title: Structural analysis of haemin demetallation by L-chain apoferritins.
abstract: There are extensive structural similarities between eukaryotic and prokaryotic ferritins. However, there is one essential difference between these two types of ferritins: bacterioferritins contain haem whereas eukaryotic ferritins are considered to be non-haem proteins. In vitro experiments had shown that horse spleen apoferritin or recombinant horse L chain apoferritins, when co-crystallised with haemin, undergoes demetallation of the porphyrin. In the present study a cofactor has been isolated directly from horse spleen apoferritin and from crystals of the mutant horse L chain apoferritin (E53Q, E56Q, E57Q, E60Q and R59M) which had been co-crystallised with haemin. In both cases the HPLC/ESI-MS results confirm that the cofactor is a N-ethylprotoporphyrin IX. Crystal structures of wild type L chain horse apoferritin and its three mutants co-crystallised with haemin have been determined to high resolution and in all cases a metal-free molecule derived from haemin was found in the hydrophobic pocket, close to the two-fold axis. The X-ray structure of the E53Q, E56Q, E57Q, E60Q+R59M recombinant horse L-chain apoferritin has been obtained at a higher resolution (1.16Å) than previously reported for any mammalian apoferritins. Similar evidence for a metal-free molecule derived from haemin was found in the electron density map of horse spleen apoferritin (at a resolution of 1.5Å). The out-of-plane distortion of the observed porphyrin is clearly compatible with an N-alkyl porphyrin. We conclude that L-chain ferritins are capable of binding and demetallating haemin, generating in the process N-ethylprotoporphyrin IX both in vivo and in vitro.
authors.affiliation: Institute of Life Sciences, University of Louvain, Place Croix du Sud, 1348 Louvain-la-Neuve, Belgium. natalia.de-val@pasteur.fr
authors.forename: Natalia
authors.initials: N
authors.lastname: de Val
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Declercq
authors.forename: Chang Kee
authors.initials: CK
authors.lastname: Lim
authors.forename: Robert R
authors.initials: RR
authors.lastname: Crichton
mesh_terms: D000818:Animals; D001052:Apoferritins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D018360:Crystallography, X-Ray; D006427:Hemin / Q000737:chemistry*; D006736:Horses; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011524:Protoporphyrins / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013154:Spleen / Q000737:chemistry / Q000378:metabolism
chemical_list: D008670:Metals; D011524:Protoporphyrins; D011994:Recombinant Proteins; D006427:Hemin; C030827:N-ethylprotoporphyrin IX; D001052:Apoferritins