Kinetic studies of iron deposition catalyzed by recombinant human liver heavy, and light ferritins and Azotobacter vinelandii bacterioferritin using O2 and H2O2 as oxidants
| Autor: | Stuart Lindsay, Garrett Davis, Thomas Lowry, David Brosnahan, Jared Bunker, Gerald D. Watt, Robert C. Costen, Paolo Arosio, Bo Zhang, Sang Choi |
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| Rok vydání: | 2005 |
| Předmět: |
Iron
Inorganic chemistry Biophysics Biochemistry Medicinal chemistry Catalysis Chemical kinetics Reaction rate constant Bacterial Proteins Humans Azotobacter vinelandii biology Chemistry Organic Chemistry Temperature Hydrogen Peroxide Bacterioferritin Cytochrome b Group biology.organism_classification Recombinant Proteins Oxygen Ferritin Kinetics Ferritins Mutagenesis Site-Directed biology.protein Titration Steady state (chemistry) Stoichiometry |
| Zdroj: | Biophysical Chemistry. 114:235-244 |
| ISSN: | 0301-4622 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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