Kinetics of iron passage through subcellular compartments of rabbit reticulocytes

Summary The kinetics of the separate processes of Fe₂(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and⁵⁹Fe,¹²⁵I-labeled rabbit transferrin. The binding of⁵⁹Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2×10⁵ m ^–1 sec^–1. The rate of internalization of⁵⁹Fe-transferrin was directly measured at 520±100 molecules of Fe₂(III)-transferrin internalized/sec/cell with 250±43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe₂(III)-transferrin internalization the flux of⁵⁹Fe was followed through three compartments: internalized transferrin, membrane, and cytosol.A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17±2 sec and 34±5 sec, respectively. Apparent rate constants of 0.0075±0.002 sec^–1 and 0.0343±0.0118 sec^–1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112±0.0025 sec^–1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe₂(III)-transferrin that are distinct from the handling of transferrin.     

Complexation of ytterbium to human transferrin and its uptake by K562 cells.

There is an increasing interest in the use of lanthanides in medicine. However, the mechanism of their accumulation in cells is not well understood. Lanthanide cations are similar to ferric ions with regard to transferrin binding, suggesting transferrin-receptor mediated transport is possible; however, this has not yet been confirmed. In order to clarify this mechanism, we investigated the binding of Yb3+ to apotransferrin by UV-Vis spectroscopy and stopped-flow spectrophotometry, and found that Yb3+ binds to apotransferrin at the specific iron sites in the presence of bicarbonate. The apparent binding constants of these sites showed that the affinity of Yb3+ is lower than that of Fe3+and binding of Yb3+ in the N-lobe is kinetically favored while the C-lobe is thermodynamically favored. The first Yb3+ bound to the C-lobe quantitatively with a Yb/apotransferrin molar ratio of < 1, whereas the binding to the other site is weaker and approaches completeness by a higher molar ratio only. As demonstrated by 1H NMR spectra, Yb3+ binding disturbed the conformation of apotransferrin in a manner similar to Fe3+. Flow cytometric studies on the uptake of fluorescein isothiocyanate labeled Yb3+-bound transferrin species by K562 cells showed that they bind to the cell receptors. Laser scanning confocal microscopic studies with fluorescein isothiocyanate labeled Yb3+-bound transferrin and propidium iodide labeled DNA and RNA in cells indicated that the Yb3+ entered the cells. The Yb3+-transferrin complex inhibited the uptake of the fluorescein labeled ferric-saturated transferrin (Fe2-transferrin) complex into K562 cells. The results demonstrate that the complex of Yb3+-transferrin complex was recognized by the transferrin receptor and that the transferrin-receptor-mediated mechanism is a possible pathway for Yb3+ accumulation in cells.     

The nonspecific binding of Fe3+ to transferrin in the absence of synergistic anions.

An obligatory role for barbonate (or other synergistic anions) in the specific binding of Fe3+ by transferrin has been a point of controversy for two decades. There are an equal number of confirmatory and negative reports of specific Fe3+-transferrin binary complexes. A criticism of previous studies is the use of only one synthetic route, and limited product testing. This study reports the development of several preparative routes aimed at the formation of a specific Fe3+-transferrin complex, and the characterization of the products by spectrophotometry and chemical reactivity. The preparative routes described include: (a) displacement of carbonate from Fe3+-transferrin-CO32- at low pH followed by removal of CO2 by several techniques; (b) addition of FeCl3 to apotransferrin under CO2-free conditions; (c) oxidation of Fe2+ in the presence of apotransferrin under CO2-free conditions; (d) reaction of apotransferrin with nonsubstituting Fe3+ complexes in the absence of CO2; and (e) attempts to displace anions from weak Fe3+-transferrin-anion complexes. The product were examined with regard to their visible spectra, and their examined with regard to their visible spectra, and their reactivity with: (a) NaHCO3, (b) Fe3+-nitrilotriacetic acid in NaHCO3, and (c) citrate. The results are compared with the characteristics of Fe3+-transferrin-anion complexes and nonspecific Fe3+, transferrin mixtures. The data indicate that in the absence of synergistic anions the affinity of the specific metal binding sites of transfe-rin for Fe3+ is so low as to not compete favorably with hydrolytic polymerization and nonspecific binding effects.     

The effect of pH on the kinetics of iron release from human transferrin

The rate of iron release from the N-terminal and C-terminal monoferrictransferrins (Fe_N-transferrin and Fe_C-transferrin, respectively) has been studied at 37°C over the pH range 3.5–10.6 using EDTA as the accepting chelate. Fe_N-transferrin is the more facile except above pH 8.2. Plots of log₁₀k_obs against pH showed a deviation for both monoferrictransferrins between pH 5.6 and 6.0 and studies above and below this transition point indicated that iron release occures by different mechanisms. At low pH (< 5.6) the rate of release from Fe_N-transferrin is independent of the presence of EDTA or NaClO₄, whereas Fe_c-transferrin shows a small but significant increase with increasing EDTA concentration. Rapid protonation of both monoferrictransferrins is followed by relatively slow release of Fe³⁺ which is subsequently chelated by EDTA. The slower release from Fe_c-transferrin is probably due to its greater binding strength for iron and the greater conformational stability of the C-terminal domain. Above pH 6.0 iron release from both monoferrictransferrins increases as the concentration of EDTA is increased. Direct attacks of EDTA probably occurs giving Fe-transferrin (HCO₃). EDTA as a transition state or intermediate. The factors which may lead to the observed pH dependence of the rate include (i) protonation of groups directly bound to the iron, (ii) conformers which differ in degree of protonation and (iii) the degree of protonation of the attacking chelating agent. It is suggested that an increase in conformational fluctuations as the pH is lowered may play a very important role. Studies with differrictransferrin at pH 4.53 and 7.40 showed that when iron is released to EDTA the rate is independent of the occupancy of the other site; that is, the two sites are exhibiting non-co-operativity.     

Correlation of proton release and ultraviolet difference spectra associated with metal binding by transferrin

A variety of metal ions can bind to the iron-transport protein, transferrin, at two specific sites. For each metal ion, a carboxylate anion is concomitantly bound. Six metal ions which were examined fall into two classes based on proton release and ultraviolet spectral changes which accompany binding to the protein. Class II ions, which include Cu²⁺ and Zn²⁺, release approximately 2 H⁺/metal bond. Class III ions, which include Fe³⁺, Ga³⁺, Al³⁺, and VO²⁺, release approximately 3 H⁺/metal bound. The increase in absorbance near 242 nm, characteristic of tyrosine ionization, has the ratio 0.55–0.75 for class II:class III ions. Both Fe³⁺ and Cu²⁺ form metal-transferrin-oxalate complexes in the presence of excess C₂O₄^2?. Fe³⁺ releases close to 3 H⁺/metal whether forming oxalate or bicarbonate complexes with transferrin. Binding of Cu²⁺ to transferrin releases 2 H⁺/metal in the presence of C₂O^2?₄ or HCO₃^?. Since equal numbers of H⁺/metal are released for both anions, it is likely that the bicarbonate ion does not lose its proton, and remains as HCO₃^? in transferrin. These results are interpreted in terms of possible combinations of ligands at the metal binding sites.     

Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: Catalytic requirements and oxygen dependence

The ability of paraquat radicals (PQ^+.) generated by xanthine oxidase and glutathione reductase to give H₂O₂-dependent hydroxyl radical production was investigated. Under anaerobic conditions, paraquat radicals from each source caused chain oxidation of formate to CO₂, and oxidation of deoxyribose to thiobarbituric acid-reactive products that was inhibited by hydroxyl radical scavengers. This is in accordance with the following mechanism derived for radicals generated by γ-irradiation [H. C. Sutton and C. C. Winterbourn (1984) Arch. Biochem. Biophys.235, 106–115] PQ^+. + Fe³⁺ (chelate) → Fe²⁺ (chelate) + PQ⁺⁺ H₂O₂ + Fe²⁺ (chelate) → Fe³⁺ (chelate) + OH^? + OH.. Iron-(EDTA) and iron-(diethylenetriaminepentaacetic acid) (DTPA) were good catalysts of the reaction; iron complexed with desferrioxamine or transferrin was not. Extremely low concentrations of iron (0.03 μm) gave near-maximum yields of hydroxyl radicals. In the absence of added chelator, no formate oxidation occurred. Paraquat radicals generated from xanthine oxidase (but not by the other methods) caused H₂O₂-dependent deoxyribose oxidation. However, inhibition by scavengers was much less than expected for a reaction of hydroxyl radicals, and this deoxyribose oxidation with xanthine oxidase does not appear to be mediated by free hydroxyl radicals. With O₂ present, no hydroxyl radical production from H₂O₂ and paraquat radicals generated by radiation was detected. However, with paraquat radicals continuously generated by either enzyme, oxidation of both formate and deoxyribose was measured. Product yields decreased with increasing O₂ concentration and increased with increasing iron(DTPA). These results imply a major difference in reactivity between free and enzymatically generated paraquat radicals, and suggest that the latter could react as an enzyme-paraquat radical complex, for which the relative rate of reaction with Fe³⁺ (chelate) compared with O₂ is greater than is the case with free paraquat radicals.     

Binding of vanadate to human serum transferrin

Human serum transferrin specifically and reversibly binds 2 equiv of vanadate at the two metal-binding sites of the protein. The vanadium(V)-transferrin complex can be formed either by the addition of vanadate to apotransferrin or by the air oxidation of the vanadyl(IV)-transferrin complex. The formation of the vanadium complex can be blocked by loading the apotransferrin with iron(III), and bound vanadium can be displaced from the protein by the subsequent addition of either gallium(III) or iron(III). The binding constant for the second equiv of vanadate is 10(6.5) in 0.1 M hepes, pH 7.4 at 25 degrees C. The binding constant for the first equiv of vanadate is probably very similar, although no quantitative value could be determined. Although transferrin reacts with the vanadate anion, studies on the transferrin model compound ethylenebis(o-hydroxyphenylglycine) indicate that at pH 9.5, the vanadium is binding at the metal-binding site as a dioxovanadium(V) cation coordinated to two phenolic residues at each binding site. This bound cation appears to be protonated over the pH range 9.5-6.5, as shown by changes in the difference uv spectrum of the transferrin complex, to produce an oxohydroxo species. Further decreases in the pH lead to dissociation of the vanadium-transferrin complex.     

Process for biological oxidation and control of dissolved iron in bioleach liquors

Iron has a central role in bioleaching and biooxidation processes. Fe²⁺ produced in the dissolution of sulfidic minerals is re-oxidized to Fe³⁺ mostly by biological action in acid bioleaching processes. To control the concentration of iron in solution, it is important to precipitate the excess as part of the process circuit. In this study, a bioprocess was developed based on a fluidized-bed reactor (FBR) for Fe²⁺ oxidation coupled with a gravity settler for precipitative removal of ferric iron. Biological iron oxidation and partial removal of iron by precipitation from a barren heap leaching solution was optimized in relation to the performance and retention time (τ_FBR) of the FBR. The biofilm in the FBR was dominated by Leptospirillum ferriphilum and “Ferromicrobium acidiphilum.” The FBR was operated at pH 2.0 ± 0.2 and at 37 °C. The feed was a barren leach solution following metal recovery, with all iron in the ferrous form. 98–99% of the Fe²⁺ in the barren heap leaching solution was oxidized in the FBR at loading rates below 10 g Fe²⁺/L h (τ_FBR of 1 h). The optimal performance with the oxidation rate of 8.2 g Fe²⁺/L h was achieved at τ_FBR of 1 h. Below the τ_FBR of 1 h the oxygen mass transfer from air to liquid limited the iron oxidation rate. The precipitation of ferric iron ranged from 5% to 40%. The concurrent Fe²⁺ oxidation and partial precipitative iron removal was maximized at τ_FBR of 1.5 h, with Fe²⁺ oxidation rate of 5.1 g Fe²⁺/L h and Fe³⁺ precipitation rate of 25 mg Fe³⁺/L h, which corresponded to 37% iron removal. The precipitates had good settling properties as indicated by the sludge volume indices of 3–15 mL/g but this step needs additional characterization of the properties of the solids and optimization to maximize the precipitation and to manage sludge disposal.     

Iron release from transferrin induced by mixed ligand complexes of copper(II)

Summary Copper(II) complexes CuL₁L₂ with the ligand pairs 3-phosphoglycerate (PG)/ethylenediamine (en), phosphoserine (PS)/ethylenediamine, phosphoserine/malonate (mal) are shown to be effective in inducing the release of both iron atoms from di-ferric transferrin (Fe2Tf; human serum transferrin) at pH 7.3 in 1 M NaCl at 25°C. Half-times of the reaction with Cu(PG)(en)^– were less than 1 min at 0.02 M concentration. The iron(III) products are polynuclear hydroxo complexes. There is weaker interaction with Cu(PS) ₂ ^4– and virtually none with Cu(serine)(en) nor Cu(PS)(2,2-bipyridyl)^–, revealing crucial effects of the combined ligand sphere including the phosphomonoester group. The results suggest that the release of iron from Fe2Tf, or from either monoferric transferrins, occurred due to the breakdown of the stability of iron binding in conjunction with the expulsion of the synergistic anion carbonate (or oxalate). The active copper(II) complexes are postulated to be models of membrane components that could liberate iron from transferrin succeeding its uptake at the receptor sites of cells.Abbreviations PG phosphoglycerate - PS phosphoserine - en ethylenediamine - Fe2Tf diferric transferrin - Fe_cTf and Fe_NTf transferrin with iron bound to the lobe containing the C- or N-terminus, respectively - apoTf apotransferrin - K-3 all-cis-1,3,5-tris(trimethylammonio)-2,4,6-cyclo-hexanetriol - NTA nitrilotriacetic acid; bipy, 2,2-bipyridine; mal, malonate     

The influence of pH on OH scavenger inhibition of damage to deoxyribose by Fenton reaction

Summary Hydroxyl radicals (OH') can be formed in aqueous solution by direct reaction of hydrogen peroxide (H₂O₂) with ferrous salt (Fenton reaction). OH' damage to deoxyribose, measured as formation of thiobarbituric acid-reactive material, was evaluated at different pHs to study the mechanism of action of classical OH' scavengers. OH' scavenger effect on Fe²⁺ oxidation was also evaluated in the same experimental conditions. In the absence of OH' scavengers, OH' damage to deoxyribose is higher at acidic compared to neutral and moderately basic pH. At acidic pH deoxiribose is per se able to inhibit Fe²⁺ oxidation by H₂0₂. Most of OH' scavengers tested inhibit deoxyribose damage and Fe²⁺ oxidation in a similar manner: both inhibitions are most relevant at acidic pH and decrease by increasing the pH. These results are not due to OH' scavenger inhibition of Fenton reaction. The influence of pH on the parameters studied appears to be due to the competition of deoxyribose and OH' scavengers for iron. These results suggest the prominent role of iron binding in the degradation of deoxyribose and in the OH' scavenging ability of different compounds. Results obtained with triethylenetetramine, a iron chelator with a low rate constant with OH', confirm that both deoxyribose and the OH' scavengers interact with iron bringing about a site specific Fenton reaction; that the OH' formed at these sites oxidize these molecules to their radical forms which in turn reduce the Fe^3– produced by Fenton reaction. The results presented indicate that most of classical OH' scavengers exert their effect predominantly by preventing the site specific reaction between Fe²⁺ and H₂0₂ on the deoxyribose molecule.     

Ferroxidase activity of recombinant Desulfovibrio vulgaris rubrerythrin

 Rubrerythrin (Rr) is the trivial name given to a non-heme iron protein of unknown function which has been found in anaerobic sulfate-reducing bacteria. Rr is unique in containing both rubredoxin-type FeS₄ and diiron-oxo sites in the same protein. The results described here demonstrate for the recombinant protein that: (a) Rr catalyzes oxidation of Fe²⁺ to Fe³⁺ by O₂, i.e., Rr has ferroxidase activity, (b) both FeS₄ and diiron domains of the Rr protein are required for ferroxidase activity, (c) with excess Fe²⁺ and O₂ the initial rate of this oxidation appears to be first order in [Rr] and independent of starting [Fe²⁺] above 30 μM, (d) the Fe³⁺ is produced in a form which is capable of rapid incorporation into the iron-binding site of ovotransferrin, and (e) the ferroxidase activity of Rr is comparable to that of published ferroxidase activities of apoferritins on a subunit basis. Ferroxidase activity of Rr was monitored either by the rate of increase in absorbance at 315 nm (which lies near an isosbestic point for oxidized and reduced Rr) or by using apoovotransferrin as Fe³⁺ acceptor, and measuring the rate and extent of diferric transferrin formation at 460 nm. No polyironoxyhydroxide aggregates appeared to associate with Rr after the ferroxidase reaction. A truncated form of Rr containing only the diiron domain had little or no ferroxidase activity. Rr could function as one component of a set of enzymes which channels the reaction products of O₂ and Fe²⁺ onto a non-toxic pathway during transient exposure of the bacteria to air.     

Competitive binding of Fe<Superscript>3+</Superscript>, Cr<Superscript>3+</Superscript>, and Ni<Superscript>2+</Superscript> to transferrin

Competitive binding of Fe³⁺, Cr³⁺, and Ni²⁺ to transferrin (Tf) was investigated at various physiological iron to Tf concentration ratios. Loading percentages for these metal ions are based on a two M ⁿ⁺ to one Tf (i.e., 100% loading) stoichiometry and were determined using a particle beam/hollow cathode–optical emission spectroscopy (PB/HC-OES) method. Serum iron concentrations typically found in normal, iron-deficient, iron-deficient from chronic disease, iron-deficient from inflammation, and iron-overload conditions were used to determine the effects of iron concentration on iron loading into Tf. The PB/HC-OES method allows the monitoring of metal ions in competition with Fe³⁺ for Tf binding. Iron-overload concentrations impeded the ability of chromium (15.0 μM) or nickel (10.3 μM) to load completely into Tf. Low Fe³⁺ uptake by Tf under iron-deficient or chronic disease iron concentrations limited Ni²⁺ loading into Tf. Competitive binding kinetic studies were performed with Fe³⁺, Cr³⁺, and Ni²⁺ to determine percentages of metal ion uptake into Tf as a function of time. The initial rates of Fe³⁺ loading increased in the presence of nickel or chromium, with maximal Fe³⁺ loading into Tf in all cases reaching approximately 24%. Addition of Cr³⁺ to 50% preloaded Fe³⁺–Tf showed that excess chromium (15.0 μM) displaced roughly 13% of Fe³⁺ from Tf, resulting in 7.6 ± 1.3% Cr³⁺ loading of Tf. The PB/HC-OES method provides the ability to monitor multiple metal ions competing for Tf binding and will help to understand metal competition for Tf binding.     

An in vitro study on the binding of Al(III) to human serum transferrin with the isoelectric focusing technique

Transferrin saturated with Al³⁺ subjected to isoelectric focusing (IEF) in a pH gradient can be separated into four fractions, representing the apotransferrin, transferrin with aluminum at the metal binding site in the C- or N-terminal lobe, or both. The electrophoretic mobilities of these four fractions are identical to those of the iron-transferrin counterparts. Simultaneous binding of aluminum and iron to transferrin can also be demonstrated. The decreased saturation after IEF indicates that the affinity of transferrin for aluminum is low compared with its affinity for iron. This effect is particularly evident when bicarbonate is used as the synergistic anion in the loading procedure. In contrast, loading of transferrin with aluminum in the presence of oxalate produces a di-aluminum-transferrin complex that is stable during IEF.     

Iron Autoxidation in Mops and Hepes Buffers

Iron autoxidation in Mops and Hepes buffers is characterized by a lag phase that becomes shorter with increasing FeCl₂ concentration and pH. During iron oxidation in these buffers a yellow colour develops in the solution. When the reaction is conducted in the presence of nitro blue tetrazolium (NBT), blue formazan is formed. Of the many OH' scavengers tested, mannitol and sorbitol are most effective in inhibiting Fe²⁺ oxidation, yellow colour development and NBT reduction. Some inhibition was also noted with catalase. The iron product of the oxidative reaction differs from Fe³⁺ in its absorption spectrum and its low reactivity with thiocyanate. Similar results are obtained when iron autoxidation is studied in unbuffered solutions brought to alkaline pH with NaOH. In phosphate buffer, no lag phase is evident and the absorption spectrum of the final solution is identical to that of Fe³⁺ in this buffer. The iron product reacts immediately with thiocyanate. When iron oxidation is conducted in the presence of NBT the formation of formazan is almost undetectable. Of the many compounds tested only catalase inhibits iron autoxidation in this buffer. The sequence of reactions leading to iron autoxidation in Good-type buffers¹ thus resembles that occurring in unbuffered solutions brought to alkaline pH with NaOH and greatly differs from that occurring in phosphate buffer. These results are in agreement with the observation that these buffers have very low affinity for iron.¹ The data presented define experimental conditions where Fe²⁺ is substantially stable for a considerable length of time in Mops buffer.     

Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

Chemical evolution 40. clay-mediated oxidation of diaminomaleonitrile

Summary The previously reported inhibition of the oligomerization of HCN by montmorillonite clays was investigated. The inhibition is due to the oxidation of diaminomaleonitrile (DAMN) by the Fe³⁺ in the clay lattice. Fe²⁺ and oxalic acid were shown to be the reaction products. From these reaction products and the previous report that two equivalents of HCN are formed per equivalent ofDAMN, it was established that diiminosuccinonitrile (DISN) is the initial reaction product, which is rapidly hydrolyzed to oxalic acid and HCN. The same oxidative transformations are effected by Fe³⁺ bound to Dowex 50, Fe³⁺ in solution and Ni(NH₃)₆ ²⁺. The rate of reaction of DAMN decreased in the order Fe³⁺ > Fe³⁺-Dowex > montmorillonite, indicating no catalytic role for the clay in the oxidation of DAMN. Little reaction of DAMN was observed with montmorillonite in which the bulk of the iron was in the Fe²⁺ oxidation state. The possible significance of these redox reactions to chemical evolution is discussed.For the previous papers in this series see Ferris JP, Alwis KW, Edelson EH, Mount N, Hagan Jr J (1980) Origin of Life Wolman Y (ed) Reidel, Dordrecht, p 125–128 Ferris JP, Edelson EH, Auyeung JM, Joshi PC (1981) J Mol Evol 17:69-77     

Competitive Inhibition of Ferrous Iron Oxidation by Thiobacillus ferrooxidans by Increasing Concentrations of Cells

The oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺) with dioxygen (O₂) by various strains of Thiobacillus ferrooxidans was studied by measuring the rate of O₂ consumption at various Fe²⁺ concentrations and cell concentrations. The apparent K_m values for Fe²⁺ remained constant at different cell concentrations of laboratory strains ATCC 13661 and ATCC 19859 but increased with increasing cell concentrations of mine isolates SM-4 and SM-5. The latter results are explained by the competitive inhibition of the Fe²⁺-binding site of a cell by other cells in the reaction mixture. Possible mechanisms involving cell surface properties are discussed.     

Concanavalin A, a receptor protein with apparently co-operative binding characteristics.

Laser nephelometry is a suitable technique for the quantitative determination and differentiation of both lectins and glycoconjugates in the low-picomolar range. Simultaneously this method renders possible investigations on the specificity and mode of interaction between lectins and different ligands. The results demonstrate that the degree of co-operativity between concanavalin A and the respective glycoconjugate is dependent on the presence of hydrophobic binding sites and can be substantially altered by conformational changes of the ligand. The transition from apotransferrin to Fe3+-transferrin induces a transformation of the sigmoidal-shaped binding curve to a hyperbolic one. Hence, at low concentrations, Fe3+-transferrin is bound far better than apotransferrin, whereas maximal binding is nearly identical. After removal of N-acetylneuraminate, concanavalin A is less efficient in differentiating between the Fe3+-charged and Fe3+-free (apo) forms of transferrin.     

Metal binding to the tetrathiolate motif of desulforedoxin and related polypeptides

 Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)₄ metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin mutant proteins were reconstituted with Fe³⁺, Cd²⁺, and Zn²⁺ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe³⁺ bound with the highest affinity, whereas the affinity of Zn²⁺ was greater than Cd²⁺ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe³⁺ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe³⁺ is reduced to Fe²⁺ with attendant oxidation of ligand thiols. Received: 17 June 1998 / Accepted: 3 September 1998     

Biogeochemical Properties of Bacteriogenic Iron Oxides

Bacteriogenic iron oxides (BIOS) are composite materials that consist of intact and partly degraded remains of bacterial cells intermixed with variable amounts of poorly ordered hydrous ferric oxide (HFO) minerals. They form in response to chemical or bacterial oxidation of Fe²⁺, which gives rise to Fe³⁺. Once formed, Fe³⁺ tends to undergo hydrolysis to precipitate in association with bacterial cells. In acidic systems where the chemical oxidation of Fe²⁺ is slow, bacteria are capable of accelerating the reaction by several orders of magnitude. At circumneutral pH, the chemical oxidation of Fe²⁺ is fast. This requires Fe²⁺ oxidizing bacteria to exploit steep redox gradients where low pO₂ slows the abiotic reaction enough to allow the bacteria to compete kinetically. Because of their reactive surface properties, BIOS behave as potent sorbents of dissolved metal ions. Strong enrichments of Al, Cu, Cr, Mn, Sr, and Zn in the solid versus aqueous phase (log 10 K_d values range from 1.9 to 4.2) are common; however, the metal sorption properties of BIOS are not additive owing to surface chemical interactions between the constituent HFO and bacteria. These interactions have been investigated using acid-base tritrations, which show that the concentration of high pK_a sites is reduced in BIOS compared to HFO. At the same time, hydroxylamine insoluble material (i.e., residual bacterial fraction) is enriched in low pK_a sites relative to both BIOS and HFO. These differences indicate that low pK_a or acidic sites associated with bacteria in BIOS interact specifically with high pK_a or basic sites on intermixed HFO.