Differed preferential iron-binding lobe in human transferrin depending on the presence of bicarbonate detected by HPLC/high-resolution inductively coupled plasma mass spectrometry

The binding of iron (Fe) to human serum transferrin (Tf) was analyzed with an HPLC system equipped with an anion exchange column and directly connected with a high-resolution inductively coupled plasma mass spectrometer for metal detection. The (56)Fe level in the eluate was monitored at resolution m/Deltam=3000. Two monoferric Tfs were assigned based on the results of urea-PAGE and desferrioxamine experiments. When Fe was added as Fe-citrate stepwise to an apo-Tf solution in the presence of bicarbonate, the N-lobe site was the preferential Fe-binding site, while the C-lobe site was preferred in the absence of bicarbonate. In both cases, the Fe-peak areas of the preferential site and Fe(2)-Tf increased up to an Fe/Tf molar ratio of 1, and then the peak area of the monoferric Tf decreased while the peak area of Fe(2)-Tf increased. When the Fe/Tf molar ratio was below 1, the amount of Fe bound to the lobe with a weaker affinity was higher in Fe(2)-Tf than in the monoferric Tf in each case. Namely, Fe(2)-Tf was the preferential binding state of Fe to human serum Tf. The preference is reasonable for transferring Fe ions effectively to Tf-receptors.     

The soluble form of the membrane-bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue found particularly in melanoma cells. Apart from membrane-bound MTf, a soluble form of the molecule (sMTf) has been identified in vitro[Food, M.R., Rothenberger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies, W.A. (1994) J. Biol. Chem.269, 3034-3040] and in vivo in Alzheimer's disease. However, nothing is known about the function of sMTf or its role in Fe uptake. In this study, sMTf labelled with 59Fe and 125I was used to examine its ability to donate 59Fe to SK-Mel-28 melanoma cells and other cell types. sMTf donated 59Fe to cells at 14% of the rate of Tf. Analysis of sMTf binding showed that unlike Tf, sMTf did not bind to a saturable Tf-binding site. Studies with Chinese hamster ovary cells with and without specific Tf receptors showed that unlike Tf, sMTf did not donate its 59Fe via these pathways. This was confirmed by experiments using lysosomotropic agents that markedly reduced 59Fe uptake from Tf, but had far less effect on 59Fe uptake from sMTf. In addition, an excess of 56Fe-labelled Tf or sMTf had no effect on 125I-labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells. Protein-free 125I determinations demonstrated that in contrast with Tf, sMTf was markedly degraded. We suggest that unlike the binding of Tf to specific receptors, sMTf was donating Fe to cells via an inefficient mechanism involving nonspecific internalization and subsequent degradation.     

Spectroscopic and magnetic studies of wild-type and mutant forms of the Fe(II)- and 2-oxoglutarate-dependent decarboxylase ALKBH4

The Fe(II)/2OG (2-oxoglutarate)-dependent dioxygenase superfamily comprises proteins that couple substrate oxidation to decarboxylation of 2OG to succinate. A member of this class of mononuclear non-haem Fe proteins is the Escherichia coli DNA/RNA repair enzyme AlkB. In the present work, we describe the magnetic and optical properties of the yet uncharacterized human ALKBH4 (AlkB homologue). Through EPR and UV-visible spectroscopy studies, we address the Fe-binding environment of the proposed catalytic centre of wild-type ALKBH4 and an Fe(II)-binding mutant. We could observe a novel unusual Fe(III) high-spin EPR-active species in the presence of sulfide with a g(max) of 8.2. The Fe(II) site was probed with NO. An intact histidine-carboxylate site is necessary for productive Fe binding. We also report the presence of a unique cysteine-rich motif conserved in the N-terminus of ALKBH4 orthologues, and investigate its possible Fe-binding ability. Furthermore, we show that recombinant ALKBH4 mediates decarboxylation of 2OG in absence of primary substrate. This activity is dependent on Fe as well as on residues predicted to be involved in Fe(II) co-ordination. The present results demonstrate that ALKBH4 represents an active Fe(II)/2OG-dependent decarboxylase and suggest that the cysteine cluster is involved in processes other than Fe co-ordination.     

The role of the membrane-bound tumour antigen, melanotransferrin (p97), in iron uptake by the human malignant melanoma cell.

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue with several characteristics in common with serum Tf. MTf is found at high levels in melanoma cells and previous studies have shown that MTf can bind Fe. In addition, Chinese hamster ovary cells transfected with MTf transport Fe from 59Fe-citrate at greater rates than control cells. However, the role of MTf in the Fe uptake process of human melanoma cells remains unknown. In the present study we have characterized the role of MTf in Fe uptake by SK-Mel-28 melanoma cells in order to understand its function. Initial studies examined whether modulation of intracellular Fe levels using the Fe chelator desferrioxamine (DFO) or the Fe donor ferric ammonium citrate (FAC) could change MTf mRNA levels. In contrast to transferrin receptor (TfR) mRNA that increased after exposure to DFO and decreased after incubation with FAC, there was no change in MTf mRNA levels. In addition, compared to control cells, there was no alteration of 125I-labelled anti-MTf mAb-binding in cells exposed to DFO or FAC, suggesting no change in the number of MTf sites. Further studies examined the ability of DFO and FAC to modulate Fe uptake from 59Fe-citrate which is bound by MTf. In contrast to the effect of DFO or FAC at increasing and decreasing Fe uptake from 59Fe-Tf, respectively, DFO had no influence on 59Fe-citrate uptake, whereas FAC markedly increased it. Collectively, these studies suggest that MTf is not regulated in a manner similar to the TfR in response to cellular Fe levels. MTf can be removed from the membrane by phosphatidylinositol-specific phospholipase C (PtdIns-PLC). Preincubation of melanoma cells with PtdIns-PLC reduced anti-MTf mAb binding to 3% of the control, while PtdIns-PLC only slightly reduced 59Fe uptake from 59Fe-citrate. These results suggest that MTf played only a minor role in Fe uptake from 59Fe-citrate by these cells. The expression of MTf mRNA (poly A+) was also examined in 50 human tissues and found to be markedly different to Tf mRNA or TfR mRNA. Surprisingly, MTf mRNA expression was widespread in normal tissues, and was observed at its highest levels in the salivary gland. In contrast to expectations, MTf mRNA expression was generally greater in adult than fetal tissues.     

Binding of cadmium(II) and zinc(II) to human and dog serum albumins. An equilibrium dialysis and 113Cd-NMR study.

The binding of Cd(II) and Zn(II) to human serum albumin (HSA) and dog serum albumin (DSA) has been studied by equilibrium dialysis and 113Cd(II)-NMR techniques at physiological pH. Scatchard analysis of the equilibrium dialysis data indicate the presence of at least two classes of binding sites for Cd(II) and Zn(II). On analysis of the high-affinity class of sites, HSA is shown to bind 2.08 +/- 0.09 (log K = 5.3 +/- 0.6) and 1.07 +/- 0.12 (log K = 6.4 +/- 0.8) moles of Cd(II) and Zn(II) per mole of protein, respectively. DSA bound 2.02 +/- 0.19 (log K = 5.1 +/- 0.8), and 1.06 +/- 0.15 (log K = 6.0 +/- 0.2) moles of Cd(II) and Zn(II) per mole of protein, respectively. Competition studies indicate the presence of one high-affinity Cd(II) site on both HSA and DSA that is not affected by Zn(II) or Cu(II), and one high-affinity Zn(II) site on both HSA and DSA that is not affected by Cd(II) or Cu(II). 113Cadmium-HSA spectra display three resonances corresponding to three different sites of complexation. In site I, Cd(II) is most probably coordinated to two or three histidyl residues, site II to one histidyl residue and three oxygen ligands (carboxylate), while for the most upfield site III, four oxygens are likely to be involved in the binding of the metal ion. The 113Cd(II)-DSA spectra display only two resonances corresponding to two different sites of complexation. The environment around Cd(II) at sites I and II on DSA is similar to sites I and II, respectively, on HSA. No additional resonances are observed in any of these experiments and in particular in the low field region where sulfur coordination occurs. Overall, our results are consistent with the proposal that the physiologically important high-affinity Zn(II) and Cd(II) binding sites of albumins are located not at the Cu(II)-specific NH2-terminal site, but at internal sites, involving mostly nitrogen and oxygen ligands and no sulphur ligand.     

Metal binding activity of the Escherichia coli hydrogenase maturation factor HypB

The formation of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hydrogenase pleiotropy operon hypABCDEF. The insertion of Ni(II) into the precursor enzyme follows the incorporation of the iron center and is the function of HypA, a Zn(II)-binding protein, and HypB, a GTPase. The Ni(II) donor and the mechanism of transfer of Ni(II) into the hydrogenase precursor protein are not known. In this study, we demonstrate that HypB is a nickel-binding protein capable of binding 1 equiv of Ni(II) with a K(d) in the sub-picomolar range. In addition, HypB has a weaker metal-binding site that is not specific for Ni(II) over Zn(II). Examination of the isolated C-terminal GTPase domain revealed that the high-affinity metal binding capability was severely abrogated but the low-affinity site was intact. By mutating conserved cysteine and histidine residues in E. coli HypB, we have localized the high-affinity Ni(II)-binding site to an N-terminal CXXCGC motif and the low-affinity metal-binding site to the GTPase domain. A model for the function of HypB during the Ni(II) loading of hydrogenase is proposed.     

Membrane-bound transferrin-like protein (MTf): structure, evolution and selective expression during chondrogenic differentiation of mouse embryonic cells.

Mouse membrane-bound transferrin-like protein (MTf) cDNA was cloned to examine its expression during chondrogenic differentiation in the mouse embryonic cell line ATDC5, and to analyze the phylogenetic relationships among the MTfs of four animal species and 23 other transferrin members. Phylogenetic analysis indicated that the MTf gene diverged from the common ancestor gene earlier than the genes of the other transferrins such as serum transferrin, lactoferrin and ovotransferrin, and that the divergence occurred after the divergence of vertebrates and invertebrates. MTf, as well as the other transferrins, consists of two repeated domains. The similarity between the N-terminal and the C-terminal domains of MTf is much higher than that of the other transferrins, although the five amino acid residues required for iron binding were not conserved in the C-terminal domain of MTf in contrast to the conservation of these residues in both domains of the other transferrins. Among various adult mouse tissues, MTf mRNA was expressed at the highest level in cartilage and at a moderate level in the testis. MTf mRNA was expressed only at very low levels in the brain, spleen, thymus, muscle, lung, skin and intestine, and hardly detected in the heart, kidney, stomach and liver. In cultures of the mouse ATDC5 cell line, MTf is developmentally expressed in parallel with the expression of type II collagen and aggrecan, in the pattern commensurate with the onset of chondrogenesis to form cartilage nodules. The structural characteristics and the expression pattern suggest that during development and in adult tissues, MTf has some functions that are different from those of other transferrins.     

Kinetics and mechanisms of the oxidation of myoglobin by Fe(III) and Cu(II) complexes

Two distinct mechanisms by which sperm whale myoglobin reduces, respectively, complexes of Fe(III) and Cu(II) and, in turn, is oxidized to metmyoglobin have been characterized. For both mechanisms, deoxymyoglobin is the active reductant. An outer sphere electron transfer, probably at the edge of the heme, is involved for Fe(III)NTA (NTA is nitrilotriacetic acid). This pathway does not involve ionic binding of the Fe(III) complex to the protein. The most reactive species of Fe(III)NTA is uncharged. No inhibition is observed with Ni(II) or Zn(II). An outer sphere site specific electron transfer is operative for reduction of Cu(II) complexes. The site has been characterized using NMR spectroscopy and involves one or more histidines. There is an initial binding of the Cu(II) chelate. The ternary complex of chelator-Cu(II)-deoxymyoglobin is a mandatory intermediate. Ni(II) and Zn(II) compete with Cu(II) for the binding site. A scheme for the participation of either or both of these mechanisms in reduction reactions of heme proteins is proposed. Both the overall redox potential, delta E0, and the stability constant for the ternary complex, K, govern the pathway and the reaction rate.     

Fe(III) and Cu(II) conalbumin visible circular dichroism spectra.

The single polypeptide chain of conalbumin strongly binds two Fe(III) or two Cu(II) ions to yield intense absorption in the visible region similar to that shown by the related protein transferrin. Comparison of the metal-ion-binding sites in the two proteins is made by exploiting the sensitivity to ligand geometry of circular dichroism (CD). For the Fe(III) proteins strong similarities of the CD spectra outweigh marginal differences. For Cu(II) conalbumin an additional negative extremum near 506 nm appears between two positive ones at 634 and 410 nm suggesting greater subtraction of oppositely signed CD components leading to lesser magnitudes for the two positive peaks than are found in Cu(II)-transferrin. The two Fe(III)-binding sites within conalbumin are compared by noting the strong similarities of the CD and MCD of proteins with Fe(III) in one site and Ga(III) in the other site, and vice versa, with the protein containing Fe(III) in both sites. Due to features of the amino acid sequences of the single protein chains, the four strong metal ion binding sites in conalbumin and transferrin cannot be identical in all particulars, yet CD spectra of their metal ion complexes are closely similar. From a study of model phenolate complexes and the wavelength maxima of visible absorption in the Fe(III), Cu(II), and Co(III) proteins near 465, 440, and 405 nm, respectively, these strong absorption bands are identified as ligand to metal ion electron-transfer transitions. It is suggested that tyrosyl residues are the donors in the electron transfer transitions and that they lock in the metal ions after being keyed into position by binding of bicarbonate or other anions.     

Iron uptake by melanoma cells from the soluble form of the transferrin homologue,melanotransferrin

Melanotransferrin (MTf) is a membrane-bound transferrin (Tf) homologue that can also exist in a soluble form (sMTf). Considering the high homology of MTf to Tf, it is possible to suggest that sMTf could bind to the high affinity transferrin receptor 1 (TfR1) or lower affinity TfR2. We have used sMTf labelled with 59Fe to examine its ability to donate Fe to cells. Our experiments demonstrate that sMTf is far less effective than Tf at donating Fe to cells and this does not occur via specific receptors. Indeed, the uptake of sMTf by cells occurred via a non-specific process (e.g. adsorptive pinocytosis).     

pH Dependence of the Efficiency of Binding of Iron Cations to the Donor Side of Photosystem II

Light-induced interaction of Fe(II) cations with the donor side of Mn-depleted photosystem II (PS II(–Mn)) results in the binding of iron cations and blocking of the high-affinity (HA_Z) Mn-binding site. The pH dependence of the blocking was measured using the diphenylcarbazide/2,6-dichlorophenolindophenol test. The curve of the pH dependence is bell-shaped with pK ₁ = 5.8 and pK ₂ = 8.0. The pH dependence of the O₂-evolution mediated by PS II membranes is also bellshaped (pK ₂ = 7.6). The pH dependence of the process of electron donation from exogenous donors in PS II(–Mn) was studied to determine the location of the alkaline pH sensitive site of the electron transport chain. The data of the study showed that the decrease in the iron cation binding efficiency at pH > 7.0 during blocking was determined by the donor side of the PS II(–Mn). Mössbauer spectroscopy revealed that incubation of PS II(–Mn) membranes in a buffer solution containing ⁵⁷Fe(II) + ⁵⁷Fe(III) was accompanied by binding only Fe(III) cations. The pH dependence of the nonspecific Fe(III) cation binding is also described by the same bell-shaped curve with pK ₂ = 8.1. The treatment of the PS II(–Mn) membranes with the histidine modifier diethylpyrocarbonate resulted in an increase in the iron binding strength at alkaline pH. It is suggested that blocking efficiency at alkaline pH is determined by competition between OH^– and histidine ligand for Fe(III). Because the high-affinity Mn-binding site contains no histidine residue, this fact can be regarded as evidence that histidine is located at another (other than high-affinity) Fe(III) binding site. In other words, this means that the blockage of the high-affinity Mn-binding site is determined by at least two iron cations. We assume that inactivation of oxygen-evolving complex and inhibition of photoactivation in the alkaline pH region are also determined by competition between OH^– and a histidine residue involved in coordination of manganese cation outside the high-affinity site.     

Synthesis and properties of different metal complexes of the siderophore desferriferricrocin

Desferriferricrocin is a cyclic hexa-peptide siderophore with three hydroxamates as primary coordination groups. It forms metal complexes with Fe(III), Cr(III), Al(III), Ga(III), Cu(II), and Zn(II). These complexes were prepared and characterized using UV–vis, circular dichroism spectroscopy (CD), nuclear magnetic resonance spectroscopy (NMR), and electrospray ionization mass spectroscopy (ESI-MS). The mononuclear trivalent metal complexes of desferriferricrocin were stable in aqueous solutions, and their coordination centers primarily adopted the Λ configuration. The formation of multinuclear complexes of desferriferricrocin was determined by ESI-MS. Desferriferricrocin was able to bind up to three Cu(II) and two Zn(II) respectively. Heteronuclear complexes containing one trivalent and one divalent were also determined. In these complexes, amide nitrogens were utilized as alternative binding groups of desferriferricrocin in addition to the primary binding groups, the hydroxamates. Published online December 2004     

Effects of overexpression of membrane-bound transferrin-like protein (MTf) on chondrogenic differentiation in Vitro

Membrane-bound transferrin-like protein (MTf) is expressed in parallel with the expression of cartilage-characteristic genes during differentiation of chondrocytes, and the MTf level is much higher in cartilage than in other tissues. To investigate the role of MTf in cartilage, we examined the effects of growth factors on MTf expression in mouse prechondrogenic ATDC5 cells and the effect of MTf overexpression on differentiation of ATDC5 and mouse pluripotent mesenchymal C3H10T1/2 cells. In ATDC5 cultures, bone morphogenetic protein-2 and transforming growth factor-beta as well as insulin induced MTf mRNA expression when these peptides induced chondrogenic differentiation. Forced expression of rabbit MTf in ATDC5 cells induced aggrecan, type II collagen, matrilin-1, type X collagen mRNAs, and cell-shape changes from fibroblastic cells to spherical chondrocytes. Accordingly, the synthesis and accumulation of proteoglycans were higher in MTf-expressing cultures than in control cultures. These effects of MTf overexpression correlated with the MTf protein level on the cell surface and decreased in the presence of anti-MTf antibody. However, the aggrecan mRNA level in the ATDC5 cells overexpressing MTf was lower than that in wild type ATDC5 cells exposed to 10 microg/ml insulin. MTf overexpression in C3H10T1/2 cells also induced aggrecan and/or type II collagen mRNA but not the spherical phenotype. These findings suggest that the expression of MTf on the cell surface facilitates the differentiation of prechondrogenic cells, although MTf overexpression alone seems to be insufficient to commit pluripotent mesenchymal cells to the chondrocyte lineage.     

Neither human hephaestin nor ceruloplasmin forms a stable complex with transferrin

Iron homeostasis is essential for maintaining the physiological requirement for iron while preventing iron overload. Cell toxicity is caused by the generation of hydroxyl-free radicals that result from redox reactions involving Fe(II). Multicopper ferroxidases regulate the oxidation of Fe(II) to Fe(III), circumventing the generation of these harmful by-products. Ceruloplasmin (Cp) is the major multicopper ferroxidase in blood; however, hephaestin (Hp), a membrane-bound Cp homolog, was recently discovered and has been implicated in the export of iron from duodenal enterocytes into blood. In the intracellular milieu, it is likely that iron exists as reduced Fe(II), yet transferrin (Tf), the plasma iron transporter, is only capable of binding oxidized Fe(III). Due to the insoluble and reactive nature of free Fe(III), the oxidation of Fe(II) upon exiting the duodenal enterocyte may require an interaction between a ferroxidase and the iron transporter. As such, it has been suggested that as a means of preventing the release of unbound Fe(III), a direct protein-protein interaction may occur between Tf and Hp during intestinal iron export. In the present study, the putative interaction between Tf and both Cp and a soluble form of recombinant human Hp was investigated. Utilizing native polyacrylamide gel electrophoresis, covalent cross-linking and surface plasmon resonance (SPR), a stable interaction between the two proteins was not detected. We conclude that a stable complex between these ferroxidases and Tf does not occur under the experimental conditions used. We suggest alternative models for loading Tf with Fe(III) during intestinal iron export.     

Metal content of metallo-beta-lactamase L1 is determined by the bioavailability of metal ions

In an effort to probe whether the metal content of metallo-beta-lactamase L1 is affected by metal ion bioavailability, L1 was overexpressed as mature protein (M-L1) and full-length (FL-L1) analogues, and the analogues were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of Escherichia coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was overexpressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analogue was obtained. On the other hand, M-L1, lacking the putative leader sequence, was localized in the cytoplasm of E. coli and shown to bind various amounts of Fe and Zn(II), and like FL-L1, the metal content of the resulting enzyme could be affected by the amount of metal ions in the growth medium. L1 was refolded in the presence of Fe, and a dinuclear Fe-containing analogue of L1 was obtained, although this analogue is catalytically inactive. EPR spectra demonstrate the presence of an antiferromagnetically coupled Fe(III)Fe(II) center in Fe-containing L1 and suggest the presence of a Fe(III)Zn(II) center in M-L1. Metal analyses on the cytoplasmic and periplasmic fractions of E. coli showed that the concentration of metal ions in the periplasm is not tightly controlled and increases as the concentration of metal ions in the growth medium increases. In contrast, the concentration of Zn(II) in the cytoplasm is tightly controlled while that of Fe is less so.     

Novel cytotoxic chelators that bind iron(II) selectively over zinc(II) under aqueous aerobic conditions

To achieve cellular iron deprivation by chelation, it is important to develop chelators with selective metal-binding properties. Selectivity for iron has long been the province of certain oxygen-donor chelators such as desferrioxamine, which target Fe(III) and exploit the strength of a relatively ionic Fe(III)-O interaction. We have been studying novel chelators that possess mechanisms to selectively chelate +2 biometals, particularly tachpyr [N,N',N"-tris(2-pyridylmethyl)-1,3,5-cis,cis-triaminocyclohexane] and derivatives from N,N',N"-trialkylation and pyridine ring alkylation. Metal-exchange and metal-binding competition reactions have been conducted at pH 7.4, 37 degrees C and time periods until no further change was observed (generally 24-48 h). Under anaerobic conditions, tachpyr is strongly selective for iron, binding 95+/-5% Fe(II) versus 5+/-5% Zn(II) in the forms [Fe(tachpyr)](2+) and [Zn(tachpyr)](2+) respectively. Under aerobic conditions, tachpyr complexes Fe(II) more effectively than Fe(III), forming iminopyridyl complexes [Fe(tachpyr-ox-n)](2+) (n=2, 4) by O(2)-induced and iron-mediated oxidative dehydrogenation. Complexes [Fe(tachpyr-ox-n)](2+) are also strongly bound forms of iron that are unaffected by an excess of Zn(II) (75 mol zinc:1 mol iron complex). The preference of tachpyr for iron over zinc under aerobic conditions appears to be hindered by oxidation of Fe(II) to Fe(III), such that the proportions bound are 44+/-10% Fe(II) versus 56+/-10% Zn(II), in the respective forms [Fe(tachpyr-ox-n)](2+) and [Zn(tachpyr)](2+). However, upon addition of the reducing agent Na(2)S(2)O(4) that converts Fe(III) to Fe(II), the binding proportions shift to 76+/-10% Fe(II) versus 24+/-10% Zn(II), demonstrating a clear preference of tachpyr for Fe(II) over Zn(II). Iron(II) is in the low-spin state in [Fe(tachpyr)](2+) and [Fe(tachpyr-ox-n)](2+) (n=2, 4), which is a likely cause of the observed selectivity. N-methylation of tachpyr [giving (N-methyl)(3)tachpyr] results in the loss of selectivity for Fe(II), which is attributed to the steric effect of the methyl groups and a resulting high-spin state of Fe(II) in [Fe(N-methyl)(3)tachpyr)](2+). The relationship of chelator selectivity to cytotoxicity in the tach family will be discussed.     

Complexation of desferricoprogen with trivalent Fe, Al, Ga, In and divalent Fe, Ni, Cu, Zn metal ions: effects of the linking chain structure on the metal binding ability of hydroxamate based siderophores

Complexes of the natural siderophore, desferricoprogen (DFC), with several trivalent and divalent metal ions in aqueous solution were studied by pH-potentiometry, UV-Vis spectrophotometry and cyclic voltammetry. DFC was found to be an effective metal binding ligand, which, in addition to Fe(III), forms complexes of high stability with Ga(III), Al(III), In(III), Cu(II), Ni(II) and Zn(II). Fe(II), however, is oxidized by DFC under anaerobic conditions and Fe(III) complexes are formed. By comparing the results with those of desferrioxamine B (DFB), it can be concluded that the conjugated beta-double bond slightly increases the stability of the hydroxamate chelates, consequently increases the stability of mono-chelated complexes of DFC. Any steric effect by the connecting chains arises only in the bis- and tris-chelated complexes. With metal ions possessing a relatively big ionic radius (Cu(II), Ni(II), Zn(II), In(III)) DFC, containing a bit longer chains than DFB, forms slightly more stable complexes. With smaller metal ions the trend is the opposite. Also a notable difference is that stable trinuclear complex, [Cu(3)L(2)], is formed with DFC but not with DFB. Possible bio-relevance of the Fe(II)/Fe(III) results is also discussed in the paper.     

Probing of metal-binding domains of RNA hairpin loops by laser-induced lanthanide(III) luminescence

Direct laser excitation of aqueous Eu(III) bound to specific RNA fragments was used to probe the metal-binding sites of the anticodon loop of tRNA(Phe) from E. coli and of a tetraloop containing a GNRA consensus sequence. Binding of Mg(II) or Eu(III) to either RNA fragment resulted in a higher melting transition, but no global change in structure was observed. Aqueous Eu(III) exhibits a single weak excitation peak at 17273 cm(-1), the intensity of which increased upon addition of the tRNA loop fragment. Analysis of incremental increases in the luminescence intensity upon complexation with the tRNA loop indicated a stoichiometry of one high-affinity Eu(III)-binding site per loop fragment, with a Kd of 1.3 +/- 0.2 microM. Competition experiments between Eu(III) and Mg(II) were consistent with the two metal ions binding to a common site and with an approximately 30-fold lesser affinity of the tRNA loop for Mg(II) than for Eu(III). The rate of luminescence decay following excitation of Eu(III) bound to the tRNA loop corresponded to displacement of up to 4-5 (of a possible 9) waters of hydration on binding to the tRNA loop. By comparison, Eu(III) binds to the DNA analogue of the tRNA loop with an 8-fold lesser affinity and one fewer direct coordination site than to the RNA sequence, suggesting that a 2'OH of RNA is one of the direct ligands. In contrast with the absence of a shift in the excitation peak of aqueous Eu(III) upon formation of the tRNA loop complex, direct excitation of Eu(III) bound to a GNRA tetraloop fragment resulted in a substantially blue-shifted excitation peak (17290 cm(-1)). The tetraloop fragment also has a single Eu(III)-binding site, with a Kd of 12 +/- 3 microM. The bound Eu(III) was competed by Mg(II), although the relative affinity for Mg(II) was approximately 150-450-fold less than that for Eu(III). The Eu(III)-binding site of the tetraloop site is highly dehydrated, with approximately 7 water molecules displaced upon binding by RNA ligands, suggesting that the blue-shift of the excitation peak is the result of Eu(III) specifically bound in a nonpolar site within the GNRA loop structure.