance spectroscopy
  • Ferritin
  • Site-directed mutagenesis
  • Vanadyl ion

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

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