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Iron binding to horse spleen ferritin. A vanadyl endor spin probe study
METALREGOGNITION,STORAGE,ANDTRANSPORT
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B()l() IRON BINDING TO HORSE SPLEEN FERRITIN. A VANADYL ENDOR SPIN PROBE STUDY P.M. Hanna,a N.D. Chasteenla G.A. Rottman, P. Aisen,b
aDepartment of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, and bDepartment of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. The role of the protein shell in the formation of the hydrous ferric oxide core of ferritin is poorly understood. A V02+ spin probe study [l] was undertaken to characterize the initial complex of Fe2+ with horse spleen apoferritin. The ferritin used in these studies was composed of 96% L-subunits. A competitive binding study of V02+ and Fe2+ showed that the two metals compete 1:l for binding at the same site or region of the protein. Curve fitting of the data showed that the vanadyl affinity for the protein was 16 times that of iron. Electron nuclear double resonance (ENDOR) measurements on the V02+-apoferritin complex showed couplings from two nitrogen nuclei having A, = 7.14 and Q, = 0.24 MHz, and A, = 6.36 and Q, = 0.85 MHz, tentatively assigned to the Nl and N3 nitrogens, respectively, of an imidazole ligand of histidine [Z]. An exchangeable proton with a coupling of approximately 1 MHz is assigned to the Nh proton of the coordinated nitrogen [2]. A 60-70% reduction in the intensity of the 'H matrix ENDOR line upon 'H,O-H,O exchange indicates that the metal binding site is accessible to solvent and, therefore, to molecular oxygen as well. The ENDOR data provide the first evidence for a principle iron binding site 'with The nitrogen coordination in an L-subunit ferritin. site may be important in Fe(I1) oxidation during the beginning stages of core formation. 1. N.D. Chasteen and E.C. Theil, J. Biol. Chem. 257, 7672 (1982). 2. C. F. Mulks, B. Kirste, and H. van Willigen, J. Am. Chem. Sot., 104, 5906 (1982).
133
B()l() IRON BINDING TO HORSE SPLEEN FERRITIN. A VANADYL ENDOR SPIN PROBE STUDY P.M. Hanna,a N.D. Chasteenla G.A. Rottman, P. Aisen,b
aDepartment of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, and bDepartment of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. The role of the protein shell in the formation of the hydrous ferric oxide core of ferritin is poorly understood. A V02+ spin probe study [l] was undertaken to characterize the initial complex of Fe2+ with horse spleen apoferritin. The ferritin used in these studies was composed of 96% L-subunits. A competitive binding study of V02+ and Fe2+ showed that the two metals compete 1:l for binding at the same site or region of the protein. Curve fitting of the data showed that the vanadyl affinity for the protein was 16 times that of iron. Electron nuclear double resonance (ENDOR) measurements on the V02+-apoferritin complex showed couplings from two nitrogen nuclei having A, = 7.14 and Q, = 0.24 MHz, and A, = 6.36 and Q, = 0.85 MHz, tentatively assigned to the Nl and N3 nitrogens, respectively, of an imidazole ligand of histidine [Z]. An exchangeable proton with a coupling of approximately 1 MHz is assigned to the Nh proton of the coordinated nitrogen [2]. A 60-70% reduction in the intensity of the 'H matrix ENDOR line upon 'H,O-H,O exchange indicates that the metal binding site is accessible to solvent and, therefore, to molecular oxygen as well. The ENDOR data provide the first evidence for a principle iron binding site 'with The nitrogen coordination in an L-subunit ferritin. site may be important in Fe(I1) oxidation during the beginning stages of core formation. 1. N.D. Chasteen and E.C. Theil, J. Biol. Chem. 257, 7672 (1982). 2. C. F. Mulks, B. Kirste, and H. van Willigen, J. Am. Chem. Sot., 104, 5906 (1982).