Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin.
Ferritin is a ubiquitous iron-storage protein that has 24 subunits. Each subunit of ferritins that exhibit high Fe(II) oxidation rates has a diiron binding site, the so-called ferroxidase center (FC). The role of the FC appears to be essential for the iron-oxidation catalysis of ferritins. Studies of the iron oxidation by mammalian, bacterial, and archaeal ferritin have indicated different mechanisms are operative for Fe(II) oxidation, and for inhibition of the Fe(II) oxidation by Zn(II). These differences are presumably related to the variations in the amino acid residues of the FC and/or transport channels. We have used a combination of UV-vis spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry to study the inhibiting action of Zn(II) ions on the iron-oxidation process by apoferritin and by ferritin aerobically preloaded with 48 Fe(II) per 24-meric protein, and to study a possible role of phosphate in initial iron mineralization by Pyrococcus furiosus ferritin (PfFtn). Although the empty FC can accommodate two zinc ions, binding of one zinc ion to the FC suffices to essentially abolish iron-oxidation activity. Zn(II) no longer binds to the FC nor does it inhibit iron core formation once the FC is filled with two Fe(III). Phosphate and vanadate facilitate iron oxidation only after formation of a stable FC, whereupon they become an integral part of the core. These results corroborate our previous proposal that the FC in PfFtn is a stable prosthetic group, and they suggest that its formation is essential for iron-oxidation catalysis by the protein.
Honarmand Ebrahimi
Kourosh
K
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.
Hagedoorn
Peter-Leon
PL
Hagen
Wilfred R
WR
D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis; D005293:Ferritins / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D010710:Phosphates; D005293:Ferritins; D007501:Iron; D015032:Zinc