title: Functionality of the three-site ferroxidase center of Escherichia coli bacterial ferritin (EcFtnA).
abstract: At least three ferritins are found in the bacterium Escherichia coli : the heme-containing bacterioferritin (EcBFR) and two nonheme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A and B sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA was further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV-vis, fluorescence, and EPR spectroscopic measurements on both the wild-type protein and site-directed variants of the A, B, and C sites. The data reveal that although H2O2 is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe(2+)/O2 ∼ 3:1 most of the H2O2 produced is consumed in subsequent reactions with a 2:1 Fe(2+)/H2O2 stoichiometry, thus suppressing hydroxyl-radical formation. Although the A and B sites are essential for rapid iron oxidation, the C site slows oxidation and suppresses iron turnover at the ferroxidase center. A tyrosyl radical, assigned to Tyr24 near the ferroxidase center, is formed during iron oxidation, and its possible significance to the function of the protein is discussed. Taken as a whole, the data indicate that there are multiple iron-oxidation pathways in EcFtnA with O2 and H2O2 as oxidants. Furthermore, our data do not support a universal mechanism for iron oxidation in all ferritins whereby the C site acts as transit site, as has been recently proposed.
authors.affiliation: Department of Chemistry, State University of New York , Potsdam, New York 13676, United States.
authors.forename: F
authors.initials: F
authors.lastname: Bou-Abdallah
authors.forename: H
authors.initials: H
authors.lastname: Yang
authors.forename: A
authors.initials: A
authors.lastname: Awomolo
authors.forename: B
authors.initials: B
authors.lastname: Cooper
authors.forename: M R
authors.initials: MR
authors.lastname: Woodhall
authors.forename: S C
authors.initials: SC
authors.lastname: Andrews
authors.forename: N D
authors.initials: ND
authors.lastname: Chasteen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002570:Ceruloplasmin / Q000737:chemistry* / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005293:Ferritins / Q000737:chemistry* / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D019159:Nonheme Iron Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D005296:Ferrous Compounds; D019159:Nonheme Iron Proteins; C534454:ferritin A, E coli; D005293:Ferritins; C018032:bacterioferritin; D006861:Hydrogen Peroxide; D002570:Ceruloplasmin; D010100:Oxygen