Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q).
An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin is found in a corresponding pocket to haem in bacterioferritins [Précigoux, G., Yariv, J., Gallois, B., Dautant, A., Courseille, C. and Langlois, d'Estaintot B. (1994) A crystallographic study of haem binding to ferritin. Acta Cryst. D 50, 739-743]. A mechanism of demetallation of haemin by L-chain apoferritin was proposed [Crichton, R.R., Soruco, J.A., Roland, F., Michaux, M.A., Gallois, B., Précigoux, G., Mahy, J.P. and Mansuy. (1997) Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH. J. P. Biochem. 36, 49, 15049-15054]: this involved four Glu residues (53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared to be favoured by acidic conditions. To verify this mechanism, we have mutated these four Glu to Gln and examined demetallation in both acidic and basic conditions. In this paper, we report the mass spectrometry studies of L-chain apoferritin and its mutant incubated with haemin and analysed after different times of incubation: 15 days, 2 months, 6 months, 9 months and 12 months. These studies show that the recombinant L-chain apoferritin and its mutant are able to demetallate haemin to give a hydroxyethyl protoporphyrin IX derivative in a dimeric form [Macieira, S., Martins, B. M. and Huber, R. (2003) Oxygen-dependent coproporphyrinogen IX oxidase from Escherichia coli: one-step purification and biochemical characterization. FEMS. Microbiology Letters 226, 31-37].
de Val
Natalia
N
Department of Biochemistry, Université Catholique de Louvain, Bâtiment Lavoisier, 1 Place Louis Pasteur, 1348 Louvain-la-Neuve, Belgium. deval@bioc.ucl.ac.be
Herschbach
Haiko
H
Potier
Noëlle
N
Dorsselaer
Alain Van
AV
Crichton
Robert R
RR
D000818:Animals; D001052:Apoferritins / Q000737:chemistry* / Q000235:genetics*; D018360:Crystallography, X-Ray; D006427:Hemin / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008670:Metals / Q000737:chemistry*; D020125:Mutation, Missense; D011485:Protein Binding; D011524:Protoporphyrins / Q000737:chemistry; D011994:Recombinant Proteins; D013154:Spleen
D008670:Metals; D011524:Protoporphyrins; D011994:Recombinant Proteins; D006427:Hemin; D001052:Apoferritins; C028025:protoporphyrin IX