Iron Binding and Oxidation Kinetics in Frataxin CyaY of Escherichia coli

Bou-Abdallah, Fadi; Adinolfi, Salvatore; Pastore, Annalisa; Laue, Thomas M.; Chasteen, N. Dennis

doi:10.1016/j.jmb.2004.05.072

Cited by 116 publications

(157 citation statements)

References 43 publications

(46 reference statements)

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“…This interpretation is in agreement with the X-ray crystallographic data of the Zn 2+ derivative of EcFtnA, showing full occupancy of the A and B sites of the protein by Zn 2+ (11,29) with no Zn 2+ found at the C site. This is also consistent with Zn 2+ being an inhibitor of iron oxidation in EcFTnA presumably because Zn 2+ competes with Fe 2+ ions for the dinuclear ferroxidase sites (10,26 (14,15,18,19). The B-site variant E94A is the only variant to exhibit both favorable enthalpy and entropy changes (Table 1).…”

Section: Discussionsupporting

confidence: 76%

“…Isothermal titration calorimetry (ITC) has been shown to be an excellent tool for studying metal ion binding to amino acids, peptides, and proteins in solution (14)(15)(16)(17)(18)(19). In a recent investigation, we used ITC to determine the number and location of the primary binding sites for Fe 2+ in recombinant H-chain ferritin and their affinity for iron (14).…”

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Thermodynamic Analysis of Ferrous Ion Binding toEscherichia coliFerritin EcFtnA

et al. 2005

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Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe 2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe 2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe 2+ -binding stoichiometries at the unaltered sites. The data imply some degree of inter-and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe 2+ , both A and B sites in EcFtnA bind Fe 2+ , implying a role for the C site in influencing the binding of Fe 2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.

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Section: Discussionsupporting

confidence: 76%

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Thermodynamic Analysis of Ferrous Ion Binding toEscherichia coliFerritin EcFtnA

et al. 2005

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“…1). The stability of the spin-adducts and the well characterized electron spin resonance spectra make these compounds the best choice to trap ROS such as O 2 •− and hydroxyl radicals in biological systems [21,22,24,25]. Spectrum A of Figure 1 is a control experiment where O 2 •− produced by the hypoxanthine/xanthine oxidase generating system is added to phosphate buffer, pH 7.4 in the presence of EMPO.…”

Section: Resultsmentioning

confidence: 99%

Quenching of superoxide radicals by green fluorescent protein

Bou-Abdallah

Chasteen

Lesser

2006

Biochimica et Biophysica Acta (BBA) - General Subjects

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Green fluorescent protein (GFP) is a widely used in vivo molecular marker. These proteins are particularly resistant, and maintain function, under a variety of cellular conditions such as pH extremes and elevated temperatures. Green fluorescent proteins are also abundant in several groups of marine invertebrates including reef-forming corals. While molecular oxygen is required for the post-translational maturation of the protein, mature GFPs are found in corals where hyperoxia and reactive oxygen species (ROS) occur due to the photosynthetic activity of algal symbionts. In vitro spin trapping electron paramagnetic resonance and spectrophotometric assays of superoxide dismutase (SOD)-like enzyme activity show that wild type GFP from the hydromedusa, Aequorea victoria, quenches superoxide radicals (O 2 •− ) and exhibits SOD-like activity by competing with cytochrome c for reaction with O 2 •− . When exposed to high amounts of O 2 •− the SOD-like activity and protein structure of GFP are altered without significant changes to the fluorescent properties of the protein. Because of the distribution of fluorescent proteins in both the epithelial and gastrodermal cells of reef-forming corals we propose that GFP, and possibly other fluorescent proteins, can provide supplementary antioxidant protection.

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“…4D). The frataxin structure can be adapted to oligomerize as evidenced by the ability of bacterial CyaY to form tetramers and of yeast Yfh1 to form trimers at low iron to protein ratios (Յ2:1) (28,59), and by the ability of both CyaY and Yfh1 to form higher order oligomers at increasing iron to protein ratios (5,27,56). Unlike CyaY or Yfh1, FXN oligomerizes in an iron-independent manner in human, yeast, and bacterial cells (7), which is also an intrinsic property of the frataxin structure because certain point mutations result in iron-independent oligomerization of Yfh1 both in yeast and bacterial cells (29).…”

Section: Discussionmentioning

confidence: 99%