Ferroxidase kinetics of horse spleen apoferritin.
Protein ferroxidase site(s), which catalyze the reaction between ferrous ion and dioxygen, have long been thought to play a role in core formation in ferritin; however, the mechanism of the reaction has never been studied in detail. In the present work, the enzymatic activity of ferritin was examined using oximetry, the net Fe2+ oxidation reaction being as follows. [formula: see text] The reaction exhibits saturation kinetics with respect to both Fe2+ and O2 (apparent Michaelis constants: Km,Fe = 0.35 +/- 0.01 mM and Km,O2 = 0.14 +/- 0.03 mM). The enzyme has a turnover number kcat = 80 +/- 3 min-1 at 20 degrees C with maximal activity at pH 7. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe(II) oxidation occurs in 1-electron steps. Zinc(II) is a competitive inhibitor of iron(II) oxidation at Zn2+/apoprotein ratios > or = 6 (inhibitor constant KI,Zn = 0.067 +/- 0.011 mM) but appears to be a noncompetitive inhibitor at lower ratios (< or = 2), indicating the presence of more than one type of zinc binding site on the protein. At increments of 50 Fe2+/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. However, when larger increments are employed, some iron oxidation appears to occur on the surface of the mineral core. The results of these studies emphasize the role of the protein shell in all phases of core growth and confirm the presence of a functionally important catalytic site in ferritin in addition to other binding sites on the protein for iron.
Sun
S
S
Department of Chemistry, University of New Hampshire, Durham 03824.
Chasteen
N D
ND
D000818:Animals; D001052:Apoferritins / Q000378:metabolism*; D002570:Ceruloplasmin / Q000378:metabolism*; D006736:Horses; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D016018:Least-Squares Analysis; D008433:Mathematics; D008962:Models, Theoretical; D010101:Oxygen Consumption; D013154:Spleen / Q000201:enzymology*; D013816:Thermodynamics
D001052:Apoferritins; D007501:Iron; D002570:Ceruloplasmin