Effect of chaotropes on the kinetics of iron release from ferritin by flavin nucleotides

BackgroundFerritins are ubiquitous multi-subunit iron storage and detoxification proteins that play a critical role in iron homeostasis. Ferrous ions that enter the protein's shell through hydrophilic channels are rapidly oxidized at dinuclear centers on the H-subunit before transfer to the protein's cavity for storage. The mechanisms of iron loading have been extensively studied, but little is known about iron mobilization. Fe(III) reduction can occur via rapid reduction by suitable reducing agents followed by chelation of Fe(II) ions or via direct and slow Fe(III) chelation. Here, the iron release kinetics from ferritin by FMNH₂ in the presence of various chaotropic agents are studied and their in-vivo physiological significance discussed.MethodsThe iron release kinetics from horse and human ferritins by FMNH₂ were monitored at 522 nm where the Fe(II)–bipyridine complex absorbs. The experiments were performed in the presence of different concentrations of three chaotropic agents, urea, guanidine HCl, and triton.Results and conclusionsUnder our experimental conditions, iron reductive mobilization by the non-enzymatic FMN/NAD(P)H system is limited by the concentration of FMNH₂ and is independent on the type or amount of chaotropes present. Diffusion of FMNH₂ through the ferritin pores is an unlikely mechanism for ferritin iron reduction. An iron mobilization mechanism involving rapid electron transfer through the protein shell is discussed.General significanceCaution must be exercised when interpreting the kinetics of iron mobilization from ferritin using the FMN/NAD(P)H system. The kinetics are highly dependent on the amount of dissolved oxygen and the concentration of reagents used.     

Oxidation of caffeic acid by Fe(III) trapped in a Ca-polygalacturonate network.

With the aim to verify if Fe(III) ions accumulated in a network of Ca-polygalacturonate (PGA) may promote the oxidation of caffeic acid (CAF) the interaction at pH 5.0 between CAF and Fe(III) ions trapped in a PGA was studied. The sorption kinetics evidenced a great affinity of CAF towards the Fe-PGA matrix. Chromatographic tests showed that the interaction leads to the formation of products which can be considered as CAF oligomers characterized by FT-IR spectra similar to those of natural humic acids. Tests carried out under nitrogen suggest that at pH 5.0 oxygen does not affect the nature of these oxidation products. Oxygen was hypothesized to exert a direct action on the redox process by oxidizing the Fe(II) ions, produced by oxidation of CAF, to Fe(III) thus regenerating oxidizing sites. A possible mechanism of formation of the polymers was proposed that implies that the CAF oxidation leads to highly reactive species such as semiquinones which give rise, by an oxidative coupling reaction, to the formation of oligomers that can aggregate through secondary bonds to produce more complex structures as those that characterize humic acids.     

Sorption efficiency of chitosan nanofibers toward metal ions at low concentrations

Chitosan fibers showing narrow diameter distribution with a mean of 42 nm were produced by electrospinning and utilized for the sorption of Fe(III), Cu(II), Ag(I), and Cd(II) ions from aqueous solutions. The ion concentrations in the supernatant solutions were determined using inductively coupled plasma-mass spectrometry (ICP-MS). The filtration efficiency of the fibers toward these ions was studied by both batch and microcolumn methods. High efficiency in sorption of the metal ions was obtained in the both methods. The effects of sorbent amount (0.10-0.50 mg), shaking time (15-120 min), initial metal ion concentration (10.0-1000.0 μg·L(-1)), and temperature (25 and 50 °C) on the extent of sorption were examined. The sorbent amount did not significantly alter the efficiency of sorption; however, shaking time, temperature, and metal ion concentration were found to have a strong influence on sorption. By virtue of its mechanical integrity, the applicability of the chitosan mat in solid phase extraction under continuous flow looks promising.     

Role of the Ca-pectates on the accumulation of heavy metals in the root apoplasm

In order to better understand the processes that regulate the accumulation in the apoplasm of heavy metals and their mobilization by the plant metabolites it is essential to study the mechanisms that regulate the interactions between metal ions and pectins. In such a context, the sorption of Cd(II), Zn(II), Cu(II) and Pb(II) from single and multi-metal solutions, by a Ca-polygalacturonate gel with a degree of esterification of 18.0 (PGAM₁) and 65.5% (PGAM₂) was studied in the 3.0–6.0 pH range in the presence of CaCl₂ 2.5 mM. The sorption of Cr(III) from single metal solution was also considered. The results show that the amount of each metal ion sorbed increases with increasing the initial metal ion concentration and pH. The data from the single metal solution tests show that at pH 6.0 the affinity of the metal ions towards the PGAM₁ matrix follows the order: Cr(III) > Cu(II) ? Pb(II) ? Zn(II) ? Cd(II). The simultaneous sorption of the bivalent metal ions by the PGAM₁ gels indicates that Pb(II) is selectively sorbed. The FT-IR spectra show that the carboxylate groups are mainly responsible for the metal ion coordination. The ability of PGAM₂ to accumulate Cr(III), Cu(II), and Pb(II) was lower than that found in the PGAM₁ systems whereas the sorption of Zn(II) and Cd(II) was negligible.     

Identification of a carbon-oxygen lyase activity cleaving the ether linkage in carboxymethyloxysuccinic acid

Cell-free extracts of an organism capable of degrading the synthetic ether carboxymethyloxysuccinic acid were used to study the mechanism of degradation of this compound. The initial degradation products were acidic and could be separated by high-voltage electrophoresis. Extended incubation of [¹⁴C]carboxymethyloxysuccinate with the cell-free extracts gave rise to radioactive carbon dioxide and to glycolic, malic, fumaric, and pyruvic acids which were identified by electrophoresis. Identification of the malic and glycolic acids was confirmed by the addition of non-radioactive carrier malic and glycolic acids and recrystallization to constant specific radioactivity. The identification of pyruvate was substantiated by thin-layer chromatography of the 2,4-dinitrophenylhydrazine derivative. The presence of fumaric acid was substantiated by its ultraviolet absorbance.Shorter incubations give rise to only malic, glycolic, and fumaric acids. When the time course of the reaction was followed carefully over the first 15 min of the reaction, it was found that initially nearly equimolar quantities of glycolic and fumaric acids were produced. The early production of fumaric acid was also demonstrated by following the increase in 245-nm absorbance of the degradation mixture. The fumaric acid was subsequently hydrated to malic acid, and the quantity of fumaric acid present decreased slightly until it reached a steady-state level. The presence of fumarase in the extract was demonstrated. It was concluded that the initial attack on the carboxymethyloxysuccinic acid was a cleavage of the ether linkage by an elimination mechanism catalyzed by a carbon-oxygen lyase giving rise to glycolic and fumaric acids.     

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.     

Reverse reaction of malic enzyme for HCO3- fixation into pyruvic acid to synthesize L-malic acid with enzymatic coenzyme regeneration

Malic enzyme [L-malate: NAD(P)(+) oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)(+)). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO(3)(-) fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD(+), and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO(3)(-) and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 degrees C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD(+).     

The Effect of Acetate on Euglena gracilis var. bacillaris as a Function of Environmental Conditions

SYNOPSIS. Acetate, a widely used substrate for the growth of Euglena gracilis var. bacillaris , becomes lethal to these organisms at pH's below 5.0. The kinetics, as well as the extent of acetate-induced death, depend upon both the hydrogen ion and acetate concentrations. Formic, propionic, butyric, and fluoroacetic acids also kill E. gracilis at low pH's, whereas fumaric, malic, succinic and pyruvic acids do not under these conditions.
It is suggested that death is caused by a lowering of the intra-cellular pH, due to the release of hydrogen ion brought into the cell by freely penetrating undissociated acetate molecules.     

Microbially Available Phosphorus in Boreal Forests: Effects of Aluminum and Iron Accumulation in the Humus Layer

Soil microorganisms play an important role in the mobilization of phosphorus (P), and these activities may be beneficial for plant P utilization. We investigated the effects on microbial P availability of different combinations of aluminum and iron (Al + Fe) concentrations and different P pools in humus soils from boreal forest ecosystems. We measured respiration rates in laboratory incubations before and after additions of glucose plus (NH₄)₂SO₄ (Glu+N), with or without a small dose of KH₂PO₄. Glu+N was added in excess so that the availability of the inherent soil P would be growth-limiting for the microorganisms. The exponential increases observed in microbial growth after substrate additions (Glu+N) was slower for humus soils with high Al+Fe concentrations than for humus soils with low Al+Fe concentrations. Adding a small dose of KH₂PO₄ to humus soils with high Al+Fe concentrations did, however, increase the exponential growth, measured as the slope of the log-transformed respiration rates, by more than 200%. By contrast, the average increase in exponential growth was only 6% in humus soils with low Al+Fe concentrations. Almost eight times more carbon dioxide (CO₂) was evolved between the substrate additions and the point at which the respiration rate reached 1 mg CO₂ h^–1 for soils with high Al+Fe concentrations compared to humus soils with low Al+Fe concentrations. The amount of CO₂ evolved was positively related to the Al+Fe concentration of the humus soils (r ² = 0.86, P < 0.001), whereas the slope was negatively related to Al+Fe concentration (r ² = 0.70, P < 0.001). Easily available P forms were negatively related to the Al+Fe concentration, whereas organic P showed a strong positive relationship to Al+Fe (r ² = 0.85, P < 0.001), suggesting that other forms of P, as well as inorganic P, are affected by the increased sorption capacity. The results indicate that P mobilization by microorganisms is affected by the presence of sorption sites in the humus layer, and that this capacity for sorption may relate not only to phosphate but also to organic P compounds.     

Spectroscopic studies on the binding of iron, terbium, and zinc by apoferritin

Ultraviolet difference spectroscopy has been used to study Fe (III)-apoferritin complexes formed after addition of Fe (II) to apoferritin in air. At constant iron, the recorded spectra varied with time after Fe (II) addition and with the number of iron atoms/molecule (protein concentration). The results indicate that after production of an initial complex, rearrangement or migration of Fe (III) atoms occurs, with polynuclear species forming as end-product, probably by hydrolytic polymerization. The presence of Tb3+ or Zn2+ ions affected the Fe (III) spectra and their development in different ways. The combined data suggest that more than one site, or processes, are involved in ferritin iron-core formation and that some of the metal sites are clustered.     

Interactions of Cu (II) and Fe (III) with mangal humic substances studied by synchronous fluorescence spectroscopy and potentiometric titration

Humic (HA) and fulvic (FA) acids isolated from mangrove sediments of Sundarban, the largest delta on earth in the estuarine phase of the river Ganges, were studied and attempts were made to characterize their binding sites by quenching of Synchronous fluorescence (SyF) bands with Fe (III) and Cu (II). A modified Stern-Volmer relationship applicable for static quenching was applied for the determination of conditional stability constants and the data were compared with those determined by potentiometric titration. In the excited state HA and FA showed different acidity constant compared to the ground state. Values of the conditional stability constant (log K_c) for Fe (III) and Cu (II) indicated that binding sites were bidentate in nature. FA were better chelators than the HA fractions. High energy binding sites of both FA & HA were occupied by Fe(III) and the low energy binding sites, mainly responsible for mobilization and immobilization of metal, were occupied by Cu(II).     

Heme-peptide/protein ions and phosphorous ligands: search for site-specific addition reactions

High-resolution Fourier transform ion cyclotron resonance mass spectrometry is employed to gain thorough kinetics and thermodynamics information on the reaction of free and ligated heme-type ions with selected ligands, with the aim of obtaining an insight into the coordination environment of the prosthetic group in a variety of biomolecular ions. Adopting a stepwise approach towards systems of increasing complexity, we examined the reactivity of free gaseous iron(III) protoporphyrin IX ions, Fe(III)-heme(+), of the charged species from microperoxidase-11 (MP11) (covalently peptide bound heme), and of the multiply charged ions from heme proteins, namely, cytochrome c (cyt c) and myoglobin (examples of noncovalently protein bound hemes). Among an array of test compounds allowed to react with Fe(III)-heme(+), OP(OMe)(3) and P(OMe)(3) proved to be similarly efficient ligands in the first addition step, yet displayed markedly distinct reactivity towards heme iron already engaged in axial coordination. The ease with which P(OMe)(3) acts as a second axial ligand is exploited to probe structural and conformational features of biomolecular ions. In this way, circumstantial evidence is gained of a folded conformation of +2 charge state ions from MP11 and an elongated one for the +3 charge state ions. Similarly, both the general reaction pattern and detailed kinetics and thermodynamics data point to a regiospecific addition reaction of P(OMe)(3) directed at the heme iron within multiply charged ions from cyt c. This unprecedented example of ion-molecule reaction which specifically involves a prosthetic group belonging to protein ions stands in contrast to the multiple, nonspecific interactions established by OP(OMe)(3) molecules with the protonated sites of multiply charged cyt c and apomyoglobin ions. This finding may develop and provide sensitive probes of the structure and bonding features of protein ions in the gas phase.     

Adsorption behaviour of Fe(II) and Fe(III) ions in aqueous solution on chitosan and cross-linked chitosan beads

A batch adsorption system was applied to study the adsorption of Fe(II) and Fe(III) ions from aqueous solution by chitosan and cross-linked chitosan beads. The adsorption capacities and rates of Fe(II) and Fe(III) ions onto chitosan and cross-linked chitosan beads were evaluated. Chitosan beads were cross-linked with glutaraldehyde (GLA), epichlorohydrin (ECH) and ethylene glycol diglycidyl ether (EGDE) in order to enhance the chemical resistance and mechanical strength of chitosan beads. Experiments were carried out as function of pH, agitation period, agitation rate and concentration of Fe(II) and Fe(III) ions. Langmuir and Freundlich adsorption models were applied to describe the isotherms and isotherm constants. Equilibrium data agreed very well with the Langmuir model. The kinetic experimental data correlated well with the second-order kinetic model, indicating that the chemical sorption was the rate-limiting step. Results also showed that chitosan and cross-linked chitosan beads were favourable adsorbers.     

Organic acids production byStreptomyces spp. isolated from soil,rhizosphere and mycorrhizosphere of pine (Pinus sylvestris L.)

Summary The streptomycetes studied released into the medium the following organic acids: pyruvic, α-ketoglutaric, lactic, malic (or citric), succinic and oxalic. Soil streptomycetes produced more α-ketoglutaric-, lactic- and succinic acid than the root zone microorganisms. Mean indices of the total production of organic acids were in the following order: soil>rhizosphere>mycorrhizosphere. Amounts of pyruvic acid excreted by the soil streptomycetes were inversely proportional to their biomass, whereas those of α-ketoglutaric acid were directly proportional to dry weight. Small repeatability of the results of α-keto acids determinations in these organisms was stated.     

A model of factors controlling orthophosphate removal in planted vertical flow wetlands

Orthophosphate removal from wastewater by planted vertical-flow wetlands (VFWs) occurs through three parallel paths, with reaction rates of: sorption to substratum>biofilm assimilation⪢macrophyte uptake. Short term (one or two loadings) plant removal of phosphorus (P) is small but irreversible, whereas P removed by substratum sorption, or non-reactive P (NRP) formation, can be returned as reactive phosphorus (RP). The quantity of P removed by the three paths is substratum>macrophyte ⪢biofilm, in the short term, but macrophyte>substratum⪢biofilm, over months. Rhizosphere hydrology restricts P removal, the rate is limited by mass transfer without liquid mixing, but trebled by mixing. Evapotranspirational mixing alone is small and erratic. In small, above-ground, systems environmental temperature changes cause daily mixing, but prevailing soil temperature gradients limit below-ground mixing. A planted wetland, conceptual model, explains: (1) retention times: determined by initial RP removal rates, and operationally dependent on RP concentration and mixing. Aqueous phase cycling reduces retention times several fold; (2) minimum outflow concentrations: controlled by the gravel–PO₄ sorption equilibrium; (3) sustainable annual P removal: the quantity harvested in the macrophytes. Substratum Fe(III) oxide–hydroxide sorption provides additional assimilation for some years. Specific, model derived, VFW design and operation recommendations are made.     

Scanning electron microscopic examination of Thiobacillus ferrooxidans on different support matrix materials in packed bed and fluidized bed bioreactors

Summary Thiobacillus ferrooxidans was used in fixed-film bioreactors to oxidize ferrous iron to ferric iron. The test support matrix materials included activated carbon particles, glass beads, and ion-exchange resin particles. The experimental systems included a fluidized bed approach, which was evaluated with activated carbon only, and a packed bed approach which was tested with each of the support matrix materials. The colonization of the matrix surface was examined with scanning electron microscopy. There were contrasting differences in the bacterial colonization and accumulation of Fe(III) precipitates on the matrix surface among the test materials. The packed bed activated carbon bioreactor displayed the fastest kinetics and the highest amount of cell sorption as well as the roughest and most porous matrix surface.     

Effect of temperature on phosphorus sorption to sediments from shallow eutrophic lakes

The availability of phosphorus (P) in lakes is dependent on the sorption characteristics of the underlying sediments. Temperature is a crucial factor affecting the P sorption in sediments. The objective of this study was to evaluate the effect of temperature on sorption of P by sediments from two eutrophic lakes. The study was carried out using short-term batch experiments at 4, 20 and 30 °C. Phosphorus sorption kinetics, isotherms, fractionation and desorption were investigated. The P sorption was dependent on sediment type and temperature (p < 0.001). The Mei sediments showed a higher sorption rate and sorption capacity than Hua sediments. The P sorption kinetics were best described by a pseudo second order model (R² > 0.97). Activation energies derived from the kinetics rate constant indicated that P sorption onto the two sediments was controlled by a diffusion process. For both sediments, Freundlich model fit the P sorption isotherms well and the calculated apparent sorption heat was 6.37 kJ mol⁻¹ for Mei sediments and 8.67 kJ mol⁻¹ for Hua sediments. This indicated that P sorption onto both sediments was endothermic. Adding P significantly increased the soluble and loosely bound P (S/L-P), aluminum-bound P (Al-P) and iron-bound P (Fe-P) (p < 0.05). The amount of Al-P and Fe-P was markedly higher at 30 °C than at 4 °C (p < 0.05). Subsequent P desorption indicated that adsorbed P was highly labile, in particular for Hua sediment. The degree of P mobility that occurred during sediment sorption was inversely related to the temperature at the time of sorption. A significant relationship (R² = 0.978) between phosphorus sorption maximum and oxalate-extractable Fe and Al at different temperatures reflects that the amorphous contents of Fe and Al are responsible for the temperature effect on P sorption.     

Improved experimental and computational methodology for determining the kinetic equation and the extant kinetic constants of Fe(II) oxidation by Acidithiobacillus ferrooxidans

The variety of kinetics expressions encountered in the literature and the unreasonably broad range of values reported for the kinetics constants of Acidithiobacillus ferrooxidans underscore the need for a unifying experimental procedure and for the development of a reliable kinetics equation. Following an extensive and critical review of reported experimental techniques, a method based on batch pH-controlled kinetics experiments lasting less than one doubling time was developed for the determination of extant kinetics constants. The Fe(II) concentration in the experiments was measured by a method insensitive to Fe(III) interference. Kinetics parameters were determined by nonlinear fitting of the integrated form of the Monod equation to yield a K(S) of 31 +/- 4 mg Fe(2+) liter(-1) (mean +/- standard deviation), a K(P) of 139 +/- 20 mg Fe(3+) liter(-1), and a mu(max) of 0.082 +/- 0.002 h(-1). The corresponding kinetics equation was as follows: dSdt=-0.0822.3.10(7)S.X31(1+P(0)+S(0)-S139)+S where S represents the Fe(II) concentration in mg liter(-1), P(0) represents the initial Fe(III) concentration in mg liter(-1), X represents the suspended bacterial cell concentration in cells ml(-1), and t represents time in hours. The measured data fit this equation exceptionally well, with an R(2) of >0.99. Fe(III) inhibition was found to be of a competitive nature. Contrary to previous reports, the results show that the concentration of Acidithiobacillus ferrooxidans cells has no affect on the kinetics constants. The kinetics equation can be considered applicable only to A. ferrooxidans cells grown under environmental conditions similar to those of the inoculum tested in the study. In contrast, the experimental and computational procedure is completely general and can be applied to A. ferrooxidans irrespective of the culture history.     

Organic acids promote the uptake of lanthanum by barley roots

Organic acids play an important role in metal uptake by, and accumulation in, plants. However, the relevant mechanisms remain obscure. Acetic, malic and citric acids increased the uptake of lanthanum (La) by barley (Hordeum vulgare) roots and enhanced La content in shoots under hydroponic conditions. Concentration-dependent net La influx in the absence and presence of organic acids yielded nonsaturating kinetic curves that could be resolved into linear and saturable components. The saturable component followed Michaelis-Menten kinetics. The K(m) values were similar; however, the V(max) values in the presence of acetic, malic and citric acids were 4.3, 2.8, 1.5-times that of the control, respectively. Enhanced uptake of La by organic acids was mediated mainly, but not solely, by Ca(2+) channels. X-ray absorption spectroscopic techniques provided evidence of La-oxygen environment and established that La(III) was coordinated to 11 oxygen atoms that are likely to be involved in the binding of La(III) to barley roots via carboxylate groups and hydration of La(III).     

Antibiotic activity and siderophores of Pseudomonas cepacia

The ability of 46 strains of Pseudomonas cepacia to inhibit phytopathogenic fungi and the effect of iron on their antifungal activity were studied. The antifungal effect of the bacteria and the antimicrobial activity of their crude yellow and violet pigments showed a 4-5-fold decrease in the presence of Fe(III). The addition of 100 micrograms/ml of FeCl3 to the medium decreased the biosynthesis of violet and yellow pigments; the complex of the yellow pigment with Fe(III) promoted the growth of the P. cepacia producing strain under iron-deficient conditions. The data obtained suggest a participation of some P. cepacia pigments in iron transport. The resistance of the P. cepacia strains to the synthetic chelating agents hydroxyethylenediphosphonic and diethylenediaminepentaacetic acids was demonstrated, which may indicate a high Fe(III)-binding constant of P. cepacia siderophores.