51V NMR as a Probe of Vanadium(V) Coordination to Human Apotransferrin

5'V NMR is a highly sensitive tool to probe the nature of the metal binding sites of human transferrin (Tf). At a field strength of 11.7 T, the two vanadium(V) binding sites in V,-Tf are characterized by 51V chemical shifts at -529.5 and -53 1.5 ppm versus VOCl,. These shifts are assigned to the Cand N-terminal sites, respectively, based on the 51V NMR spectra of vanadate addition to FeN-Tf and Fec-Tf. Tight binding of V(V) to the metal binding site is further ascertained by a linear increase of signal area with V(V) concentration up to an approximate 2:l stoichiometry, stoichiometric displacement of protein-bound V(V) during titration with Fe(III), and the absence of the -529.5/-531.5 ppm resonance upon addition of V(V) to Fe,Tf and Ga2Tf. The chemical shift of the Vc-Tf resonance is independent of pH (5.8-9.0) and temperature (275-310 K), whereas the VN-Tf resonance varies slightly (1-2 ppm) with pH and temperature. At relatively high V(V) concentrations and at high ratios of V(V)/Tf, conditions under which a large fraction of the V(V) is present as V oligomers, the 51V resonance of Tf-bound V(V) is not observed, possibly owing to interference by the oligomeric species. Under conditions of tight V(V) binding, the sharpness of the protein-bound resonances is a consequence of the motional characteristics of transferrin, which place the Tf-bound vanadium(V) outside the extreme narrowing limit but within the motional narrowing limit. Several theoretically predicted consequences for an I = 7/2 nucleus are observed experimentally, some for the first time in an aqueous protein system. (1) The observed signal intensity of transferrin-bound vanadium(V) compared to that of an equimolar aqueous vanadate sample is substantially reduced, since only one out of four spinspin relaxation components (Le., + I / * I / * transition) is observed. (2) At 7.05 T the line width is substantially greater than at 11.7 T, and a 5-ppm upfield shift is observed which is attributed to a dynamic frequency shift. (3) The line width of Tf-bound V(V) is not affected by solvent viscosity up to 50% v/v glycerol/buffer. (4) Measurements of signal intensity as a function of pulse length reveal increased 51V precession frequencies in the radio-frequency field, as frequently observed for studies of half-integer quadrupolar nuclei in the solid state. The excitation spectrum is found to be identical for Vc and VN sites and independent of the V/Tf ratio. At small pulse angles, the fraction of the signal observed is constant and amounts to ca. 20% of that of an equimolar solution of free vanadate, in close agreement with the theoretical prediction. The present study confirms that 51V NMR is a powerful tool to probe V(V) binding to apotransferrin and should also become widely applicable to characterize V(V) binding to other macromolecules. Transferrins are glycoproteins whose primary function is to bind and transport iron. Transferrins also coordinate a wide variety of other metal ions (e.g., Cu(II), V02+, Cr(III), Ga(III), TI(III), etc.),' including v a n a d i ~ m ( V ) . ~ ~ Human transferrin (Tf) is a single polypeptide chain with two homologous regions each of which binds one metal atom.' Elucidation of the similarities and differences between the two metal binding sites continues to be an area of active interest, the results of which should increase our understanding of the functional differences of the two binding sites. The X-ray crystal structure of human lactoferrin shows that the iron binding sites are separated by 42 8, and that the metal binding ( l ? (a) Chasteen, N. D. Adu. Inorg. Biochem. 1983, 58 201. (b) Aisen, P.; Listowsky, I . Annu. Reo. Biochem. 1980, 49, 357. (c) Brock, J. H. in Metalloproteins; Harrison, P. M., Ed.; Verlag Chemie: Weinheim, West Germany, 1985; Part 2, p 183 and references therein. (2) Harris, W. R.; Carrano, C. J . J . Inorg. Biochem. 1984, 22, 201. (3) Chasteen, N. D.; Grady, J. K.; Holloway, C. E. Inorg. Chem. 1986, (4) Butler, A.; Danzitz, M. J.; Eckert, H. J . Am. Chem. Sot. 1987, 1/39, 25, 2754. 1864. ligands at both binding sites are two tyrosine residues, one histidine residue, one aspartate residue, a H,O (or OH-) molecule, and an anion (COj2or HCO;).5 The fact that human transferrin and lactoferrin share a high degree of sequence homology (30%),Ic that all the metal binding amino acids in lactoferrin are conserved in human transferrin, and that much spectroscopic evidence indicates that the binding sites of lactoferrin and human transferrin are similar, suggests that the metal binding ligands are the same between transferrin and lactoferrin and between the two metal binding sites in human transferrin. A wide variety of experimental results, however, suggest the two metal binding sites in human transferrin are not equiualent, even though the ligands of both sites may be identical. Structural differences have been inferred from the ESR spectra of the Fe(HI)-: V02+-,7 Cu(11)-, and Cr(III)-bound8 transferrin derivatives ( 5 ) Anderson, B. F.; Baker, H. M.; Dodson, E. J.; Norris, G. E.; Rumball, S. V.; Waters, J. M.; Baker, E. D. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, (6) Aisen, P.; Liebman, A.; Zweier, J. J . Eiol. Chem. 1978, 253, 1930. 1769-1773. 0002-7863/89/1511-2802$01.50/0