Biomedical subjects

N D Chasteen

Publications and source records attributed to N D Chasteen.

At least 19 recordsLinked to original sources

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↗

PubMed1988

Vanadium.

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Animals↗

PubMed1987

Electron paramagnetic resonance and magnetic susceptibility studies of dimanganese concanavalin A. Evidence for antiferromagnetic exchange coupling.

The double Mn2+ complex of concanavalin A with bound saccharide (SMMPL) was examined by electron paramagnetic resonance (EPR) spectroscopy and magnetic susceptibility measurements. A room temperature X-band (9 GHz) EPR spectrum of SMMPL revealed a relatively weak, broad resonance in contrast to the spectrum with a six-line hyperfine-split pattern observed for the mononuclear, high-spin Mn2+ complex found in Ca2+-Mn2+-concanavalin A with saccharide present (SCMPL). The EPR spectrum of SMMPL at 77 K, however, consisted of a series of overlapping patterns of 11 hyperfine-split lines near g = 2.0 with members of each pattern separated by 47 G, half the value of the hyperfine splitting of SCMPL. These 11-line patterns are preserved at Q-band (35 GHz), indicating that the manganese ions in SMMPL form a spin-coupled, binuclear center. As expected for an exchange-coupled system, the EPR signal of SMMPL at 77 K saturates at a higher microwave power than those for SCMPL or Mn2+ aquoion. There is also a marked loss of EPR signal intensity for SMMPL between 4.2 and 1.4 K, which supports the view that the pair of manganese ions is exchanged-coupled. The temperature dependence of both the magnetic susceptibility and the low-temperature EPR spectral intensity can be explained by a model in which the two high-spin Mn2+ ions of SMMPL are antierromagnetically exchanged-coupled with an isotropic coupling constant J = 1.8 cm-1 (for the spin Hamiltonian Hex = JS1.S2). Zero-field splitting D' was estimated to be 375 G from the EPR spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Concanavalin A↗

PubMed1986

Vanadium complexes of transferrin and ferritin in the rat.

Vanadium associates with serum transferrin of rats administered vanadyl(IV) sulfate or ammonium metavanadate(V) by gastric intubation. Low molecular weight species account for only 3% of the vanadium present in plasma. The element distributes between the two major isotransferrins in proportion to their concentrations. Rat apotransferrin binds both vanadium(IV) and vanadium(V), forming 2:1 metal-protein complexes in both instances. Although the two isotransferrins apparently differ in their physiological properties, they exhibit identical vanadyl(IV) (VO2+) EPR spectra, indicating identical or very similar metal binding sites for both proteins. In contrast to other transferrins, the two sites of the rat protein are spectroscopically indistinguishable and exhibit a VO2+ EPR spectrum similar to that of the C-terminal metal binding site of human serum transferrin. VO2+ EPR signals are observed with liver, spleen, and kidney tissue samples from animals maintained on a vanadium-supplemented diet. These signals arise from a specific intracellular VO2+ complex with the iron storage protein ferritin.

Animals↗

PubMed1986

The influence of uncoordinated histidines on iron release from transferrin. A chemical modification study.

Histidine residues that influence the chelate-mediated removal of iron from transferrin have been investigated. Diferric human serum transferrin was chemically modified to various extents using ethoxyformic anhydride, a reagent for histidines. A kinetic analysis of the modification reaction revealed the presence of a fast reacting pool of 9 +/- .8 histidine residues and a slow reacting pool of 5.8 +/- .6 residues. There are 18 histidine residues in transferrin. The rates of modification of the two pools differed by a factor of 5. The pyrophosphate-mediated removal of iron from the two binding sites of native and partially modified transferrins was studied at pH 6.9 using desferrioximine B as a terminal iron acceptor. Under these conditions, the rate of iron removal from the NH2-terminal site was about six times faster than from the COOH-terminal site. Both rates were significantly reduced, i.e. by a factor of approximately 6-8, upon complete ethoxyformylation of all reactive histidines on the protein. The kinetic data of partially modified transferrins were analyzed by the Tsou Chen-Lu statistical method; the results are consistent with the hypothesis that modification of a single uncoordinated histidine in each of the two iron binding domains stabilizes the protein kinetically against loss of iron. The dependence of the iron removal reaction on pH is consistent with such an interpretation. The putative histidines, although not ligands, may be close to the metal in both binding sites, thus influencing the rate of iron removal by pyrophosphate. These histidines belong to the pool of rapidly modified residues and thus are readily accessible to solvent and chelators.

Diethyl Pyrocarbonate↗

PubMed1986

Metal ion complexes of apoferritin. Evidence for initial binding in the hydrophilic channels.

Metal binding to the iron storage protein apoferritin is the first step in the process by which iron accumulates within the protein shell. In the present study, the stoichiometry of metal binding to apoferritin in solution has been examined using the probe ions Mn(II), VO(IV), and Cd(II) in conjunction with EPR spectroscopic and cadmium ion selective electrode measurements. Binding studies were carried out with the individual ions, in competition with one another, and in competition with Fe(II), Fe(III), and Tb(III). All three probe ions show binding stoichiometries near 0.3 and 0.7 metal ion per subunit, close to the theoretically predicted values of 0.33 and 0.67 for the binding of one and two metal ions, respectively, per three subunits. These results in conjunction with other data are consistent with the binding of one, and possibly two, metal ions within each of the eight hydrophilic channels which are located on 3-fold axes leading to the interior of the protein. Pairs of cadmium binding sites have been located in these channels by x-ray crystallography (Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A., and Harrison, P. M. (1983) Adv. Inorg. Biochem. 5, 39-49). The possibility that some metal binding occurs elsewhere on the protein is not precluded by the present data, however. In competition experiments between various metal ions, approximately 0.3 metal ion per subunit is readily displaced implying common binding sites in the channels for all of them. The stoichiometry of Mn(II) displacement by Fe(II) is less clear. Oxidation of Fe(II) to Fe(III) by molecular oxygen in the presence of Mn(II) regenerates some Mn(II) binding on the protein, suggesting migration of iron(III) to other protein sites, or perhaps to core.

Animals↗

PubMed1986

The effects of salts and amino group modification on the iron binding domains of transferrin.

The origins of the effects of salts on the properties of the iron binding sites of transferrin have been investigated. The chaotropically distinct salts NaCl and NaClO4 each induce characteristic changes in the EPR lineshapes of the N- and C-terminal Fe3+ binding domains, respectively. To a good approximation the perturbed EPR spectrum of diferric transferrin in the presence of salts is the sum of the EPR spectra of the N- and C-terminal monoferric proteins. Acetylation of amino groups causes spectral and kinetic changes in the protein similar to those induced by NaClO4. Thus, both acetylation and NaClO4 cause a loss of structure in the g' = 4.3 EPR signal of the N-terminal domain, and both retard iron removal from this domain. In contrast, iron removal from the C-terminal domain is accelerated by acetylation or the presence of NaClO4. These observations are ascribed to charge effects of lysine residues which are probably in the vicinity of the iron binding sites.

Acetylation↗

PubMed1986

The exchange of Fe3+ between pyrophosphate and transferrin. Probing the nature of an intermediate complex with stopped flow kinetics, rapid multimixing, and electron paramagnetic resonance spectroscopy.

A detailed study of the exchange of Fe3+ between pyrophosphate and human serum transferrin was undertaken to test the hypothesis of a generalized reaction route for exchange of Fe3+ between transferrin and chelators. The initial rate of Fe3+ transfer from pyrophosphate to apotransferrin-CO2-3 is highly sensitive to the pyrophosphate to iron ratio with a maximal rate being observed at a ratio of 3:1, consistent with the presence of slowly reactive polymeric species at ratios less than 3:1 as revealed by EPR and kinetic measurements. At a ratio of 4:1 the reaction is distinctly biphasic. The rapid first phase results in the formation of an intermediate postulated as a mixedligand complex of the type PPi-Fe3+-transferrin-CO2-3. The intermediate has a distinct EPR spectrum and an absorption spectrum similar to that of Fe3+-transferrin-CO2-3, but with a spectral maximum at 450 nm rather than 465 nm. The second phase principally arises from the slow reaction of polymeric iron-pyrophosphate with the apoprotein and has contributions from the breakdown of the intermediate formed in the first phase. The rate of formation of the intermediate shows a hyperbolic dependence on NaHCO3 and apotransferrin concentrations, the latter suggesting a rate-limiting labilization of Fe3+(PPi)3, perhaps to form species of the type Fe3+(PPi)2, prior to attack by apotransferrin-CO2-3. Multimixing stopped flow spectrophotometry was employed to test the chemical reactivity of the Fe3+ to reduction at various times during the first phase. Surprisingly, a diminution of reactivity of 1000-fold was noted after only 2% of the first phase was completed, indicating a fast initial reaction which is not observed by normal rapid flow spectrophotometry. This initial reaction may involve the binding of iron-pyrophosphate to allosteric sites on the protein. The kinetics of iron removal from Fe3+-transferrin-CO2-3 by PPi are consistent with a rate-limiting conformational change in the protein as proposed earlier.

Apoproteins↗

PubMed1985

Fe(III).ATP complexes. Models for ferritin and other polynuclear iron complexes with phosphate.

Polynuclear iron complexes of Fe(III) and phosphate occur in seawater and soils and in cells where the iron core of ferritin, the iron storage protein, contains up to 4500 Fe atoms in a complex with an average composition of (FeO.OH)8FeO.OPO3H2. Although phosphate influences the size of the ferritin core and thus the availability of stored iron, little is known about the nature of the Fe(III)-phosphate interaction. In the present study, Fe-phosphate interactions were analyzed in stable complexes of Fe(III).ATP which, in the polynuclear iron form, had phosphate at interior sites. Such Fe(III).ATP complexes are important not only as models but also because they may play a role in intracellular iron transport and in iron toxicity; the complexes were studied by extended x-ray absorption fine structure, EPR, NMR spectroscopy, and measurement of proton release. Mononuclear iron complexes exhibiting a g' = 4.3 EPR signal were formed at Fe:ATP ratios less than or equal to 1:3, and polynuclear iron complexes (Fe greater than or equal to 250, EPR silent at g' = 4.3) were formed at an Fe:ATP ratio of 4:1. No NMR signals due to ATP were observed when Fe was in excess (Fe:ATP = 4:1). Extended x-ray absorption fine structure analysis of the polynuclear Fe(III).ATP complex was able to distinguish an Fe-P distance at 3.27 A in addition to the octahedral O at 1.95 A and 4-5 Fe atoms at 3.36 A. The Fe-O and Fe-Fe distances are the same as in ferritin, and the Fe-P distance is analogous to that in another metal-ATP complex. An observable Fe-P environment in such a large polynuclear iron cluster as the Fe(III).ATP (4:1) complex indicates that the phosphate is distributed throughout rather than merely on the surface, in contrast to earlier models of chelate-stabilized iron clusters. Complexes of Fe(III) and ATP similar to those described here may form in vivo either as normal components of intracellular iron metabolism or during iron excess where the consequent alteration of free nucleotide triphosphate pools could contribute to the observed toxicity of iron.

Adenosine Triphosphate↗

PubMed1985

Iron deposition in apoferritin. Evidence for the formation of a mixed valence binuclear iron complex.

A preliminary EPR investigation of iron accumulation in apoferritin has identified paramagnetic species generated during the early stage of iron deposition within the apoprotein shell. A featureless resonance at g' = 4.3, attributable to solitary high spin Fe3+ ions bound to the protein, is generated when Fe(II) is added to apoferritin at a level of 0.5 Fe/subunit (12 Fe/molecule) followed by air oxidation. This resonance accounts for 36% of the added iron. The remainder is EPR-silent and is probably present as oligomeric Fe3+ species. The intensity of the g' = 4.3 signal is reduced 3-fold upon anaerobic addition of 5 Fe(II)/subunit as a new iron resonance with g' values of 1.94, 1.87, and 1.80 is generated. This signal is observable only at temperatures near that of liquid helium and resists saturation at power levels of 100 milliwatts. Its distinctive g-factors, temperature dependence, and saturation characteristics suggest that it arises from a spin-coupled Fe(II)-Fe(III) dimer having a net electron spin of 1/2. In accord with this idea, the signal disappears when air is admitted, presumably because of oxidation of the Fe(II). The proposed mixed valence dimer may be an important intermediate formed during the initiation of core formation within the protein shell.

Animals↗