A crystallographic study of haem binding to ferritin

Abstract

Ferritin, the iron-storage protein, binds porphyrins, metalloporphyrins and the fluorescent dyes ANS (8-anilino-1-naphthalenesulfonic acid) and TNS (2-p-toluidinyl-6-naphthalenesulfonic acid), similarly to apo-myoglobin. Octahedral crystals of horse-spleen apo-ferritin (HSF; 174 amino acids) complexes prepared by the addition of haem, hematoporphyrin or Sn-protoporphyrin IX to a solution of apo-ferritin crystallize in space group F432 with cell parameter a = 184.0/~,. X-ray crystallographic analysis of single crystals prepared from a mixture containing haem or Sn-protoporphyrin IX shows that the haem-binding sites in these crystals are occupied by protoporphyrin IX, which is free of metal, rather than by the original metalloporphyrin. The present paper describes the structure of horse-spleen apo-ferfitin cocrystallized with Sn-protoporphyrin IX. The 6797 reflections up to 2.6 A resolution used in the refinement were obtained from a data set recorded on a Nicolet/Xentronics area detector with Cu Ks radiation from a Rigaku RU 200 rotating anode. The final structure comprises 1613 non-H atoms, two Cd atoms and 170 solvent molecules. Four residues are described as disordered. The root-meansquare deviations from ideal bond lengths and angles are 0.013 A and 2.88 °, respectively. Protoporphyrins are observed in special positions on the twofold axes of the ferritin molecule with a stoichiometry of 0.4 per subunit.

Beyond the detergent effect: a binding site for sodium dodecyl sulfate (SDS) in mammalian apoferritin

“…This site has also been reported to bind other amphipathic molecules (Niemeyer et al, 2008;Trikha et al, 1995). In the evolutionarily related bacterioferritins the corresponding site serves to bind heme (Carrondo, 2003), and horse spleen apoferritin retains the ability to bind porphyrin in this site (Pré cigoux et al, 1994). In the horse ferritinporphyrin complex structure the hydrophobic ring structure binds in the heart of the cavity, while the propionic acid side chains extend out from the openings to this cavity, where they can interact with the two symmetry-related copies of Arg59 in a manner similar to that seen with SDS.…”

Binding of Mammalian and Avian Ferritins with Biotinylated Hemin: Demonstration of Preferential Binding of the H Subunit to Heme

“…The binding of mammalian ferritin with hemin has been studied with commercial horse spleen apoferritin in spectroscopic and crystallographic studies [6,23], indicating that mammalian ferritins bind heme. Human apolipoprotein B has been revealed to bind to horse spleen, bovine spleen and canine liver holoferritins through heme-mediated binding, suggesting that these ferritins bind hemin because heme is oxidized during these preparations [27].…”

“…On the other hand, once biotinylated hemin binds ferritin, the bound hemin is unlikely to dissociate from the heme-binding site on the surface of the ferritin molecule. Horse spleen apoferritin has been revealed to possess a heme-binding pocket [6,12,23], and reducing agents that remove iron cause a simultaneous release of heme from the heme-binding site on the ferritin surface [27]. This can explain why mammalian apoferritins show higher binding activity with biotinylated hemin than their respective holoferritins.…”

“…Horse spleen apoferritin and probably all mammalian ferritins not only bind iron but also heme, as does bacterial ferritin [11,12,16,23]. Interestingly, horse spleen apoferritin not only binds hemin but also demetallates hemin and remetallates released iron to protoporphyrin IX (PPIX) based on spectroscopic data as a function of pH [6].…”

“…The ferritin L subunit, but not the H subunit, has been reported to be involved in heme binding based on a comparison of their amino acid sequences and a predicted heme binding pocket [5,6,23]. However, it remains to be demonstrated whether or not the H subunit binds heme.…”