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

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.

Iron-binding properties of plant phenolics and cranberry's bio-effects.

The health benefits of cranberries have long been recognized. However, the mechanisms behind its function are poorly understood. We have investigated the iron-binding properties of quercetin, the major phenolic phytochemical present in cranberries, and other selected phenolic compounds (chrysin, 3-hydroxyflavone, 3',4'-dihydroxy flavone, rutin, and flavone) in aqueous media using UV/vis, NMR and EPR spectroscopies and ESI-Mass spectrometry. Strong iron-binding properties have been confirmed for the compounds containing the "iron-binding motifs" identified in their structures. The apparent binding constants are estimated to be in the range of 10(6) M(-1) to 10(12) M(-2) in phosphate buffer at pH 7.2. Surprisingly, quercetin binds Fe(2+) even stronger than the well known Fe(2+)-chelator ferrozine at pH 7.2. This may be the first example of an oxygen-based ligand displaying stronger Fe(2+)-binding affinity than a strong nitrogen-based Fe(2+)-chelator. The strong Fe-binding properties of these phenolics argue that they may be effective in modulating cellular iron homeostasis under physiological conditions. Quercetin can completely suppress Fenton chemistry both at micromolar levels and in the presence of major cellular iron chelators like ATP or citrate. However, the radical scavenging activity of quercetin provides only partial protection against Fenton chemistry-mediated damage while Fe chelation by quercetin can completely inhibit Fenton chemistry, indicating that the chelation may be key to its antioxidant activity. These results demonstrate that quercetin and other phenolic compounds can effectively modulate iron biochemistry under physiologically relevant conditions, providing insight into the mechanism of action of bio-active phenolics.

Is hydrogen peroxide produced during iron(II) oxidation in mammalian apoferritins?

The ferritins are a class of iron storage and detoxification proteins that play a central role in the biological management of iron. These proteins have a catalytic site, "the ferroxidase site", located on the H-type subunit that facilitates the oxidation of Fe(II) to Fe(III) by O(2). Measurements during the past 10 years on a number of vertebrate ferritins have provided evidence that H(2)O(2) is produced at this diiron ferroxidase site. Recently reported experiments using three different analytical methods with horse spleen ferritin (HoSF) have failed to detect H(2)O(2) production in this protein [Lindsay, S., Brosnahan, D., and Watt, G. D. (2001) Biochemistry 40, 3340-3347]. These findings contrast with earlier results reporting H(2)O(2) production in HoSF [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965-19970]. Here a sensitive fluorescence assay and an assay based on O(2) evolution in the presence of catalase were used to demonstrate that H(2)O(2) is produced in HoSF as previously reported. However, because of the relatively few H-chain ferroxidase sites in HoSF and the reaction of H(2)O(2) with the protein, H(2)O(2) is more difficult to detect in this ferritin than in recombinant human H-chain ferritin (HuHF). The proper sequence of addition of reagents is important for measurement of the total amount of H(2)O(2) produced during the ferroxidation reaction.

Copper(II) complexes of novel N-alkylated derivatives of cis,cis-1,3,5-triaminocyclohexane. 1. Preparation and structure.

Novel N,N',N' '-trialkylated derivatives of cis,cis-1,3,5-triaminocyclohexane (tach), designated tach-R(3), were prepared through alkylation of N-protected tach with subsequent acid deprotection, to afford N-methyl, N-ethyl, and N-n-propyl derivatives as their trihydrobromide salts. The tach-neopentyl(3) and tach-furan(3) derivatives were prepared by formation of the imine from tach and pivaldehyde or furan-2-carboxaldehyde, respectively, followed by reduction of the imine. Complexes [Cu(tach-R(3))Cl(2)] (R = Me, Et, n-Pr, CH(2)-2-thienyl, and CH(2)-2-furanyl) were prepared from CuCl(2) in MeOH or MeOH-Et(2)O solvent. Crystallographic characterization of [Cu(tach-Et(3))Br(0.8)Cl(1.2)] (Pnma, a = 8.2265(1) A, b = 12.5313(1) A, c = 15.3587(3) A, Z = 4) reveals a square-based pyramidal CuN(3)X(2) coordination sphere in which one nitrogen donor occupies the apical position at a slightly longer distance (Cu-N = 2.218(5) A) than those of the basal nitrogens (Cu-N = 2.053(2) A). The solution-phase (pH 7.4 buffered and methanol) and solid-phase structures of [Cu(tach-R(3))Cl(2)] have been studied extensively by EPR and visible-near-IR spectroscopies. The square-based pyramidal structure is retained in solution, according to correspondence of solution and solid-state data. In aqueous solution, halide is replaced by water, as indicated by the high-energy UV-vis spectral shifts and bonding parameters of [Cu(tach-Et(3))](2+)(aq) derived from EPR data. The proposed aqueous-phase species, in the pH range 7.4 to 10.1, is [Cu(tach-Et(3))(H(2)O)(2)](2+). The complex [Cu(tach-Me(3))](2+)(aq) does not appear to dimerize or form metal-hydroxo species at pH 7.4, in contrast to other Cu(II)-triamine complexes, e.g., [Cu(1,4,7-triazacyclononane)](2+) (aq) and [Cu(tach-H(3))](2+)(aq) (the complex of unalkylated tach). This difference is attributed to the steric effect of the N-alkyl groups in the tach-R(3) series.

Copper(II) complexes of novel N-alkylated derivatives of cis,cis-1,3,5-triaminocyclohexane. 2. Metal-promoted phosphate diester hydrolysis.

Aqueous copper(II) N,N',N' '-trimethyl-cis,cis-1,3,5-triaminocyclohexane (Cu(tach-Me(3))(2+)(aq)) promotes the hydrolysis of activated phosphate diesters in aqueous medium at pH 7.2. This complex is selective for cleavage of the phosphate diester sodium bis(p-nitrophenyl) phosphate (BNPP), the rate of hydrolysis of the monoester disodium p-nitrophenyl phosphate being 1000 times slower. The observed rate acceleration of BNPP hydrolysis is slightly greater than that observed for other Cu(II) complexes, such as [Cu([9]aneN(3))Cl(2)] ([9]aneN(3) identical with 1,4,7-triazacyclononane). The rate of hydrolysis is first-order in phosphate ester at low ester concentration and second-order in [Cu(tach-Me(3))](2+)(aq), suggesting the involvement of two metal complexes in the mechanism of substrate hydrolysis. The reaction exhibits saturation kinetics with respect to BNPP concentration according to a modified Michaelis-Menten mechanism: 2CuL + S <==> LCu-S-CuL --> 2CuL + products (K(M) = 12.3 +/- 1.8 mM(2), k(cat) = (4.0 +/- 0.4) x 10(-)(4) s(-1), 50 degrees C) where CuL (triple bond) [Cu(tach-Me(3))](2+), S (triple bond) BNPP, and LCu-S-CuL is a substrate-bridged dinuclear complex. EPR data indicate that the dicopper complex is formed only in the presence of BNPP; the active LCu-S-CuL intermediate species then slowly decays to products, regenerating monomeric CuL.

Purification and characterization of a novel calcium-binding protein from the extrapallial fluid of the mollusc, Mytilus edulis.

In the bivalve mollusc Mytilus edulis shell thickening occurs from the extrapallial (EP) fluid wherein secreted shell matrix macromolecules are thought to self-assemble into a framework that regulates the growth of CaCO(3) crystals, which eventually constitute approximately 95% of the mature shell. Herein is the initial report on the purification and characterization of a novel EP fluid glycoprotein, which is likely a building block of the shell-soluble organic matrix. This primary EP fluid protein comprises 56% of the total protein in the fluid and is shown to be a dimer of 28,340 Da monomers estimated to be 14.3% by weight carbohydrate. The protein is acidic (pI = 4.43) and rich in histidine content (11.14%) as well as in Asx and Glx residues (25.15% total). The N terminus exhibits an unusual repeat sequence of histidine and aspartate residues that occur in pairs: NPVDDHHDDHHDAPIVEHHD approximately. Ultracentrifugation and polyacrylamide gel electrophoresis demonstrate that the protein binds calcium and in so doing assembles into a series of higher order protomers, which appear to have extended structures. Circular dichroism shows that the protein-calcium binding/protomer formation is coupled to a significant rearrangement in the protein's secondary structure in which there is a major reduction in beta-sheet with an associated increase in alpha-helical content of the protein. A model for shell organic matrix self-assembly is proposed.

Vanadyl(IV) binding to mammalian ferritins. An EPR study aided by site-directed mutagenesis.

During its metabolism, vanadium is known to become associated with the iron storage protein, ferritin. To elucidate probable vanadium binding sites on the protein, VO2+ binding to mammalian ferritins was studied using site-directed mutagenesis and EPR spectroscopy. VO2+-apoferritin EPR spectra of human H-chain (100% H), L-chain (100% L), horse spleen (84% L, 16% H) and sheep spleen (45% L, 55% H) ferritins revealed the presence of alpha and beta VO2+ species in all the proteins, implying that the ligands for these species are conserved between the H- and L-chains. The alpha species is less stable than the beta species and decreases with increasing pH, demonstrating that the two species are not pH-related, a result contrary to earlier proposals. EPR spectra of site-directed HuHF variants of several residues conserved in H- and L-chain ferritins (Asp-131, Glu-134, His-118 and His-128) suggest that His-118 near the outer opening of the three-fold channel is probably a ligand for VO2+ and is responsible for the beta signals in the EPR spectrum. The data indicate that VO2+ does not bind to the Asp-131 and Glu-134 residues within the three-fold channels nor does it bind at the ferroxidase site residues Glu-62 or His-65 or at the putative nucleation site residues Glu-61,64,67. While the ferroxidase site is not a site for VO2+ binding, mutation of residues Glu-62 and His-65 of this site to Ala affects VO2+ binding at His-118, located some 17 A away. Thus, VO2+ spin probe studies provide a window on structural changes in ferritin not seen in most previous work and indicate that long-range effects caused by point mutations must be carefully considered when drawing conclusions from mutagenesis studies of the protein.

Iron oxidation and hydrolysis reactions of a novel ferritin from Listeria innocua.

Iron deposition in the unusual 12-subunit ferritin from thebacterium Listeria innocua proceeds in three phases: a rapidfirst phase in which Fe(2+) binds to the apoprotein, P(Z) of charge Z, according to the postulatedreaction 2Fe(2+)+P(Z)-->[Fe(2)-P](Z+2)+2H(+), where[Fe(2)-P](Z+2) represents adinuclear iron(II) complex formed at each of the 12 ferroxidase centresof the protein; a second phase corresponding to oxidation of thisputative complex, i.e. [Fe(2)-P](Z+2)+1/2 O(2)-->[Fe(2)O-P](Z)+2H(+);and a third phase of iron(II) oxidation/mineralization, i.e. 4Fe(2+)+O(2)+8H(2)O-->8FeOOH((s))+8H(+) [where FeOOH((s)) represents the hydrous ferric oxidemineral that precipitates from the solution], which occurs when iron isadded in excess of 24Fe(2+)/protein. In contrast with otherferritins, the ferroxidation reaction in L. innocua ferritinproceeds more slowly than the oxidation/mineralization reaction. Wateris the final product of dioxygen reduction in the 12-subunit L.innocua ferritin (the present work) and in the 24-subunit Escherichia coli bacterioferritin, whereas H(2)O(2) is produced in 24-subunit mammalian ferritins. Possible reasonsfor this difference are discussed.

Improvements in dating tooth enamel by ESR.

ESR dating requires that growth curves be determined by interpreting complex spectra. Spectra, however, can vary significantly in shape and field position between different samples, or occasionally between subsamples, even though the mineralogy remains the same. In some cases, this spectral variability does not affect the resulting accumulated dose calculation. In other cases, signal subtraction may be needed. However, some samples that until recently might have been considered unsuitable for dating are now shown to yield accurate and precise results because a broad interference peak is integral to the hydroxyapatite signal. By studying the spectrum at the Q-band frequency, it can be shown that the interfering signal in most cases is not a problem for dating. A second concern has been that artificially irradiating sample aliquots can introduce a short-lived component that is simply an unstable enhancement of the dating signal. The apparent accumulated dose from growth curves created immediately after irradiation is considerably greater than that after annealing, although the curve's shape remains unchanged. Annealing both the natural and artificially irradiated signal shows the dating signal's lifetime to be greater than 10(10) years.

The iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin.

Bacterioferritins are members of a class of spherical shell-like iron storage proteins that catalyze the oxidation and hydrolysis of iron at specific sites inside the protein shell, resulting in formation of a mineral core of hydrated ferric oxide within the protein cavity. Electrode oximetry/pH stat was used to study iron oxidation and hydrolysis chemistry in E. coli bacterioferritin. Consistent with previous UV-visible absorbance measurements, three distinct kinetic phases were detected, and the stoichiometric equations corresponding to each have been determined. The rapid phase 1 reaction corresponds to pairwise binding of 2 Fe(2+) ions at a dinuclear site, called the ferroxidase site, located within each of the 24 subunits, viz., 2Fe(2+) + P(Z) --> [Fe(2)-P](Z) + 4H(+), where P(Z) is the apoprotein of net charge Z and [Fe(2)-P](Z) represents a diferrous ferroxidase complex. The slower phase 2 reaction corresponds to the oxidation of this complex by molecular oxygen according to the net equation: [Fe(2)-P](Z) + (1)/(2)O(2) --> [Fe(2)O-P](Z) where [Fe(2)O-P](Z) represents an oxidized diferric ferroxidase complex, probably a mu-oxo-bridged species as suggested by UV-visible and EPR spectrometric titration data. The third phase corresponds to mineral core formation according to the net reaction: 4Fe(2+) + O(2) + 6H(2)O --> 4FeO(OH)((core)) + 8H(+). Iron oxidation is inhibited by the presence of Zn(2+) ions. The patterns of phase 2 and phase 3 inhibition are different, though inhibition of both phases is complete at 48 Zn(2+)per 24mer, i.e., 2 Zn(2+) per ferroxidase center.

Molecular diffusion into ferritin: pathways, temperature dependence, incubation time, and concentration effects.

The detailed kinetics of permeation and effusion of small nitroxide spin probe radicals with the protein shells of horse spleen ferritin (HoSF) and human H-chain ferritin (HuHF) and a 3-fold channel variant D131H+E134H of HuHF were studied by electron paramagnetic resonance spectroscopy and gel permeation chromatography under a variety of experimental conditions. The results confirm that the permeation of molecular species of 7-9-A diameter into ferritin is a charge selective process and that the threefold channels are the likely pathways for entry into the protein. Studies with holoHoSF show that increased temperature increases the rates of penetration and effusion and also increases the concentration of positively charged spin probe accumulated within the protein in excess of that in the external solution. The interior of HoSF is much more accessible to small molecules at physiological temperature of approximately 40 degrees C than at room temperature. The large activation energy of 63-67 kJ/mol measured for the effusion/penetration and the small diffusion coefficient, D approximately 5 x 10(-22) m(2)/s at 20 degrees C, corresponding to a time of approximately 60 min for traversing the protein shell, is consistent with the kinetics of diffusion being largely controlled by the restrictive porosity of the protein itself. An inverse dependence of the first-order rate constant for effusion out of the protein channel on the incubation time used for radical penetration into the protein is attributed to increased binding of the radical within the funnel-shaped channel.

Mutations at the histidine 249 ligand profoundly alter the spectral and iron-binding properties of human serum transferrin N-lobe.

Human serum transferrin is an iron-binding and -transport protein which carries iron from the blood stream into various cells. Iron is held in two deep clefts located in the N- and C-lobes by coordinating to four amino acid ligands, Asp 63, Tyr 95, Tyr 188, and His 249 (N-lobe numbering), and to two oxygens from carbonate. We have previously reported the effect on the iron-binding properties of the N-lobe following mutation of the ligands Asp 63, Tyr 95, and Tyr 188. Here we report the profound functional changes which result from mutating His 249 to Ala, Glu, or Gln. The results are consistent with studies done in lactoferrin which showed that the histidine ligand is critical for the stability of the iron-binding site [H. Nicholson, B. F. Anderson, T. Bland, S. C. Shewry, J. W. Tweedie, and E. N. Baker (1997) Biochemistry 36, 341-346]. In the mutant H249A, the histidine ligand is disabled, resulting in a dramatic reduction in the kinetic stability of the protein toward loss of iron. The H249E mutant releases iron three times faster than wild-type protein but shows significant changes in both EPR spectra and the binding of anion. This appears to be the net effect of the metal ligand substitution from a neutral histidine residue to a negative glutamate residue and the disruption of the "dilysine trigger" [MacGillivray, R. T. A., Bewley, M. C., Smith, C. A., He, Q.-Y., Mason, A. B., Woodworth, R. C., and Baker, E. N. (2000) Biochemistry 39, 1211-1216]. In the H249Q mutant, Gln 249 appears not to directly contact the iron, given the similarity in the spectroscopic properties and the lability of iron release of this mutant to the H249A mutant. Further evidence for this idea is provided by the preference of both the H249A and H249Q mutants for nitrilotriacetate rather than carbonate in binding iron, probably because NTA is able to provide a third ligation partner. An intermediate species has been identified during the kinetic interconversion between the NTA and carbonate complexes of the H249A mutant. Thus, mutation of the His 249 residue does not abolish iron binding to the transferrin N-lobe but leads to the appearance of novel iron-binding sites of varying structure and stability.

Mineralization in ferritin: an efficient means of iron storage.

Ferritins are a class of iron storage and mineralization proteins found throughout the animal, plant, and microbial kingdoms. Iron is stored within the protein shell of ferritin as a hydrous ferric oxide nanoparticle with a structure similar to that of the mineral "ferrihydrite." The eight hydrophilic channels that traverse the protein shell are thought to be the primary avenues by which iron gains entry to the interior of eukaryotic ferritins. Twenty-four subunits constitute the protein shell and, in mammalian ferritins, are of two types, H and L, which have complementary functions in iron uptake. The H chain contains a dinuclear ferroxidase site that is located within the four-helix bundle of the subunit; it catalyzes the oxidation of ferrous iron by O(2), producing H(2)O(2). The L subunit lacks this site but contains additional glutamate residues on the interior surface of the protein shell which produce a microenvironment that facilitates mineralization and the turnover of iron(III) at the H subunit ferroxidase site. Recent spectroscopic studies have shown that a di-Fe(III) peroxo intermediate is produced at the ferroxidase site followed by formation of a mu-oxobridged dimer, which then fragments and migrates to the nucleation sites to form incipient mineral core species. Once sufficient core has developed, iron oxidation and mineralization occur primarily on the surface of the growing crystallite, thus minimizing the production of potentially harmful H(2)O(2).

Ferroxidase activity of ferritin: effects of pH, buffer and Fe(II) and Fe(III) concentrations on Fe(II) autoxidation and ferroxidation.

It is widely accepted that iron deposition in the iron storage protein ferritin in vitro involves Fe(II) oxidation, and that ferritin facilitates this oxidation at a ferroxidase site on the protein. However, these views have recently been questioned, with the protein ferroxidase activity instead being attributed to autoxidation from the buffer alone. Ligand exchange between another protein with ferroxidase activity and ferritin has been proposed as an alternative mechanism for iron incorporation into ferritin. In the present work, a pH stat apparatus is used to eliminate the influence of buffers on iron(II) oxidation. Here we show that the recent experiments questioning the ferroxidase activity of ferritin were flawed by inadequate pH control, that buffers actually retard rather than facilitate iron(II) oxidation, and that horse spleen ferritin has ferroxidase activity when measured under proper experimental conditions. Furthermore, high pH (7.0), a high Fe(II) concentration and the presence of Fe(III) all favour Fe(II) autoxidation in the presence or absence of ferritin.

Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins.

UV-visible spectroscopy, electrode oximetry, and pH stat were used to study Fe(II) oxidation and hydrolysis in horse spleen ferritin (HoSF) and recombinant human H-chain and L-chain ferritins (HuHF and HuLF). Appropriate test reactions and electrode responses were measured, establishing the reliability of oxygen electrode/pH stat for kinetics studies of iron uptake by ferritin. Stoichiometric ratios, Fe(II)/O2 and H+/Fe(II), and rates of oxygen uptake and proton production were simultaneously measured as a function of iron loading of the protein. The data show a clear distinction between the diiron ferroxidase site and mineral surface catalyzed oxidation of Fe(II). The oxidation/hydrolysis reaction attributed to the ferroxidase site has been determined for the first time and is given by 2Fe2+ + O2 + 3H2O --> [Fe2O(OH)2]2+ + H2O2 + 2H+ where [Fe2O(OH)2]2+ represents the hydrolyzed dinuclear iron(III) center postulated to be a mu-oxo-bridged species from UV spectrometric titration data and absorption band maxima. The transfer of iron from the ferroxidase site to the mineral core has been now established to be [Fe2O(OH)2]2+ + H2O --> 2FeOOH(core) + 2H+. Regeneration of protein ferroxidase activity with time is observed for both HoSF and HuHF, consistent with their having enzymatic properties, and is facilitated by higher pH (7.0) and temperature (37 degreesC) and by the presence of L-subunit and is complete within 10 min. In accord with previous studies, the mineral surface reaction is given by 4Fe2+ + O2 + 6H2O --> 4FeOOH(core) + 8H+. As the protein progressively acquires iron, oxidation/hydrolysis increasingly shifts from a ferroxidase site to a mineral surface based mechanism, decreasing the production of H2O2.

Mutations at nonliganding residues Tyr-85 and Glu-83 in the N-lobe of human serum transferrin. Functional second shell effects.

The x-ray crystal structure of the N-lobe of human serum transferrin has shown that there is a hydrogen bond network, the so-called "second shell," around the transferrin iron binding site. Tyrosine at position 85 and glutamic acid at position 83 are two nonliganding residues in this network in the human serum transferrin N-lobe (hTF/2N). Mutation of each of these two amino acids has a profound effect on the metal binding properties of hTF/2N. When Tyr-85 is mutated to phenylalanine, iron release from the resulting mutant Y85F is much more facile than from the parent protein. Elimination of the hydrogen bond between Tyr-85 and Lys-296 appears to interfere with the "di-lysine (Lys-206-Lys-296) trigger," which affects the iron binding stability of the protein. Surprisingly, mutation of Glu-83 to alanine leads to the absence of one of the normal iron binding ligands; introduction of a monovalent anion is able to restore the normal first coordination sphere. The missing ligand appears to be His-249, as revealed by comparison of the metal binding behaviors of mutants H249Q and E83A and structural analysis. Glu-83 has a strong H bond linkage with His-249 in apo-hTF/2N, which helps to hold the His-249 in the proper position for iron binding. Disabling Glu-83 by mutation to an alanine seriously disturbs the H bond network, allowing His-249 to move away. A monovalent anion can help reestablish the normal network by providing a negative charge near the position of Glu-83 to reach charge balance, so that ligand His-249 is available again for iron binding.

Inequivalence of the two tyrosine ligands in the N-lobe of human serum transferrin.

Human serum transferrin N-lobe (hTF/2N) has four iron-binding ligands, including one histidine, one aspartate, and two tyrosines. The present report elucidates the inequivalence of the two tyrosine ligands (Tyr 95 and Tyr 188) on the metal-binding properties of hTF/2N by means of site-directed mutagenesis, metal release kinetics, and absorption and electron paramagnetic resonance (EPR) spectroscopies. When the liganding tyrosines were mutated individually to phenylalanine, the resulting mutant Y95F showed a weak binding affinity for iron and no affinity for copper, whereas, mutant Y188F completely lost the ability to bind iron but formed a stable complex with copper. Since other studies have demonstrated that mutations of the other two ligands, histidine and aspartate, did not completely abolish iron binding, the present findings suggest that the tyrosine ligand at position 188 is essential for binding of iron to occur. Replacement of Tyr 188 with phenylalanine created a favorable chemical environment for copper coordination but a fatal situation for iron binding. The positions of the two liganding tyrosines in the metal-binding cleft suggest a reason for the inequivalence.

Molecular diffusion into horse spleen ferritin: a nitroxide radical spin probe study.

Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike the others, the apolar TEMPO radical does not enter the cavity but instead binds to ferritin, presumably at a hydrophobic region of the protein. The kinetic data indicate that diffusion is not purely passive, the driving force coming not only from the concentration gradient between the inside and outside of the protein but also from charge interactions between the diffusant and the protein. A model for diffusion is derived that describes the observed kinetics. First-order half-lives for diffusion into the protein of 21-26 min are observed, suggesting that reductant molecules with diameters considerably larger than approximately 9 A would probably enter the protein cavity too slowly to mobilize iron efficiently by direct interaction with the mineral core.

The effect of salt and site-directed mutations on the iron(III)-binding site of human serum transferrin as probed by EPR spectroscopy.

The effects of site-directed mutation and salt on the iron(III)-binding site of the recombinant half-molecule of the N-terminal lobe (hTf/2N) of human transferrin was studied by EPR spectroscopy. Changes were observed in the EPR spectra of all variants investigated (D63S, D63C, G65R, K206Q, H207E, H249E, H249Q, K296E and K296Q) compared with that of the wild-type protein. The most pronounced changes in the metal site were caused by replacement of the coordinating residues, Asp-63 and His-249, and the non-coordinating residue Lys-296, which is located in the hinge region of the iron-binding cleft. The EPR spectral changes from replacement of other non-coordinating residues were more subtle, indicating small changes in Fe3+ coordination to the protein. The EPR spectrum of variant G65R suggests that it adopts two distinct conformations in solution, one in which the two domains forming the iron-binding cleft are closed and one in which they are open; in the latter instance Asp-63 is no longer coordinated to the Fe3+. Chloride-binding studies on hTf/2N, K206Q, H207E, K296Q and K296E showed similar binding isotherms, indicating that none of the hinge region residues replaced, i.e. Lys-206, His-207 or Lys-296, are the sites of chloride binding. The results show that the coordination environment of the Fe3+ is sensitive to structural changes from site-directed mutation of both remote and coordinated residues and also to chloride-binding and ionic strength effects.