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-di rected mutagenesis and EPR spectroscopy. VO2+ apoferritin EPR spectra of hu man H-chain (100% H), L-chain (100% L), horse spleen (84% L, 16% H) and she ep 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 speci es are conserved between the H- and L-chains. The alpha species is less sta ble than the beta species and decreases with increasing pH, demonstrating t hat the two species are not pH-related, a result contrary to earlier propos als. EPR spectra of site-directed HuHF variants of several residues conserv ed in H- and L-chain ferritins (Asp-131, Glu-134, His-118 and His-128) sugg est that His-118 near the outer opening of the three-fold channel is probab ly a ligand for VO2+ and is responsible for the beta signals in the EPR spe ctrum. 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 ferroxidas e site residues Glu-62 or His-65 or at the putative nucleation site residue s 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+ bin ding at His-118, located some 17 Angstrom away. Thus, VO2+ spin probe studi es provide a window on structural changes in ferritin not seen in most prev ious work and indicate that long-range effects caused by point mutations mu st be carefully considered when drawing conclusions from mutagenesis studie s of the protein. (C) 2000 Elsevier Science Inc. All rights reserved.