Optical and EPR spectroscopic studies of demetallation of hemin by L-chain apoferritins.
Earlier crystallographic and spectroscopic studies had shown that horse spleen apoferritin was capable of removing the metal ion from hemin (Fe(III)-protoporphyrin IX) [G. Précigoux, J. Yariv, B. Gallois, A. Dautant, C. Courseille, B. Langlois d'Estaintot, Acta Cryst. D50 (1994) 739-743; R.R. Crichton, J.A. Soruco, F. Roland, M.A. Michaux, B. Gallois, G. Précigoux, J.-P. Mahy, D. Mansuy, Biochemistry 36 (1997) 15049-15054]. We have carried out a detailed re-analysis of this phenomenon using both horse spleen and recombinant horse L-chain apoferritins, by electron paramagnetic resonance spectroscopy (EPR) to unequivocally distinguish between heme and non-heme iron. On the basis of site-directed mutagenesis and chemical modification of carboxyl residues, our results show that the UV-visible difference spectroscopic method that was used to establish the mechanism of demetallation is not representative of hemin demetallation. EPR spectroscopy does establish, as in the initial crystallographic investigation, that hemin demetallation occurs, but it is much slower. The signal at g=4.3 corresponding to high spin non-heme-iron (III) increases while the signal at g=6 corresponding to heme-iron decreases. Demetallation by the mutant protein, while slower than the wild-type, still occurs, suggesting that the mechanism of demetallation does not only involve the cluster of four glutamate residues (Glu 53, 56, 57, 60), proposed in earlier studies. However, the mutant protein had lost its capacity to incorporate iron, as had the native protein in which the four Glu residues had been chemically modified. Interestingly, a signal at g=1.94 is also observed. This signal most likely corresponds to a mixed-valence Fe(II)-Fe(III) cluster suggesting that a redox reaction may also be involved in the mechanism of demetallation.
Carette
Noëlle
N
Université catholique de Louvain, Unit of Biochemistry, Place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium.
Hagen
Wilfred
W
Bertrand
Luc
L
de Val
Natalia
N
Vertommen
Didier
D
Roland
Francine
F
Hue
Louis
L
Crichton
Robert R
RR
D000818:Animals; D001052:Apoferritins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006427:Hemin / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D019159:Nonheme Iron Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013154:Spleen / Q000737:chemistry
D019159:Nonheme Iron Proteins; D011994:Recombinant Proteins; D006427:Hemin; D001052:Apoferritins; D007501:Iron