title: Kinetic studies of iron deposition catalyzed by recombinant human liver heavy and light ferritins and Azotobacter vinelandii bacterioferritin using O2 and H2O2 as oxidants.
abstract: The discrepancy between predicted and measured H(2)O(2) formation during iron deposition with recombinant heavy human liver ferritin (rHF) was attributed to reaction with the iron protein complex [Biochemistry 40 (2001) 10832-10838]. This proposal was examined by stopped-flow kinetic studies and analysis for H(2)O(2) production using (1) rHF, and Azotobacter vinelandii bacterial ferritin (AvBF), each containing 24 identical subunits with ferroxidase centers; (2) site-altered rHF mutants with functional and dysfunctional ferroxidase centers; and (3) recombinant human liver light ferritin (rLF), containing no ferroxidase center. For rHF, nearly identical pseudo-first-order rate constants of 0.18 s(-1) at pH 7.5 were measured for Fe(2+) oxidation by both O(2) and H(2)O(2), but for rLF, the rate with O(2) was 200-fold slower than that for H(2)O(2) (k = 0.22 s(-1)). A Fe(2+)/O(2) stoichiometry near 2.4 was measured for rHF and its site altered forms, suggesting formation of H(2)O(2). Direct measurements revealed no H(2)O(2) free in solution 0.5-10 min after all Fe(2+) was oxidized at pH 6.5 or 7.5. These results are consistent with initial H(2)O(2) formation, which rapidly reacts in a secondary reaction with unidentified solution components. Using measured rate constants for rHF, simulations showed that steady-state H(2)O(2) concentrations peaked at 14 muM at approximately 600 ms and decreased to zero at 10-30 s. rLF did not produce measurable H(2)O(2) but apparently conducted the secondary reaction with H(2)O(2). Fe(2+)/O(2) values of 4.0 were measured for AvBF. Stopped-flow measurements with AvBF showed that both H(2)O(2) and O(2) react at the same rate (k = 0.34 s(-1)), that is faster than the reactions with rHF. Simulations suggest that AvBF reduces O(2) directly to H(2)O without intermediate H(2)O(2) formation.
authors.affiliation: Brigham Young University Undergraduate Research Program, USA.
authors.forename: Jared
authors.initials: J
authors.lastname: Bunker
authors.forename: Thomas
authors.initials: T
authors.lastname: Lowry
authors.forename: Garrett
authors.initials: G
authors.lastname: Davis
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: David
authors.initials: D
authors.lastname: Brosnahan
authors.forename: Stuart
authors.initials: S
authors.lastname: Lindsay
authors.forename: Robert
authors.initials: R
authors.lastname: Costen
authors.forename: Sang
authors.initials: S
authors.lastname: Choi
authors.forename: Paolo
authors.initials: P
authors.lastname: Arosio
authors.forename: Gerald D
authors.initials: GD
authors.lastname: Watt
mesh_terms: D016948:Azotobacter vinelandii / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D003573:Cytochrome b Group / Q000378:metabolism*; D005293:Ferritins / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D011994:Recombinant Proteins; D005293:Ferritins; C018032:bacterioferritin; D006861:Hydrogen Peroxide; D007501:Iron; D010100:Oxygen