Dataset Information

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.

SUBMITTER: Bou-Abdallah F

PROVIDER: S-EPMC3951517 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Functionality of the three-site ferroxidase center of Escherichia coli bacterial ferritin (EcFtnA).

Bou-Abdallah F F Yang H H Awomolo A A Cooper B B Woodhall M R MR Andrews S C SC Chasteen N D ND

Biochemistry 20140114 3

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 ...[more]

PMID: 24380371

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Project description:Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of ~80 Å in diameter. The main function of ferritin is to oxidize the cytotoxic Fe(2+) ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe(2+) (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe(2+), and the products were characterized by Mössbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe(2+)Fe(3+) species, and a mononuclear Fe(2+) species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a μ-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe(2+)Fe(3+) state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.

Project description:Ferritin is a widespread iron mineralizing and detoxification protein that stores iron as a hydrous ferric oxide mineral core within a shell-like structure of 4/3/2 octahedral symmetry. Iron mineralization is initiated at dinuclear ferroxidase centers inside the protein where Fe(2+) and O(2) meet and react to form a mu-1,2-peroxodiferric intermediate that subsequently decays to form mu-oxo dimeric and oligomeric iron(III) species and ultimately the mineral core. Several types of channels penetrate the protein shell and are possible pathways for the diffusion of iron and dioxygen to the ferroxidase centers. In the present study, UV/visible and fluorescence stopped-flow spectrophotometries were used to determine the kinetics and pathways of Fe(2+) diffusion into the protein shell, its binding at the ferroxidase center and its subsequent oxidation by O(2). Three fluorescence variants of human H-chain ferritin were prepared in which Trp34 was introduced near the ferroxidase center. They included a control variant no. 1 (W93F/Y34W), a "1-fold" channel variant no. 2 (W93F/Y34W/Y29Q) and a 3-fold channel variant no. 3 (Y34W/W93F/D131I/E134F). Anaerobic rapid mixing of Fe(2+) with apo-variant no. 1 quenched the fluorescence of Trp34 with a rate exhibiting saturation kinetics with respect to Fe(2+) concentration, consistent with a process involving facilitated diffusion. A half-life of approximately 3 ms for this process is attributed to the time for diffusion of Fe(2+) across the protein shell to the ferroxidase center. No fluorescence quenching was observed with the 3-fold channel variant no. 3 or when Zn(2+) was prebound in each of the eight 3-fold channels of variant no. 1, observations indicating that the hydrophilic channels are the only avenues for rapid Fe(2+) access to the ferroxidase center. Substitution of Tyr29 with glutamine at the entrance of the "1-fold" hydrophobic channel had no effect on the rate of Fe(2+) oxidation to form the mu-1,2-peroxodiferric complex (t(1/2) approximately 38 ms), a finding demonstrating that Tyr29 and, by inference, the "1-fold" channels do not facilitate O(2) transport to the ferroxidase center, contrary to predictions of DFT and molecular dynamics calculations. O(2) diffusion into ferritin occurs on a time scale that is fast relative to the millisecond kinetics of the stopped-flow experiment.

Dataset Information

Functionality of the three-site ferroxidase center of Escherichia coli bacterial ferritin (EcFtnA).

Publications

Functionality of the three-site ferroxidase center of Escherichia coli bacterial ferritin (EcFtnA).

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