EPR Studies of Recombinant Horse L-Chain Apoferritin and its Mutant (E 53,56,57,60 Q) with Haemin |
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Authors: | Natalia de Val Wilfred R Hagen Robert R Crichton |
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Institution: | (1) Unit of Biochemistry, Department of Chemistry, Université Catholique de Louvain, Batiment Lavoisier, 1 Place Louis Pasteur, 1348 Louvain-la-Neuve, Belgium;(2) Department of Biotechnology, Delft University of Technology, Julianalaan 67, Delft, 2628 BC, The Netherlands |
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Abstract: | Structural similarities between ferritins and bacterioferritins have been extensively demonstrated. However, there is an essential
difference between these two types of ferritins: whereas bacterioferritins bind haem, in-vivo, as Fe(II)-protoporphyrin IX (this haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded
by two axial Met 52 residues), eukaryotic ferritins are non-haem iron proteins. However, in in-vivo studies, a cofactor has been isolated from horse spleen apoferritin similar to protoporphyrin IX; in in-vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haemin (Fe(III)-protoporphyrin IX).
Studies of haemin incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin
is found in a pocket similar to that which binds haem in bacterioferritins (Précigoux et al. 1994 Acta Cryst
D50, 739–743). A mechanism of demetallation of haemin by L-chain apoferritins was subsequently proposed (Crichton et al. 1997 Biochem
36, 15049–15054) which involved four Glu residues (E 53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared
to be favoured by acidic conditions. To verify this mechanism, these four Glu have been mutated to Gln in recombinant horse
L-chain apoferritin. We report here the EPR spectra of recombinant horse L-chain apoferritin and its mutant with haemin in
basic and acidic conditions. These studies confirm the ability of recombinant L-chain apoferritin and its mutant to incorporate
and demetallate the haemin in acidic and basic conditions. |
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Keywords: | ferritin bacterioferritin EPR haemin demetallation |
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