Sulfate as a synergistic anion facilitating iron binding by the bacterial transferrin FbpA: the origins and effects of anion promiscuity.

The ferric binding protein, FbpA, has been demonstrated to facilitate the transport of naked Fe3+ across the periplasmic space of several Gram-negative bacteria. The sequestration of iron by FbpA is facilitated by the presence of a synergistic anion, such as phosphate or sulfate. Here we report the sequestration of Fe3+ by FbpA in the presence of sulfate, at an assumed periplasmic pH of 6.5 to form FeFbpA-SO4 with K'(eff) = 1.7 x 10(16) M(-1) (at 20 degrees C, 50 mM MES, 200 mM KCl). The iron affinity of the FeFbpA-SO4 protein assembly is 2 orders of magnitude lower than when bound with phosphate and is the lowest of any of the FeFbpA-X assemblies yet reported. Iron reduction at the cytosolic membrane receptor may be an essential aspect of the periplasmic iron-transport process, and with an E(1/2) of -158 mV (NHE), FeFbpA-SO4 is the most easily reduced of all FeFbpA-X assemblies yet studied. The variation of FeFbpA-X assembly stability (K'(eff)) and ease of reduction (E(1/2)) with differing synergistic anions X(n-) are correlated over a range of 14 kJ, suggesting that the variations in redox potentials are due to stabilization of Fe3+ in FeFbpA-X by X(n-). Anion promiscuity of FbpA in the diverse composition of the periplasmic space is illustrated by the ex vivo exchange kinetics of FeFbpA-SO4 with phosphate and arsenate, where first-order kinetics with respect to FeFbpA-SO4 (k = 30 s(-1)) are observed at pH 6.5, independent of entering anion concentration and identity. Anion lability and influence on the iron affinity and reduction potential for FeFbpA-X support the hypothesis that synergistic anion exchange may be an important regulator in iron delivery to the cytosol. This structural and thermodynamic analysis of anion binding in FeFbpA-X provides additional insight into anion promiscuity and importance.

Optimization of the lariat ether carboxylic acid host structure for ferrioxamine B: demonstration of a second coordination shell chelate effect.

Host-guest supramolecular assembly formation constants involving the second-sphere complexation of the siderophore ferrioxamine B (FeHDFB(+)) by a lariat ether carboxylic acid host (L(n+2)COOH) in wet chloroform were obtained from liquid-liquid extractions at pH values above and below the host pK(a) (approximately 5.3). The host-guest formation constants, K(a), determined at pH = 3.2 for the assemblies [FeHDFB(+),L(n+2)COOH,ClO(4)(-)] (n = 4, 7, 10, 15) in wet chloroform are similar to those of the parent crown ether, benzo-18-crown-6. At pH = 9.3, the lariat ethers are ionized, and this results in a more stable assembly, [FeHDFB(+),L(n+2)COO(-)], as measured by the host-guest formation constant, K(app). This enhanced stability is shown to be a function of the lariat ether sidearm chain length (n = 4, 7, 10, 15) and is corroborated by molecular modeling calculations. Additionally, molecular modeling and extraction data demonstrate that there is an optimum lariat ether sidearm chain length with respect to host-guest assembly stability as measured by K(app). We attribute the enhanced stability effect of the ionized lariat ether in the host-guest assembly [FeHDFB(+),L(n+2)COO(-)], relative to [FeHDFB(+),L(n)+2)COOH,ClO(4)(-)] or to [FeHDFB(+),B18C6,ClO(4)(-)], to a second coordination shell chelate effect.

Kinetics and mechanism of a catalytic chloride ion effect on the dissociation of model siderophore hydroxamate-iron(III) complexes.

Proton-driven ligand dissociation kinetics in the presence of chloride, bromide, and nitrate ions have been investigated for model siderophore complexes of Fe(III) with the mono- and dihydroxamic acid ligands R(1)C(=O)N(OH)R(2) (R(1) = CH(3), R(2) = H; R(1) = CH(3), R(2) = CH(3); R(1) = C(6)H(5), R(2) = H; R(1) = C(6)H(5), R(2) = C(6)H(5)) and CH(3)N(OH)C(=O)[CH(2)](n)C(=O)N(OH)CH(3) (H(2)L(n); n = 2, 4, 6). Significant rate acceleration in the presence of chloride ion is observed for ligand dissociation from the bis(hydroxamate)- and mono(hydroxamate)-bound complexes. Rate acceleration was also observed in the presence of bromide and nitrate ions but to a lesser extent. A mechanism for chloride ion catalysis of ligand dissociation is proposed which involves chloride ion dependent parallel paths with transient Cl(-) coordination to Fe(III). The labilizing effect of Cl(-) results in an increase in microscopic rate constants on the order of 10(2)-10(3). Second-order rate constants for the proton driven dissociation of dinuclear Fe(III) complexes formed with H(2)L(n)() were found to vary with Fe-Fe distance. An analysis of these data permits us to propose a reactive intermediate of the structure (H(2)O)(4)Fe(L(n)())Fe(HL(n))(Cl)(OH(2))(2+) for the chloride ion dependent ligand dissociation path. Environmental and biological implications of chloride ion enhancement of Fe(III)-ligand dissociation reactions are presented.

Treatment of iron deficiency anemia: are monomeric iron compounds suitable for parenteral administration?

Iron deficiency is the most common nutritional problem worldwide, especially in the developing countries. Oral iron supplementation programs have failed because of noncompliance and gastrointestinal toxicity, thereby necessitating parenteral administration of iron. For parenteral administration, only iron-carbohydrate complexes are currently used, because monomeric iron salts release free iron, thereby causing oxidant injury. However, iron-carbohydrate complexes also have significant toxicity, and they are expensive. We have proposed the hypothesis that monomeric iron salts can be safely administered by the parenteral route if iron is tightly complexed to the ligand, thereby causing clinically insignificant release of free iron, and the kinetic properties of the compound allow rapid transfer of iron to plasma transferrin. A detailed analysis of the physicochemical and kinetic properties reveals that ferric iron complexed to pyrophosphate or acetohydroxamate anions may be suitable for parenteral administration. We have demonstrated that infusion of ferric pyrophosphate into the circulation via the dialysate is safe and effective in maintaining iron balance in patients undergoing maintenance hemodialysis. Parenteral administration of monomeric iron compounds is a promising approach to the treatment of iron deficiency in the general population and merits further investigation.

Multiple-path dissociation mechanism for mono- and dinuclear tris(hydroxamato)iron(III) complexes with dihydroxamic acid ligands in aqueous solution.

Linear synthetic dihydroxamic acids ([CH3N(OH)C=O)]2(CH2)n; H2Ln) with short (n = 2) and long (n = 8) hydrocarbon-connecting chains form mono- and dinuclear complexes with Fe(III) in aqueous solution. At conditions where the formation of Fe2(Ln)3 is favored, complexes with each of the two ligand systems undergo [H+]-induced ligand dissociation processes via multiple sequential and parallel paths, some of which are common and some of which are different for the two ligands. The pH jump induced ligand dissociation proceeds in two major stages (I and II) where each stage is shown to be comprised of multiple components (Ix, where x = 1-3 for L2 and L8, and IIy, where y = 1-3 for L2 and y = 1-4 for L8). A reaction scheme consistent with kinetic and independent ESI-MS data is proposed that includes the tris-chelated complexes (coordinated H2O omitted for clarity) (Fe2(Ln)3, Fe2(L2)2(L2H)2, Fe(LnH)3, Fe(L8)(L8H)), bis-chelated complexes (Fe2(Ln)2(2+), Fe(LnH)2+, Fe(L8)+), and monochelated complexes (Fe(LnH)2+). Analysis of kinetic data for ligand dissociation from Fe2(Ln)(LnH)3+ (n = 2, 4, 6, 8) allows us to estimate the dielectric constant at the reactive dinuclear Fe(III) site. The existence of multiple ligand dissociation paths for the dihydroxamic acid complexes of Fe(III) is a feature that distinguishes these systems from their bidentate monohydroxamic acid and hexadentate trihydroxamic acid counterparts and may be a reason for the biosynthesis of dihydroxamic acid siderophores, despite higher environmental molar concentrations necessary to completely chelate Fe(III).

Kinetics and mechanism of iron(III) dissociation from the dihydroxamate siderophores alcaligin and rhodotorulic acid.

The kinetics and mechanism of siderophore ligand dissociation from their fully chelated Fe(III) complexes is described for the highly preorganized cyclic tetradentate alcaligin and random linear tetradentate rhodotorulic acid in aqueous solution at 25 degrees C (Fe2L3 + 6H+ reversible 2 Fe3+ aq + 3 H2L). At siderophore:Fe(III) ratios where Fe(III) is hexacoordinated, kinetic data for the H(+)-driven ligand dissociation from the Fe2L3 species is consistent with a singly ligand bridged structure for both the alcaligin and rhodotorulic acid complexes. Proton-driven ligand dissociation is found to proceed via parallel reaction paths for rhodotorulic acid, in contrast with the single path previously observed for the linear trihydroxamate siderophore ferrioxamine B. Parallel paths are also available for ligand dissociation from Fe2(alcaligin)3, although the efficiency of one path is greatly diminished and dissociation of the bis coordinated complex Fe(alcaligin)(OH2)2+ is extremely slow (k = 10(-5) M-1 s-1) due to the high degree of preorganization in the alcaligin siderophore. Mechanistic interpretations were further confirmed by investigating the kinetics of ligand dissociation from the ternary complexes Fe(alcaligin)(L) in aqueous acid where L = N-methylacetohydroxamic acid and glycine hydroxamic acid. The existence of multiple ligand dissociation paths is discussed in the context of siderophore mediated microbial iron transport.

Internal electron transfer between hemes and Cu(II) bound at cysteine beta93 promotes methemoglobin reduction by carbon monoxide.

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.

The release of iron from different asbestos structures by hydrogen peroxide with concomitant O2 generation.

Treatment of aqueous suspensions of different asbestos fibers (amosite, anthophyllite, chrysotile, and crocidolite) at 0-4 degrees C and pH 7.2 with H2O2 results in the consumption of H2O2 with concomitant release of iron and production of O2. During incubations, [H2O2] decreased in proportion to the mass of the suspended fiber, the duration of incubation, and the initial [H2O2]. The consumption of H2O2, production of O2 and release of iron all vary synergistically with the structure of the asbestos fiber. Release of silicon during the incubation was small relative to the decrement in [H2O2], reflecting a lack of dissolution of the fiber. The data are consistent with a redox process for the release of surface bound iron and it is significant that iron release occurs in the absence of a Fe(II) or Fe(III) chelator. The implications of iron release from the asbestos surface may be important in inflammatory disorders in which both silicate bound iron and H2O2 accumulate.

Hypothesis: iron chelation plays a vital role in neutrophilic inflammation.

Neutrophil influx into tissues occurs in many diverse diseases and can be associated with both beneficial and injurious effects. We hypothesize that the stimulus for certain neutrophilic inflammatory responses can be reduced to a series of competing reactions for iron, with either a labile or reactive coordination site available, between host chelators and chelators not indigenous to that specific living system. The iron focuses the transport of host phagocytic cells through a metal catalyzed generation of oxidant sensitive mediators including cytokines and eicosanoids. Many of these products are chemotactic for neutrophils. We also postulate that the iron increases the activity of the phagocyte associated NADPH oxidoreductase in the neutrophil. The function of this enzyme is likely to be the generation of superoxide in the host's attempt to chemically reduce and dislodge the iron from its chelate complex. After the reoxidation of Fe2+ in an aerobic environment, Fe3+ will be coordinated by host lactoferrin released by the neutrophil. When complexed by this glycoprotein, the metal does not readily undergo oxidation/reduction and is safely transported to the macrophages of the reticuloendothelial system where it is stored in ferritin. Finally, we propose that the neutrophil will attempt to destroy the chelator not indigenous to the host by releasing granular contents other than lactoferrin. Inability to eliminate the chelator allows this sequence to repeat itself, which can lead to tissue injury. Such persistence of a metal chelate in the host may be associated with biomineralization, fibrosis, and cancer.

Phagocyte-generated superoxide reduces Fe3+ to displace it from the surface of asbestos.

Injury after exposure to mineral oxide dusts is considered to be mediated by free radical generation. In vitro production of hydroxyl radical by a fibrous silicate increases with the [Fe3+] complexed to the dust surface. The study hypothesis tested was that extracellular fluids and phagocytic cells can decrease concentrations of iron complexed to the surface of a fibrous silicate by employing host chelators and reductants. Such a depletion of surface [Fe3+] would predict decrements in both oxidant generation and the resultant injury after inhalation and instillation of these mineral oxides. Crocidolite (2.0 mg) which was exposed to either 5.0 ml rat plasma or 10.0 ml rat lavage fluid for 1 h had diminished surface [Fe3+]. Similarly, incubations of crocidolite (2.0 mg) with either 10.0 ml rat alveolar macrophages (1.0 x 10(6) cells/ml) or 10.0 ml rat neutrophils (1.0 x 10(7) cells/ml) decreased concentrations of surface iron. In vivo exposures of asbestos contained in chambers allowing or precluding inflammatory cell entry revealed that the influx of phagocytes was associated with greater decreases in surface [Fe3+]. The body chelators transferrin and lactoferrin were unable to extract the metal from fiber surface in vitro. However, superoxide generated by phagocytes did displace the iron from the crocidolite surface. We conclude that extracellular fluids and phagocytic cells have a capacity to diminish [Fe3+] complexed to the surface of asbestos and therefore decrease the potential for oxidative stress and injury to a living system after exposure to these dusts.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA strand breaks following in vitro exposure to asbestos increase with surface-complexed [Fe3+].

Surface functional groups on silicate dusts complex iron cations which can cycle through reduction and oxidation states to generate free radicals. These oxidants have a capacity to produce DNA strand breaks and mutations which are primary events in cancer induction. A differential in the capacity of fibrous silicates to produce carcinoma is recognized with the amphiboles demonstrating a greater biologic effect than the serpentine fiber chrysotile. We tested the hypothesis that the differences in genotoxicity of these fibrous silicates correspond to varying concentrations of iron complexed to the surface. Relative to chrysotile, the amphibole fibers complexed greater amounts of iron cations from both inorganic and in vivo sources. Increased concentrations of surface-complexed iron were associated with greater oxidant generation, measured as thiobarbituric acid-reactive products of deoxyribose, and more covalently closed, circular DNA strand scission. These results indicate that genotoxic effects of these fibers may correspond to their capacity to complex iron at the surface.

Role of surface complexed iron in oxidant generation and lung inflammation induced by silicates.

Inhalation of silicates induces a variety of lung diseases in humans. The molecular mechanism(s) by which these dusts cause disease is not known. Because several naturally occurring mineral oxides have large amounts of transition metal ions on their surfaces, we tested the hypothesis that surface complexation of iron may be an important determinant of their ability to induce disease. Silica, crocidolite, kaolinite, and talc complexed considerable concentrations of Fe3+ onto their surfaces from both in vitro and in vivo sources. The potential biological importance of iron complexation was assessed by examining the relationship between surface [Fe3+] and the ability of silicates to mediate oxidative degradation of deoxyribose in vitro, induce a respiratory burst and elicit leukotriene B4 (LTB4) release by alveolar macrophages (AM) in vitro, and cause acute alveolitis after intratracheal insufflation. For these studies, three varieties of silicate dusts were used: iron-loaded, wetted (unmodified), and deferoxamine-treated to remove Fe3+. The ability of silicates to catalyze oxidant generation in an ascorbate/H2O2 system in vitro, to trigger respiratory burst activity and LTB4 release by AM, and to induce acute lung inflammation in the rat all increased with surface complexed Fe3+. The results of these studies suggest that surface complexation of iron may be an important determinant in the pathogenesis of disease after silicate exposure.

Hypothesis: is lung disease after silicate inhalation caused by oxidant generation?

Inhaled silicate dusts may cause lung disease through their surface coordination of iron with subsequent oxidant generation via the Fenton reaction. Pneumoconiosis, irritant bronchitis, focal emphysema, and carcinoma may be produced by oxidants either directly through lipid peroxidation and protein inactivation, or indirectly by oxidant-mediated release of cytokines such as platelet-derived growth factor. The increased incidence of tuberculosis observed among silicate workers could be explained by accumulation of iron complexed by dust particles in the lung and made available to dormant mycobacteria as a virulence factor.

Factors that influence siderophoremediated iron bioavailability: catalysis of interligand iron (III) transfer from ferrioxamine B to EDTA by hydroxamic acids.

Deferriferrioxamine B (H3DFB) is a linear trihydroxamic acid siderophore with molecular formula NH2(CH2)5[N(OH)C(O)(CH2)2C(O)NH(CH2)5]2N(OH)C(O)CH3 that forms a kinetically and thermodynamically stable complex with iron(III), ferrioxamine B. Under the conditions of our study (pH = 4.30, 25 degrees C), ferrioxamine B, Fe(HDFB)+, is hexacoordinated and the terminal amine group is protonated. Addition of simple hydroxamic acids, R1C(O)N(OH)R2 (R1 = CH3, R2 = H; R1 = C6H5, R2 = H; R1 = R2 = CH3), to an aqueous solution of ferrioxamine B at pH = 4.30, 25.0 degrees C, I = 2.0, results in the formation of ternary complexes Fe(H2DFB)A+ and Fe(H3DFB)A2+, and tris complexes FeA3, where A- represents the bidendate hydroxamate anion R1C(O)N(O)R2-. The addition of a molar excess of ethylenediaminetetraacetic acid (EDTA) to an aqueous solution of ferrioxamine B at pH 4.30 results in a slow exchange of iron(III) to eventually completely form Fe(EDTA)- and H4DFB+. The addition of a hydroxamic acid, HA, catalyzes the rate of this iron exchange reaction: (formula; see text) A four parallel path mechanism is proposed for reaction (1) in which catalysis occurs via transient formation of the ternary and tris complexes Fe(H2DFB) A+, Fe(H3DFB)A2+, and FeA3. Rate and equilibrium constants for the various reaction paths to products were obtained and the influence of hydroxamic acid structure on catalytic efficiency is discussed. The importance of a low energy pathway for iron dissociation from a siderophore complex in influencing microbial iron bio-availability is discussed. The system represented by reaction (1) is proposed as a possible model for in vivo catalyzed release of iron from its siderophore complex at the cell wall or interior, where EDTA represents the intracellular storage depot or membrane-bound carrier and HA represents a low molecular weight hydroxamate-based metabolite capable of catalyzing interligand iron exchange.

The selection and evaluation of new chelating agents for the treatment of iron overload.

A large-scale systematic evaluation of potential iron chelators for the treatment of hemosiderosis was conducted. The compounds were identified and evaluated using a hypertransfused mouse screen in which deferrioxamine B was a standard. This screen was designed to measure iron depletion in the tissues as well as iron excretion. Groups of 10 previously hypertransfused BDF1 male mice received a single daily i.p. injection of either vehicle, standard, or test compound for 7 days. Iron in daily urine pools and individual spleen and liver homogenates was determined by atomic absorption. More than 70 chelators were evaluated, including natural and synthetic hydroxamic acids, phenols, catechols and tropolones known to have a high affinity for iron (III) in vitro. Ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid) was shown to be considerably more effective than deferrioxamine B (i.p.) and, in addition, was orally active. Factors determining the efficacy of this and other chelating agents are discussed.