Tyrosyl radical formation during the oxidative deposition of iron in human apoferritin

Chen-Barrett, Y.; Harrison, Pauline M.; Treffry, Amyra; Quail, Michael A.; Arosio, Paolo; Santambrogio, Paolo; Chasteen, N. Dennis

doi:10.1021/bi00024a008

Cited by 40 publications

(41 citation statements)

References 46 publications

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“…This result indicates that the addition of iron results in damage to the protein and the production of lower molecular weight fragments as seen in Figs. 3 and 4 for HoSF, consistent with Fenton chemistry and the formation of active oxygen species (17)(18)(19)(20).…”

Section: Resultssupporting

confidence: 65%

“…2 show much smaller amounts of lower molecular weight protein species with shorter migration times in the 14 -17 min range than seen for tissue ferritins (below). The recombinant proteins have been subjected to significantly lower levels of iron loading than naturally occurring ferritins and therefore have been exposed to lower levels of potentially damaging activated oxygen species such as hydrogen peroxide and/or hydroxyl radical that are produced during iron deposition in mammalian ferritins (17)(18)(19).…”

Section: Resultsmentioning

confidence: 99%

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Characterization of the H- and L-Subunit Ratios of Ferritins by Sodium Dodecyl Sulfate–Capillary Gel Electrophoresis

Grady

Zang

Laue

et al. 2002

Analytical Biochemistry

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Section: Resultssupporting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

Characterization of the H- and L-Subunit Ratios of Ferritins by Sodium Dodecyl Sulfate–Capillary Gel Electrophoresis

Grady

Zang

Laue

et al. 2002

Analytical Biochemistry

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“…By analogy, our low measured P1 ⁄2 of 1.1 milliwatts suggests that the radical formed in YvrK is relatively isolated magnetically from any manganese center, which may have initiated the immediate reactive precursor. Free tyrosyl radical generated by ␥-irradiation of a frozen solution exhibited a P1 ⁄2 of 0.64 Ϯ 0.19 milliwatts, and approximately equal amounts of homogeneous and inhomogeneous broadening as illustrated by its inhomogeneity parameter of 1.48 Ϯ 0.17 (54). The P1 ⁄2 and inhomogeneity parameter for the radical signal observed with YvrK are comparable to these latter values, but the slightly higher P1 ⁄2 might reflect weak coupling to a paramagnet such as manganous ion.…”

Section: Nature Of the Manganese Center In Oxalate Decarboxylase-mentioning

confidence: 83%

EPR Spectroscopic Characterization of the Manganese Center and a Free Radical in the Oxalate Decarboxylase Reaction

Chang

Svedružić

Ożarowski

et al. 2004

Journal of Biological Chemistry

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Several molecular mechanisms for cleavage of the oxalate carbon-carbon bond by manganese-dependent oxalate decarboxylase have recently been proposed involving high oxidation states of manganese. We have examined the oxalate decarboxylase from Bacillus subtilis by electron paramagnetic resonance in perpendicular and parallel polarization configurations to test for the presence of such species in the resting state and during enzymatic turnover. Simulation and the position of the half-field Mn(II) line suggest a nearly octahedral metal geometry in the resting state. No spectroscopic signature for Mn(III) or Mn(IV) is seen in parallel mode EPR for samples frozen during turnover, consistent either with a large zero-field splitting in the oxidized metal center or undetectable levels of these putative high-valent intermediates in the steady state. A narrow, featureless g ‫؍‬ 2.0 species was also observed in perpendicular mode in the presence of substrate, enzyme, and dioxygen. Additional splittings in the signal envelope became apparent when spectra were taken at higher temperatures. Isotopic editing resulted in an altered line shape only when tyrosine residues of the enzyme were specifically deuterated. Spectral processing confirmed multiple splittings with isotopically neutral enzyme that collapsed to a single prominent splitting in the deuterated enzyme. These results are consistent with formation of an enzyme-based tyrosyl radical upon oxalate exposure. Modestly enhanced relaxation relative to abiological tyrosyl radicals was observed, but site-directed mutagenesis indicated that conserved tyrosine residues in the active site do not host the unpaired spin. Potential roles for manganese and a peripheral tyrosyl radical during steady-state turnover are discussed.The oxalate decarboxylase (OxDC, 1 or YvrK based on the corresponding open reading frame) from Bacillus subtilis has recently garnered attention because of its mechanistically intriguing reaction (1-4) (Scheme 1). Purified OxDC crystallizes as a hexamer of 43-kDa subunits, each of which is a member of the "bicupin" structural family (5), and contains two mononuclear manganese centers with amino acid ligands that are functionally similar to those of manganese superoxide dismutase (MnSOD) (6); namely three histidine residues and one carboxylic acid residue. Oxalate 13 C and 18 O isotope effects on V max /K m of YvrK suggest the presence of a slow, partially rate-determining step prior to the irreversible C-C bond cleavage (4), which was proposed to be a proton-coupled electron transfer.The requirement for and substoichiometric consumption of dioxygen in the YvrK catalytic process, involvement of open shell manganese (2), and chemical precedent for oxalate decarboxylation in the Kolbe electrolysis (7,8) and the BorodinHunsdiecker reaction (9 -11) have led to various mechanistic proposals involving radical intermediates, and enzymic manganese complexes with formal oxidation states of either Mn(III) (2, 4) or Mn(IV) (3). A previously published electron paramagneti...

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“…A Tyr in R2 forms a free radical, which is essential for the activity of the catalytic subunit. The role of other Tyr residues that occupy similar positions in other proteins is unknown, although tyrosyl radicals have been detected in bacterioferritin (81) and H-ferritin (82). DF1 may be an ideal system for studying the roles of these second shell residues.…”

Section: Discussionmentioning

confidence: 99%

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

Lombardi

Summa

Geremia

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

233

279

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De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 Å rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the GluXxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 Å. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation. P roteins use a limited repertoire of metal ion cofactors to help catalyze a multitude of reactions. For example, diiron sites (1-4) mediate reversible oxygen binding in hemerythrins, whereas they function as hydrolytic centers in phosphatases. Structurally similar diiron sites also mediate a number of oxygendependent oxidative processes. Ferritins serve as ferroxidases, while other diiron proteins catalyze hydroxylation, epoxidation, and desaturation reactions. Further, a diiron site in Escherichia coli ribonucleotide reductase is responsible for the formation of a Tyr radical. How do the structures of these proteins tune the chemical properties of a common diiron center to obtain such a diversity of highly specific catalysts? This question is being addressed through the study of the natural proteins as well as the study of small-molecule diiron complexes (1-4). Although impressive progress has been made on both fronts, these approaches have inherent limitations. The study of large proteins is hampered by their extreme complexity, and it is difficult to synthesize small-molecule models capable of simultaneously binding diiron, oxygen, and various substrates. Recently, we and others have sought a molecular middle ground between these two extremes through the design of small proteins and peptid...

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Tyrosyl radical formation during the oxidative deposition of iron in human apoferritin

Cited by 40 publications

References 46 publications

Characterization of the H- and L-Subunit Ratios of Ferritins by Sodium Dodecyl Sulfate–Capillary Gel Electrophoresis

Characterization of the H- and L-Subunit Ratios of Ferritins by Sodium Dodecyl Sulfate–Capillary Gel Electrophoresis

EPR Spectroscopic Characterization of the Manganese Center and a Free Radical in the Oxalate Decarboxylase Reaction

Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins

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