Vanadyl(IV) binding to mammalian ferritins. An EPR study aided by site-directed mutagenesis.
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 A 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.
Grady
J K
JK
Department of Chemistry, University of New Hampshire, Durham 03824-3598, USA.
Shao
J
J
Arosio
P
P
Santambrogio
P
P
Chasteen
N D
ND
D000818:Animals; D001052:Apoferritins / Q000378:metabolism; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005293:Ferritins / Q000378:metabolism*; D006736:Horses; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D012756:Sheep; D013154:Spleen / Q000378:metabolism*; D014639:Vanadium / Q000378:metabolism*
D011994:Recombinant Proteins; D014639:Vanadium; D005293:Ferritins; D001052:Apoferritins; D007501:Iron