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N D Chasteen

Publications and source records attributed to N D Chasteen.

At least 37 records · Page 2Linked to original sources
PubMed1995

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

The radical chemistry of ferritin is incompletely understood. The present study was undertaken to investigate the production of radicals in H-chain recombinant human ferritin (HuHF) and mixed H/L-chain horse spleen ferritin (HoSF) and the potential role of radicals in the oxidative deposition of iron in these proteins. Radical production follows distinct pathways for the two proteins; an intact H-chain ferroxidase site is required for radical generation in both of them, however. With the H-chain HuHF, an EPR spectrum characteristic of a tyrosyl radical is seen following Fe2+ oxidation by O2 and, based on measurements with site-directed variants, is suggested to arise from residue Tyr-34 located in the vicinity of the ferroxidase site. The observation of this radical correlates with the observation of a 400-600 nm absorbance seen in stopped-flow kinetics studies which seems to require the presence of Tyr-34 (Bauminger et al. (1993) Biochem. J. 296, 709-714). The data are inconsistent, however, with the Tyr-34 radical being critically important in the protein-catalyzed mechanism of iron oxidation. Unlike HuHF, the radicals observed in L-chain-rich HoSF appear to arise from hydroxyl radical damage to the protein through Fenton chemistry. These latter radicals also appear to be centered on aromatic amino acids and may be derived from histidine.

Animals↗
PubMed1994

Rapid kinetics of the EPR-active species formed during initial iron uptake in horse spleen apoferritin.

The molecular mechanism of oxidative deposition of iron in ferritin is incompletely understood. In this study, EPR-active species produced during ferritin reconstitution (10-50 Fe/protein) from the apoprotein, Fe2+, and O2 have been investigated using rapid-mixing freeze-quench techniques and EPR spectroscopy. Species studied include a monomeric Fe(3+)-protein complex (g' = 4.3), a mixed-valent Fe(2+)-Fe3+ complex (g' = 1.87), and a newly observed radical with axial symmetry (g parallel = 2.042, g perpendicular = 2.0033), all apparent intermediates formed during the first second of iron oxidation. The monomeric Fe(3+)-protein complex is the principal EPR-observable product of iron(II) oxidation and is produced quantitatively in the first phase of the reaction with the mixed-valent species and the radical formed at slower rates. The initial rate of formation of the monomeric complex (and the radical) is first-order in Fe2+ concentration, consistent with a mechanism in which iron oxidation occurs in a one-electron step(s) with H2O2 being the final product of O2 reduction. A 1:1 relationship between the disappearance of the monomeric Fe(3+)-protein complex and the formation of the mixed-valent Fe(2+)-Fe3+ species was observed in the early phase of the reaction, indicating that the latter is derived from the former and not from the one-electron oxidation of a preformed Fe(2+)-Fe2+ dimer. The g-factors and rapid EPR relaxation properties of the transient radical suggest that it is associated with an Fe2+ (or Fe3+) center but its identity and possible functional role in iron oxidation are unknown.

Animals↗
PubMed1994

Identification of the EPR-active iron-nitrosyl complexes in mammalian ferritins.

This study was undertaken to characterize the nitric oxide complexes of mammalian ferritin and their EPR properties to gain a better understanding of the interaction of NO with non-heme iron proteins within the cell. Measurements were made with horse spleen apo- and holoferritins, with chemically modified proteins, and with recombinant human H-chain apoferritin and its site-directed mutants. Three types of EPR signals (A, B, and C) have been identified and attributed to iron-nitrosyl complexes at imidazole groups of histidine, thiol groups of cysteine, and carboxylate groups of aspartate and glutamate, respectively. The C-type axial spectrum has features at g perpendicular' = 4 and g parallel' = 2 characteristic of a paramagnetic Fe(3+)-NO- complex with total spin S = 3/2 and probably arises from nonspecific binding to carboxylate groups on the protein. The S = 1/2 axial B-type signal g perpendicular' = 2.033 and g parallel' = 2.014) is formed at Cys-130 (human H-chain sequence numbering). His-128 and possibly His-118 are sites of formation of the rhombic S = 1/2 A-type complex (gx' = 2.055, gy' = 2.033, and gz' = 2.015); the former residue perhaps plays a role in the conformational stability of the protein as well as in iron binding. The data reveal that the residues Cys-130 and His-128 in the vicinity of 3-fold channels leading to the interior of the protein shell are important in iron-nitrosyl complex formation in mammalian ferritins.

Animals↗
PubMed1994

Salt effects on the physical properties of the transferrins.

Salts are known to have a pronounced effect on the spectroscopic, thermodynamic and kinetic properties of human serum transferrin. The present study was undertaken to examine the effect of NaCl on the related proteins ovotransferrin and lactoferrin. EPR difference spectroscopy was used to probe changes in the metal site of these proteins. Sodium chloride was found to perturb the g' = 4.3 EPR spectra of both ovotransferrin and lactoferrin but in different ways. The spectrum of ovotransferrin is reduced in amplitude with a broad feature appearing at g' = 4.8 whereas there is a loss of resolution of the doublet feature at the peak of the EPR derivative spectrum for lactoferrin. The increase in the amplitude of the ovotransferrin EPR difference spectrum (spectrum without NaCl minus spectrum with NaCl) as a function of NaCl concentration is suggestive of saturation binding. A Hill plot binding isotherm gave n = 1.87 +/- 0.32 and log K = 1.49 +/- 0.03 for ovotransferrin, where n is the number of C1- ions binding to either one or both iron containing lobes of the protein and K is the overall association constant. Preliminary measurements with lactoferrin gave n = 1.95 +/- 0.34 and log K = 1.41 +/- 0.06. These results are similar to those previously reported for serum transferrin and suggest that Cl- binds to all the transferrins with strong pairwise cooperativity. This binding may reflect a functional role for chloride and other physiological anions in the uptake and release of iron by the transferrins.

Chlorides↗
PubMed1993

Characterization of the non-heme iron center of human 5-lipoxygenase by electron paramagnetic resonance, fluorescence, and ultraviolet-visible spectroscopy: redox cycling between ferrous and ferric states.

Purified human 5-lipoxygenase, a non-heme iron containing enzyme, has been characterized by electron paramagnetic resonance, (EPR), ultraviolet (UV)-visible and fluorescence spectroscopy. As isolated, the enzyme is largely in the ferrous state and shows a weak X-band EPR signal extending from 0 to 700 G at 15 K, tentatively ascribed to integer spin Fe(II). Titration of the protein with 13-HPOD (13-hydroperoxyoctadecadienoic acid) generates a strong multicomponent EPR signal in the g' approximately 6 region, a yellow color associated with an increased absorption between 310 and 450 nm (epsilon 330nm = 2400 M-1 cm-1), and a 17% decrease in the intrinsic protein fluorescence. The multiple component nature of the g' approximately 6 signal indicates that the metal center in its oxidized state exists in more than one but related forms. The g' approximately 6 EPR signal and the yellow color reach a maximum when approximately 1 mol of 13-HPOD is added/mol of iron; the resultant EPR spectrum accounts quantitatively for all of the iron in the protein with a signal at g' = 4.3 representing less than 3% of the total iron in the majority of samples. Addition of a hydroxyurea reducing agent abolished the g' approximately 6 signal and yellow color of the protein and also reversed the decrease in fluorescence caused by the oxidant 13-HPOD. The results indicate that the g' approximately 6 EPR signal, the yellow color, and the decreased fluorescence are associated with the formation of the Fe(III) form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonate 5-Lipoxygenase↗
PubMed1993

Ferroxidase kinetics of human liver apoferritin, recombinant H-chain apoferritin, and site-directed mutants.

A detailed study of the kinetics of iron(II) oxidation by molecular oxygen in natural and recombinant human apoferritins has been carried out using electrode oximetry to better understand the ferroxidase activity of the protein shell. A comparative study of recombinant L-chain ferritin (rLF), recombinant H-chain ferritin (rHF), and variants has shown that (1) rLF lacks a ferroxidase activity, confirming the results of previous studies; (2) the ferroxidase site of rHF involves Glu-62 and His-65, presumably as Fe2+ ligands, since mutation of these residues abolishes most of the oxidase activity, in agreement with previous studies; and (3) mutation of both the putative ferroxidase and nucleation site ligands in rHF renders the protein totally incapable of catalyzing the oxidation of Fe2+ whereas mutation of nucleation site ligands alone (Glu-61, Glu-64, and Glu-67) decreases the activity only slightly. Analysis of the kinetics of rHF and natural human liver ferritin (HLF) (4% H-chain, 96% L-chain) gave the following apparent parameters at pH 7: Km,O2 = 6 +/- 2 microM, Km,Fe = 80 +/- 10 microM, and kcat = 201 +/- 14 min-1 for rHF and Km,O2 = 60 +/- 12 microM, Km,Fe = 50 +/- 10 microM, and kcat = 31.2 +/- 0.6 min-1 for HLF. Furthermore, Zn2+ was shown to be a noncompetitive inhibitor of Fe2+ oxidation in rHF but a mixed inhibitor in HLF. These different forms of Zn2+ inhibition in the two proteins and the higher activity of HLF than expected, based on its H-chain composition as well as differences in their enzyme kinetic parameters, suggest that H- and L-chains cooperate in modulating the ferroxidase activity of the apoferritin even though the L-subunit lacks a ferroxidase site itself.

Apoferritins↗
PubMed1992

Ferroxidase kinetics of horse spleen apoferritin.

Protein ferroxidase site(s), which catalyze the reaction between ferrous ion and dioxygen, have long been thought to play a role in core formation in ferritin; however, the mechanism of the reaction has never been studied in detail. In the present work, the enzymatic activity of ferritin was examined using oximetry, the net Fe2+ oxidation reaction being as follows. [formula: see text] The reaction exhibits saturation kinetics with respect to both Fe2+ and O2 (apparent Michaelis constants: Km,Fe = 0.35 +/- 0.01 mM and Km,O2 = 0.14 +/- 0.03 mM). The enzyme has a turnover number kcat = 80 +/- 3 min-1 at 20 degrees C with maximal activity at pH 7. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe(II) oxidation occurs in 1-electron steps. Zinc(II) is a competitive inhibitor of iron(II) oxidation at Zn2+/apoprotein ratios > or = 6 (inhibitor constant KI,Zn = 0.067 +/- 0.011 mM) but appears to be a noncompetitive inhibitor at lower ratios (< or = 2), indicating the presence of more than one type of zinc binding site on the protein. At increments of 50 Fe2+/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. However, when larger increments are employed, some iron oxidation appears to occur on the surface of the mineral core. The results of these studies emphasize the role of the protein shell in all phases of core growth and confirm the presence of a functionally important catalytic site in ferritin in addition to other binding sites on the protein for iron.

Animals↗
PubMed1991

Iron oxidation chemistry in ferritin. Increasing Fe/O2 stoichiometry during core formation.

The origin of previously observed variations in stoichiometry of iron oxidation during the oxidative deposition of iron in ferritin has been poorly understood. Knowledge of the stoichiometry of Fe(II) oxidation by O2 is essential to establishing the mechanism of iron core formation. In the present work, the amount of Fe(II) oxidized was measured by Mössbauer spectrometry and the O2 consumed by mass spectrometry. The number of protons produced in the reaction was measured by "pH stat" titration and hydrogen peroxide production by the effect of the enzyme catalase on the measured stoichiometry. For protein samples containing low levels of iron (24 Fe(II)/protein) the stoichiometry was found to be 1.95 +/- 0.18 Fe(II)/O2 with H2O2 being a product, viz. Equation 1. 2Fe2+ + O2 + 4H2O----2FeOOH + H2O2 + 4H+ (1) EPR spin trapping experiments showed no evidence of superoxide radical formation. The stoichiometry markedly increased with additional iron (240-960 Fe/protein), to a value of 4 Fe(II)/O2 as in Equation 2. 4Fe2+ + O2 + 6H2O----4FeOOH + 8H+ (2) As the iron core is progressively laid down, the mechanism of iron oxidation changes from a protein dominated process with H2O2 being the primary product of O2 reduction to a mineral surface dominated process where H2O is the primary product. These results emphasize the importance of the apoferritin shell in facilitating iron oxidation in the early stage of iron deposition prior to significant development of the polynuclear iron core.

Animals↗
PubMed1991

Iron binding to horse spleen apoferritin: a vanadyl ENDOR spin probe study.

The role of the protein shell in the formation of the hydrous ferric oxide core of ferritin is poorly understood. A VO2+ spin probe study was undertaken to characterize the initial complex of Fe2+ with horse spleen apoferritin (96% L-subunits). A competitive binding study of VO2+ and Fe2+ showed that the two metals compete 1:1 for binding at the same site or region of the protein. Curve fitting of the binding data showed that the affinity of VO2+ for the protein was 15 times that of Fe2+. Electron nuclear double resonance (ENDOR) measurements on the VO(2+)-apoferritin complex showed couplings from two nitrogen nuclei, tentatively ascribed to the N1 and N3 nitrogens of the imidazole ligand of histidine. The possibility that the observed nitrogen couplings are from two different ligands is not precluded by the data, however. A pair of exchangeable proton lines with a coupling of approximately 1 MHz is tentatively assigned to the NH proton of the coordinated nitrogen. A 30-40% reduction in the intensity of the 1H matrix ENDOR line upon D2O-H2O exchange indicates that the metal-binding site is accessible to solvent and, therefore, to molecular oxygen as well. The ENDOR data provide the first evidence for a principle iron(II)-binding site with nitrogen coordination in an L-subunit ferritin. The site may be important in Fe2+ oxidation during the beginning stages of core formation.

Animals↗
PubMed1991

Role of phosphate in initial iron deposition in apoferritin.

Ferritins from microorganisms to man are known to contain varying amounts of phosphate which has a pronounced effect on the structural and magnetic properties of their iron mineral cores. The present study was undertaken to gain insight into the role of phosphate in the early stages of iron accumulation by ferritin. The influence of phosphate on the initial deposition of iron in apoferritin (12 Fe/protein) was investigated by EPR, 57Fe Mössbauer spectroscopy, and equilibrium dialysis. The results indicate that phosphate has a significant influence on iron deposition. The presence of 1 mM phosphate during reconstitution of ferritin from apoferritin, Fe(II), and O2 accelerates the rate of oxidation of the iron 2-fold at pH 7.5. In the presence or absence of phosphate, the rate of oxidation at 0 degrees C follows simple first-order kinetics with respect to Fe(II) with half-lives of 1.5 +/- 0.3 or 2.8 +/- 0.2 min, respectively, consistent with a single pathway for iron oxidation when low levels of iron are added to the apoprotein. This pathway may involve a protein ferroxidase site where phosphate may bind iron(II), shifting its redox potential to a more negative value and thus facilitating its oxidation. Following oxidation, an intermediate mononuclear Fe(III)-protein complex is formed which exhibits a transient EPR signal at g' = 4.3. Phosphate accelerates the rate of decay of the signal by a factor of 3-4, producing EPR-silent oligonuclear or polynuclear Fe(III) clusters. In 0.5 mM Pi, the signal decays according to a single phase first-order process with a half-life near 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoferritins↗
PubMed1991

Fluorescence and kinetic properties of Ru(III) (NH3)5 modified transferrin.

Diferric transferrin was modified using aquopentaammine ruthenium(II), a reagent for surface-accessible uncoordinated histidines. Introduction of the cationic Ru(III) (NH3)3 + 5 group on the imidazole of only 5.5 of the 17 uncoordinated histidines enhances the rates of pyrophosphate-assisted iron removal from the N-terminal and C-terminal binding sites by 16- and 2-fold, respectively. This differential effect on the kinetics of the two sites may partially explain why in the native protein the N-terminal site is more labile than the C-terminal site in acidic solutions where histidine residues become positively charged through protonation. The distance between the metal site and nearby uncoordinated histidines was estimated from fluorescence energy transfer measurements using Tb (III) as the donor and pentaammine ruthenium(III)-labeled imidazole of histidine as the acceptor chromophore. A Tsou Chen-Lu statistical analysis of the fluorescence quenching data suggest that two residues in each lobe of the protein are involved in quenching the fluorescence. By using estimates for the index of refraction and the quantum yield and assuming the energy transfer follows parallel first-order kinetics, an upper limit for the donor-acceptor distance of about 1.4 nm was obtained, assuming two uncoordinated histidine residues equidistant from the metal. His-207 and His-242 in the N-terminal lobe of transferrin and His-535 and His-577 in the C-terminal lobe are within this distance, based on information from the lactoferrin crystal structure. It is postulated that His-207 in the N-terminal lobe and His-535 in the C-terminal lobe are the uncoordinated residues that, when protonated or modified with Ru(III) (NH3)3 + 5, lead to accelerated loss of iron from the two binding sites of the protein.

Binding Sites↗
PubMed1991

Initial iron oxidation in horse spleen apoferritin. Characterization of a mixed-valence iron(II)-iron(III) complex.

In ferritin, iron is stored by oxidative deposition of the ferrous ion to form a hydrous ferric oxide mineral core. Two intermediates, formed during the initial stages of iron accumulation in apoferritin, have been observed previously in our laboratory and have been identified as a mononuclear Fe3(+)-protein complex and a mixed-valence Fe2(+)-Fe3(+)-protein complex. The physical characteristics of the mixed-valence Fe2(+)-Fe3+ complex and its relationship to the mononuclear Fe3+ complex in horse spleen apoferritin samples to which 0-240 iron atoms were added was examined by EPR spectroscopy. The results indicate that the mononuclear complex is not a precursor to the formation of the mixed-valence complex. Competitive binding studies with Cd2+, Zn2+, Tb3+, and UO2+(2) suggest that the mixed-valence complex is formed on the interior of the protein in the vicinity of the 2-fold axis of the subunit dimer. The mixed-valence complex could be generated by the partial oxidation of Fe2+ in apoferritin containing 120 Fe2+ or by the addition of up to 120 Fe2+ to ferritin already containing 18 Fe3+/protein molecule. The fact that the complex is generated during early Fe2+ oxidation suggests that it may be a key intermediate during the initial oxidative deposition of iron in the protein. The unusual EPR powder lineshape at 9.3 GHz of the mixed-valence complex was simulated with a rhombic g-tensor (gx = 1.95, gy = 1.88, gz = 1.77) and large linewidths and g-strain parameters. The presence of significant g-strain in the complex probably accounts for the failure to observe an EPR signal at 35 GHz and likely reflect considerable flexibility in the structure of the metal site. The temperature dependence of the EPR intensity in the range 8-38 K was modeled successfully by an effective spin Hamiltonian including exchange coupling (-2JS1.S2) and zero-field terms, from which an antiferromagnetic coupling of J = -4.0 +/- 0.5 cm-1 was obtained. This low value for J may reflect the presence of a mu-oxo bridge(s) in the dimer.

Animals↗
PubMed1991

Stepped potential microcoulometry of ferritin.

A coulometric method for measuring the redox potentials of ferritins and the rates of reduction of the iron core is reported. The half-reduction potential, Eo', of the protein is determined from the coulomb-applied potential curve. This curve is obtained from the difference between the curves for the mediator plus protein and the mediator alone. The coulomb-applied potential curves are generated by stepping the potential in increments over the region of interest and measuring the charged passed following each step. The method is reasonably fast, enabling the redox potential to be determined in 1 day, and gives Eo', values for mediators and the protein that are in good agreement with literature values.

Electrochemistry↗
PubMed1989

Hydroxyl radical production during oxidative deposition of iron in ferritin.

The chemistry of oxidative deposition of iron(III) in ferritin and apoferritin is poorly understood. This study was undertaken to look for radicals formed as the hydrous ferric oxide core is developed from Fe(II) and O2. Radicals were observed indirectly by using the spin-trapping reagent N-tert-butyl-alpha-phenylnitrone (PBN) at room temperature and directly by measuring ESR spectra of frozen solutions at 77 K. In both instances, radical production was inhibited by the hydroxyl radical scavenging agents dimethyl sulfoxide, thiourea, and mannitol and enhanced by the addition of hydrogen peroxide. These findings strongly suggest that hydroxyl radical, produced from the iron-catalyzed Haber-Weiss reaction, is a by-product of core formation in ferritin and is a precursor to the observed radicals. The yield of ESR-observable and spin-trapped radicals is quite low, being at the micromolar level when millimolar concentrations of ferrous ion are employed. Furthermore, radical production appears to be confined to the interior of the ferritin molecule, where cellular components would be protected from the oxygen-derived toxic effects of iron. It is postulated that hydroxyl radical-medicated oxidative damage to the protein, a process that may contribute to the formation of hemosiderin from ferritin, leads to the observed radicals. By serving as a sink for hydroxyl radical, the protein shell may therefore efficiently minimize damage to other biomolecules in the cell.

Apoferritins↗
PubMed1988

Radicals from "Good's" buffers.

Oxidative deposition of iron in ferritin or the autoxidation of iron in the absence of protein produces radicals from Good's buffers. Radical species are formed from the piperazine ring-based buffers Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Epps 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, and Pipes 1,4-piperazinediethanesulfonic acid, but not from Mes (4-morpholineethanesulfonic acid) which contains a morpholine ring. The radicals all have half-lives around 10 min and display very similar electron paramagnetic resonance spectra consisting of at least 30 lines. The Hepes radical can be formed by the addition of potassium superoxide directly to the buffer and its production during iron(II) autoxidation is inhibited by superoxide dismutase (EC 1.15.1.1). Catalase (EC 1.11.1.6) accelerates the decay of the EPR spectrum. Thus the buffer radicals are secondary radical species produced from oxygen radicals formed during the iron catalyzed Haber-Weiss process. The deoxyribose/thiobarbituric acid assay for hydroxyl radical production shows that Hepes is an effective hydroxyl radical scavenging agent. The Hepes radical can also be formed electrolytically at a potential of +0.8 V (vs standard hydrogen electrode). Oxidation of Hepes at pH 10 during the autoxidation of iron(II) or by the addition of hydrogen peroxide produces a nitroxide radical. These results indicate that piperazine ring Good buffers should be avoided in studies of redox processes in biochemistry.

Buffers↗