Speciation 98: Abstracts

Most bacterioferritins contain iron-protoporphyrin IX, typically with stoichiometries of 12 haem/ 24 subunits, and with the iron bound by bis-Met axial ligands (1). Horse spleen apoferritin has been shown to bind haemin at binding stoichiometries of between 12-24 haemins/apoferritin molecule (2,3). However, when crystalline horse spleen apoferritin was incubated with haemin, only the iron-free PPIX-apoferritin complex without its iron atom was found (4,5). There are some obvious questions about this discrepancy between solution studies and X-ray crystallography, and it was with a view to resolving some of these questions that solution studies were undertaken which showed that horse spleen apoferritin is capable of demetallating haemin and of remetallating protoporphyrin IX (2). A mechanism was proposed based on the involvement of a cluster of carboxylates at the surface of a hydrophobic pocket within which protoporphyrin IX could be bound. Protonation of the tetrapyrrole nitrogens and complexation of the iron atom by the carboxyl groups would favorise demetallation, and iron-free protoporphyrin IX would then be able to bind in the preformed hydrophoric pocket (2).

These observations have been extended to recombinant human H chain apoferritins and to rat liver apoferritin. We demonstrate that recombinant human H chain apoferritin can demetallate haemin at acidic pH values, while at alkaline pH protopophyrin IX can be remetallated. Taken together with previous solution studies on horse spleen apoferritin (predominantly L-chain), this suggests that demetallation requires the presence of both a cluster of carboxyl functions and a site within which protoporphyrin IX can be bound. The importance of the carboxyl functions is underlined by the observation that recombinant human H chain apoferritin in which Glu 58 has been replaced by Lys, which additionally allows the formation of a salt bridge with Glu 103, demetallates haemin much more slowly that the native recombinant H chain protein. The observation that rat liver apoferritin is also able to demetallate haemin and to remetallate protoporohyrin IX indicates that this is a general property of mammalian apoferritins. The potential role for haem binding in both mammalian and bacterial ferritins will be reviewed.

References

1. George, GN et al. (1993) J. Am. Chem. Soc. 115, 7716-7718.
2. Crichton, RR et al. (1997) Biochemistry 36, 15049-15054.
3. Kadir, FHA and Moore, GR (1990) FEBS Letts. 276, 81-84.
4. Précigoux, G et al. (1994) Acta Cryst. D50, 739-743.
5. Michaux, M-A et al. (1996) Proteins; Struct. Funct. Genet. 24, 314-321.

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