Ferroxidase kinetics of human liver apoferritin, recombinant H-chain apoferritin, and site-directed mutants.
A detailed study of the kinetics of iron(II) oxidation by molecular oxygen in natural and recombinant human apoferritins has been carried out using electrode oximetry to better understand the ferroxidase activity of the protein shell. A comparative study of recombinant L-chain ferritin (rLF), recombinant H-chain ferritin (rHF), and variants has shown that (1) rLF lacks a ferroxidase activity, confirming the results of previous studies; (2) the ferroxidase site of rHF involves Glu-62 and His-65, presumably as Fe2+ ligands, since mutation of these residues abolishes most of the oxidase activity, in agreement with previous studies; and (3) mutation of both the putative ferroxidase and nucleation site ligands in rHF renders the protein totally incapable of catalyzing the oxidation of Fe2+ whereas mutation of nucleation site ligands alone (Glu-61, Glu-64, and Glu-67) decreases the activity only slightly. Analysis of the kinetics of rHF and natural human liver ferritin (HLF) (4% H-chain, 96% L-chain) gave the following apparent parameters at pH 7: Km,O2 = 6 +/- 2 microM, Km,Fe = 80 +/- 10 microM, and kcat = 201 +/- 14 min-1 for rHF and Km,O2 = 60 +/- 12 microM, Km,Fe = 50 +/- 10 microM, and kcat = 31.2 +/- 0.6 min-1 for HLF. Furthermore, Zn2+ was shown to be a noncompetitive inhibitor of Fe2+ oxidation in rHF but a mixed inhibitor in HLF. These different forms of Zn2+ inhibition in the two proteins and the higher activity of HLF than expected, based on its H-chain composition as well as differences in their enzyme kinetic parameters, suggest that H- and L-chains cooperate in modulating the ferroxidase activity of the apoferritin even though the L-subunit lacks a ferroxidase site itself.
Sun
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Department of Chemistry, University of New Hampshire, Durham 03824.
Arosio
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P
Levi
S
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Chasteen
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ND
D001052:Apoferritins / Q000235:genetics / Q000378:metabolism*; D002570:Ceruloplasmin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D015032:Zinc / Q000494:pharmacology
D011994:Recombinant Proteins; D001052:Apoferritins; D007501:Iron; D002570:Ceruloplasmin; D015032:Zinc