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kinetic studies of iron deposition catalyzed by recombinant human liver heavy, and light ferritins and azotobacter vinelandii bacterioferritin using o2 and h2o2 as oxidantsThe discrepancy between predicted and measured H2O2 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 H2O2 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) rccombinant human liver light ferritin (rLF), containing 110 ferroxidase center. For rHF, nearly identical pseudo-first-order rate constants of 0.18 per second at pH 7.5 were measured for Fe(2+) oxidation by both O2 and H2O2, but for rLF, the rate with O2 was 200-fold slower than that for H2O2 (k-0.22 per second). A Fe(2+)/O2 stoichiometry near 2.4 was measured for rHF and its site altered forms, suggesting formation of H2O2. Direct measurements revealed no H2O2 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 H2O2 formation, which rapidly reacts in a secondary reaction with unidentified solution components. Using measured rate constants for rHF, simulations showed that steady-state H2O2 concentrations peaked at 14 pM at approx. 600 ms and decreased to zero at 10-30 s. rLF did not produce measurable H2O2 but apparently conducted the secondary reaction with H2O2. Fe(2+)/O2 values of 4.0 were measured for AvBF. Stopped-flow measurements with AvBF showed that both H2O2 and O2 react at the same rate (k=0.34 per second), that is faster than the reactions with rHF. Simulations suggest that AvBF reduces O2 directly to H2O without intermediate H2O2 formation.
Document ID
20060045804
Document Type
Reprint (Version printed in journal)
Authors
Bunker, Jared
(Brigham Young Univ. Provo, UT, United States)
Lowry, Thomas
(Brigham Young Univ. Provo, UT, United States)
Davis, Garrett
(Brigham Young Univ. Provo, UT, United States)
Zhang, Bo
(Brigham Young Univ. Provo, UT, United States)
Brosnahan, David
(Brigham Young Univ. Provo, UT, United States)
Lindsay, Stuart
(Brigham Young Univ. Provo, UT, United States)
Costen, Robert
(NASA Langley Research Center Hampton, VA, United States)
Choi, Sang
(NASA Langley Research Center Hampton, VA, United States)
Arosio, Paolo
(Brescia Univ. Italy)
Watt, Gerald D.
(Brigham Young Univ. Provo, UT, United States)
Date Acquired
August 23, 2013
Publication Date
January 7, 2005
Publication Information
Publication: Biophysical Chemistry
Volume: 114
Subject Category
Life Sciences (General)
Funding Number(s)
CONTRACT_GRANT: NCC1-02005
Distribution Limits
Public
Copyright
Other