The main objective of this thesis is the elucidation of the mechanism of demetallation of haemin by L-chain apoferritins by different techniques: Crystallographic studies can give us information about the nature of the porphyrin binding sites. In order to attain maximum occupation of these sites we have used 24 haemin /apoferritin molecules. In contrast, for the EPR studies, which allow us to distinguish between haem and non-haem iron, we have used less than saturating values, typically 8 haemin / apoferritin. For the mass spectrometry studies, which give us information on the molecular weight of the bound porphyrin derivative, we have chosen, after preliminary experiments to use 12 haemin / apoferritin. The same conditions were employed for the EXAFS studies, which give us information concerning the ligand environment of the metal ion. We have analysed the wild type L-chain apoferritin (rHoLF) and its three mutants ( rHoLF E53,56,57,60Q; rHoLF R59M and rHoLF E53,56,57,60Q + R59M) incubated with haemin in acidic and basic conditions and after different times of incubation.

The main objective of this thesis is the elucidation of the mechanism of demetallation of haemin by L-chain apoferritins by different techniques: Crystallographic studies can give us information about the nature of the porphyrin binding sites. In order to attain maximum occupation of these sites we have used 24 haemin /apoferritin molecules. In contrast, for the EPR studies, which allow us to distinguish between haem and non-haem iron, we have used less than saturating values, typically 8 haemin / apoferritin. For the mass spectrometry studies, which give us information on the molecular weight of the bound porphyrin derivative, we have chosen, after preliminary experiments to use 12 haemin / apoferritin. The same conditions were employed for the EXAFS studies, which give us information concerning the ligand environment of the metal ion. We have analysed the wild type L-chain apoferritin (rHoLF) and its three mutants ( rHoLF E53,56,57,60Q; rHoLF R59M and rHoLF E53,56,57,60Q + R59M) incubated with haemin in acidic and basic conditions and after different times of incubation.