Fur Protein Research Articles

The ferric uptake regulation (Fur) repressor is a zinc metalloprotein.

The Fur protein regulates the expression of a wide variety of iron-responsive genes; however, the interaction of this repressor with its cognate metal ion remains controversial. The iron-bound form of Fur has proved difficult to obtain, and conflicting results have been published using Mn(II) as a probe for in vitro DNA-binding studies. We report here that the purified protein contains tightly bound zinc and propose that Zn(II) is bound to the protein in vivo. Upon purification, Fur retains ca. 2.1 mol of Zn(II)/mol of Fur monomer (Zn2Fur). One zinc is easily removed by treatment of Zn2Fur with zinc chelating agents, resulting in Zn1Fur with ca. 0.9 mol of Zn(II)/mol of protein. The remaining zinc in Zn1Fur can only be removed under denaturing conditions to yield apo-Fur with ca. 0.1 mol of Zn(II)/mol of protein. Our results suggest that many literature descriptions of purified Fur protein do not correspond to the apo-protein, but to Zn1Fur or Zn2Fur. Dissociation constants (Kd) of protein-DNA complexes are ca. 20 nM for both Zn2Fur and Zn1Fur as determined by electrophoretic mobility shift assays and DNase I footprinting assays. The two metalated forms, however, show qualitative differences in the footprinting assays while apo-Fur does not bind specifically to the operator. The existence of these Zn(II) binding sites in Fur may resolve some discrepancies in the literature and have implications concerning Zur, a Fur homologue in E. coli that regulates zinc-responsive genes.

Spectroscopic and saturation magnetization properties of the manganese- and cobalt-substituted Fur (ferric uptake regulation) protein from Escherichia coli.

The Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These samples have been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetization measurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. A saturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 and Co(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (|J| < 0.1 cm-1 ). The UV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and 540(br) nm with epsilon540 nm = 65 M-1 cm-1. The EPR spectrum gives the following g values: gx = 5.0(5), gy = 4.0(2), and gz = 2. 3(1), which are in accordance with a nearly axial (E/D < 0.11) site. The value of 55 cm-1 for the splitting (Delta) between the ground and the first excited state has been derived from an EPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicate a metal environment comprising five nitrogen/oxygen atoms at 2.11 A, the absence of sulfur, and the presence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeable signals coming from histidine NH protons and shows the signature of carboxylate group(s). The combined spectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least two histidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distorted octahedral environment.

Iron regulation and pathogenicity in Erwinia chrysanthemi 3937: role of the Fur repressor protein.

Low iron availability is a triggering signal for coordinated expression of the genes encoding pectate lyases PelB, PelC, PelD, and PelE, and chrysobactin iron transport functions, which are two main determinants of phytopathogenicity of the Erwinia chrysanthemi strain 3937. The possible implication of the ferric uptake regulation (Fur) protein in this process was investigated. The E. chrysanthemi fur gene was cloned by functional complementation of an Escherichia coli fur mutant and sequenced. The 444-bp open reading frame identified was found to code for a protein highly similar to the E. coli Fur regulator. An E. chrysanthemi fur null mutant was constructed by reverse genetics. This mutant showed altered growth capacity and reduced pathogenicity on African violets. In a fur background, transcriptional lacZ fusions to genes belonging to the E. chrysanthemi high affinity iron transport systems were constitutively expressed. Transcription of the pelA, pelD, and pelE genes was analyzed, using fusions to the uidA reporter gene. Iron availability and a fur mutation did not influence the expression of pelA. In the presence of iron, pelD and pelE transcription levels were higher in the fur mutant than in the parental strain. Furthermore, iron deficiency stimulated the expression of both fusions in the fur mutant. These findings indicate that, in E. chrysanthemi 3937, (i) Fur negatively controls iron transport and genes encoding PelD and PelE, and (ii) additional factor(s) mediate iron regulation of the pel genes.

Characterization of the Acinetobacter baumannii Fur regulator: cloning and sequencing of the fur homolog gene.

Growth kinetics, siderophore activity and iron-regulated bacterial proteins of Acinetobacter baumannii BM2580 were studied in iron-restricted and iron-supplemented chemically defined media. Iron-regulated outer membrane proteins of 75 kDa and 80 kDa were expressed under iron-restricted conditions. Cloning and sequencing of the complete iron-uptake regulatory (fur) gene from A. baumannii BM2580 is reported for the first time. This gene is preceded by a single autoregulated promoter whose -10 region overlaps the Fur binding site. The open reading frame identified encodes a polypeptide consisting of 145 amino acids. The fur gene is followed by a divergent open reading frame coding for the C-terminus of a putative PilU protein. Sequence analysis indicates that the Fur protein of A. baumannii was 63% identical to the Escherichia coli Fur protein.

Characterization of the Acinetobacter baumannii Fur regulator: cloning and sequencing of the fur homolog gene

Growth kinetics, siderophore activity and iron-regulated bacterial proteins of Acinetobacter baumannii BM2580 were studied in iron-restricted and iron-supplemented chemically defined media. Iron-regulated outer membrane proteins of 75 kDa and 80 kDa were expressed under iron-restricted conditions. Cloning and sequencing of the complete iron-uptake regulatory ( fur) gene from A. baumannii BM2580 is reported for the first time. This gene is preceded by a single autoregulated promoter whose −10 region overlaps the Fur binding site. The open reading frame identified encodes a polypeptide consisting of 145 amino acids. The fur gene is followed by a divergent open reading frame coding for the C-terminus of a putative PilU protein. Sequence analysis indicates that the Fur protein of A. baumannii was 63% identical to the Escherichia coli Fur protein.

Identification of fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iron by combined two-dimensional gel electrophoresis and mass spectrometry.

Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 microM) and high-iron (70 microM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance.

Characterization of the Interaction between Fur and the Iron Transport Promoter of the Virulence Plasmid in Vibrio anguillarum

The expression of iron transport genes fatDCBA in Vibrio anguillarum strain 775 is negatively regulated by two iron-responsive repressors, the Fur protein and the antisense RNA, RNAalpha. Here we report the identification of the promoter for the iron transport genes and studied the interaction between the V. anguillarum Fur protein and this promoter. The iron transport promoter was localized in a region approximately 300 base pairs upstream of fatD by both primer extension and S1 mapping analysis. High activity of the promoter was measured in response to iron depletion in the wild-type strain when a promoter-lacZ fusion was examined, whereas the promoter was constitutive in the Fur-deficient strain. Gel retardation and DNase I footprint analysis showed that Fur binds specifically to two contiguous sites comprising the promoter region and the region downstream of the transcription start site. The identified Fur binding sites showed a low degree of homology to each other as well as to the consensus sequence for the Escherichia coli Fur protein. DNase I footprints pattern suggested a sequential interaction of Fur with these two sites that renders a protection in the template strand and a hypersensitivity to the nuclease in the nontemplate strand. The periodicity of the hypersensitive sites suggested that the promoter DNA undergoes a structural change upon binding to Fur, which might play a role in the repression of gene expression.

Iron-responsive gene regulation in a campylobacter jejuni fur mutant.

The expression of iron-regulated systems in gram-negative bacteria is generally controlled by the Fur protein, which represses the transcription of iron-regulated promoters by using Fe2+ as a cofactor. Mutational analysis of the Campylobacter jejuni fur gene was carried out by generation of a set of mutant copies of fur which had a kanamycin or chloramphenicol resistance gene introduced into the regions encoding the N and C termini of the Fur protein. The mutated genes were recombined into the C. jejuni NCTC 11168 chromosome, and putative mutants were confirmed by Southern hybridization. C. jejuni mutants were obtained only when the resistance genes were transcribed in the same orientation as the fur gene. The C. jejuni fur mutant grew slower than the parental strain. Comparison of protein profiles of fractionated C. jejuni cells grown in low- or high-iron medium indicated derepressed expression of three iron-regulated outer membrane proteins with molecular masses of 70, 75, and 80 kDa. Characterization by N-terminal amino acid sequencing showed the 75-kDa protein to be identical to CfrA, a Campylobacter coli siderophore receptor homologue, whereas the 70-kDa protein was identified as a new siderophore receptor homologue. Periplasmic fractions contained four derepressed proteins with molecular masses of 19, 29, 32, and 36 kDa. The 19-kDa protein has been previously identified, but its function is unknown. The cytoplasmic fraction contained two iron-repressed and two iron-induced proteins with molecular masses of 26, 55, 31, and 40 kDa, respectively. The two iron-repressed proteins have been previously identified as the oxidative stress defense proteins catalase (KatA) and alkyl hydroperoxide reductase (AhpC). AhpC and KatA were still iron regulated in the fur mutant, suggesting the presence of Fur-independent iron regulation. Further analysis of the C. jejuni iron and Fur regulons by using two-dimensional gel electrophoresis demonstrated the total number of iron- and Fur-regulated proteins to be lower than for other bacterial pathogens.

Binding of the Fur (ferric uptake regulator) repressor of Escherichia coli to arrays of the GATAAT sequence

The mode of DNA binding of the Fur (ferric uptake regulator) repressor which controls transcription of iron-responsive genes in Escherichia coli, has been re-examined. Using as a reference the known sites at the promoter of the aerobactin operon of Escherichia coli, we have compared in detail the patterns of interaction between the purified Fur protein and natural or synthetic DNA targets. DNase I and hydroxyl radical footprinting, as well as missing-T assays, consistently revealed that functional Fur sites are composed of a minimum of three repeats of the hexameric motif GATAAT rather than by a palindromic 19 bp target sequence. Extended binding sites, constructed by stepwise addition of one or two direct repeats of the same sequence, were occupied co-operatively by Fur with the same pattern of interactions as those observed with the core of three repeats. This indicated that functional sites with a range of affinities can be formed by the addition of discrete GATAAT extensions to a minimal recognition sequence. The fashion in which Fur binds its target, virtually unknown in prokaryotic transcriptional regulators, accounts for the observed helical wrapping of the protein around the DNA helix.

Regulation of iron uptake minimizes iron-mediated oxidative stress

Sequential accommodation of single electrons by the unpaired orbitals of dioxygen yields oxygen radicals Open image in new window , hydrogen peroxide (H2O2), hydroxide radicals (OH·), and finally water (H2O). Fe2+ catalyses the formation of the most reactive hydroxide radical from hydrogen peroxide and thus contributes substantially to the toxicity of oxygen. Insolubility of Fe3+ demands the incorporation of iron into transferrin, lactoferrin, ferritin, iron-sulphur clusters, and heme. Bacteria and fungi synthesize low molecular weight compounds, termed siderophores, which are secreted and used to transport Fe3+ into the microbial cells. Iron is economically used and iron toxicity is minimized by the synthesis of siderophores and ferric siderophore transport systems, and by induction of transport gene transcription by certain Fe3+-loaded siderophores. When cells contain sufficient iron, Fe2+-loaded Fur protein and Fe2+-loaded DtxR protein repress gene transcription in Gram-negative bacteria and in most Gram-positive bacteria, respectively. In a recently discovered novel transcription control mechanism, ferric citrate and ferric pseudobactins induce transcription of the iron transport systems by binding to cell surface receptor proteins without entering the cells. Cytoplasmic sigma factors are activated by a signaling device that involves a protein in the outer membrane and a protein in the cytoplasmic membrane. Both proteins extend into the periplasm to transduce the signal through the space between the two membranes. Intracellular iron homeostasis secured by regulation of iron uptake prevents excessive oxidative stress, which could otherwise overcome the cellular defence and repair systems and kill the cells.

Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors.

Fur (ferric uptake regulator) proteins control iron uptake in many Gram-negative bacteria. Although Fur homologues have been identified in Gram-positive bacteria, their roles in gene regulation are unknown. Genome sequencing has revealed three fur homologues in Bacillus subtilis: yqkL, yqfV and ygaG. We demonstrate that yqkL encodes an iron uptake repressor: both siderophore biosynthesis and transcription of ferri-siderophore uptake genes is constitutive in the yqkL mutant. Thus, yqkL encodes a repressor that is functionally as well as structurally related to Fur. B. subtilis peroxide stress genes are induced by either H2O2 or by metal ion limitation. Previous genetic studies defined a regulatory locus, perR, postulated to encode the peroxide regulon repressor. We demonstrate that a ygaG mutant has the perR phenotype: It is highly resistant to peroxides and overexpresses catalase, alkyl hydroperoxide reductase and the DNA binding protein MrgA. Nine spontaneous perR mutations, isolated by virtue of their ability to derepress mrgA transcription in the presence of managanous ion, all contain sequence changes in the ygaG locus and can be complemented by the cloned ygaG gene. Thus, ygaG encodes the peroxide regulon repressor and is allelic with perR.

Coordinated repression in vitro of the divergent fepA-fes promoters of Escherichia coli by the iron uptake regulation (Fur) protein.

The mechanism involved in transcriptional repression of the fepA-fes divergent promoters of Escherichia coli by the Fur (ferric uptake regulation) protein has been examined in vitro. This DNA region includes a suboptimal and single Fur-binding site with two divergent and overlapped -35/-10 hexamers. Comparison of transcription patterns generated with runoff experiments in either the presence or the absence of heparin showed that access of the RNA polymerase to the principal -35/-10 hexamers was fully prevented by Fur-Mn2+ bound to its target site within the divergent promoter region. Neither RNA polymerase bound to the fes and fepA promoters could be displaced by Fur-Mn2+, nor could the bound repressor be outcompeted by an excess of the enzyme. However, the repressor blocked reinitiation as soon as the polymerase moved away from the fes promoter during transcription. The spatial distribution of regulatory elements within the DNA region allowed the simultaneous binding of the RNA polymerase to the fes and fepA promoters and their coordinate regulation regardless of their different transcriptional activities. Comparisons with other iron-regulated systems support a general mechanism for Fur-controlled promoters that implies a direct competition between the polymerase and the regulator for overlapping target sites in the DNA.

Characterization of the cvaA and cvi promoters of the colicin V export system: iron-dependent transcription of cvaA is modulated by downstream sequences.

Secretion of the Escherichia coli toxin colicin V was previously determined to be iron regulated via the Fur (ferric uptake regulator) protein, based on studies in fur mutants. The iron dependence of transcription and expression of cvaA, which encodes a transporter accessory protein, and cvi, encoding the colicin V immunity protein, was assessed under conditions of iron excess or depletion. Immunoblots showed that production of both Cvi and CvaA is iron dependent. The iron-dependent transcriptional start for cvaA identified by primer extension and S1 nuclease analysis, P1, lies 320 bp upstream of the translational start and is associated with a newly identified Fur binding site. Beta-galactosidase activity in transcriptional lacZ fusions with the P1 promoter alone is higher than with downstream sequences present and is induced 10-fold by iron depletion. Including immediate downstream regions with P1 enhances activity from P1 even more but reduces the induction by iron depletion fivefold. Including subsequent downstream sequences, however, down-modulates overall transcription from P1 almost fourfold. Deletion of a long stem-loop structure in this region alleviates the down-modulation by increasing transcription, indicating that the sequences or structure of this element may contribute to this down-regulation. Characterization of the cvi promoter by primer extension showed that it resides where predicted, about 50 bp upstream of cvi associated with a previously identified Fur binding site. The cvi promoter is also inducible by iron depletion. The modulating sequences from cvaA were placed downstream of the cvi promoter to test their effects in transcriptional fusions of the cvi promoter to lacZ. The fusion results showed that these sequences also modulate transcription of the cvi promoter in a manner similar to that of the cvaA promoter. The potential for up- and down-regulation within the long untranslated region downstream of the cvaA promoter suggests a novel mechanism that fine-tunes expression of the colicin V secretion genes.

An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli SAB11.

The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The approximately 21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

Cloning and characterization of the fur gene from Helicobacter pylori

The fur homologue of Helicobacter pylori was isolated by screening a plasmid-based, genomic DNA library using the Fur titration assay (FURTA). The analysis of the DNA sequence revealed significant homology with Fur proteins from various other bacterial species. The highest degree of homology was observed for the Fur protein from Campylobacter jejuni. The H. pylori fur gene on a plasmid could partially complement the fur mutation in Escherichia coli strain H1681. The repressor activity depended on addition of iron to the medium indicating that iron acts as a co-repressor for the H. pylori protein similar to Fur from other bacteria. Comparison of Fur from H. pylori strain NCTC11638 with the recently published genomic DNA sequence of another strain (26695) confirmed the identity of the fur homologue and revealed that the fur locus is highly conserved in both strains.

Cloning and characterization of the fur gene from Helicobacter pylori.

The fur homologue of Helicobacter pylori was isolated by screening a plasmid-based, genomic DNA library using the Fur titration assay (FURTA). The analysis of the DNA sequence revealed significant homology with Fur proteins from various other bacterial species. The highest degree of homology was observed for the Fur protein from Campylobacter jejuni. The H. pylori fur gene on a plasmid could partially complement the fur mutation in Escherichia coli strain H1681. The repressor activity depended on addition of iron to the medium indicating that iron acts as a co-repressor for the H. pylori protein similar to Fur from other bacteria. Comparison of Fur from H. pylori strain NCTC11638 with the recently published genomic DNA sequence of another strain (26695) confirmed the identity of the fur homologue and revealed that the fur locus is highly conserved in both strains.

Metalloregulation in vitro of the aerobactin promoter of Escherichia coli by the Fur (ferric uptake regulation) protein.

The mechanism of transcriptional repression of the aerobactin operon of Escherichia coli by the Fe2+-responsive Fur (ferric uptake regulation) protein has been investigated. In the presence of a divalent metal, such as Mn2+, the Fur protein sequentially occupies two defined sites at the aerobactin promoter region, followed by a looser occupation of upstream DNA sequences. However, binding to the primary target site suffices for the entire repression effect. Comparison of transcription patterns generated with run-off experiments in the presence and absence of heparin showed that access of the RNA polymerase to the principal -35/-10 hexamers of the promoter region was fully prevented by Fur-Mn2+ bound to its primary site. Similarly, promoter-bound RNA polymerase could not be competed out from the DNA even in the presence of a large Fur-Mn2+ excess, although the repressor could immediately bind its target sequence at the region as soon as RNA polymerase moved away from the promoter during transcription. The high affinities of either protein for the promoter produce, in practice, a first-come, first-served effect that helps the system to respond instantly to changes in the iron status of the cells.

SigX of Bacillus subtilis replaces the ECF sigma factor fecI of Escherichia coli and is inhibited by RsiX.

Analysis of the Bacillus subtilis genome sequence revealed two open reading frames, designated sigX and ypuN (now termed rsiX), that are homologous to fecI and fecR, respectively, of Escherichia coli. fecI encodes a sigma 70-type factor that is necessary for transcription of the ferric citrate transport genes fecABCDE. fecR encodes a cytoplasmic transmembrane protein that is required for the induction of fec transport gene transcription by ferric citrate binding to the FecA outer membrane receptor protein. Investigation of the SigX and RsiX activities disclosed that they are not involved in ferric citrate utilization--since ferric citrate did not serve as an iron source for B. subtilis SG64--or in the regulation of any other ferric siderophore transport system tested. Strains deleted for sigX or rsiX displayed no phenotype under aerobic or anoxic conditions. However, cloned sigX complemented an E. coli fecI mutant, and the Fur box upstream of sigX responded to the E. coli iron regulatory protein Fur. The purified SigX protein was required for in vitro transcription of a sigX-containing DNA fragment by the E. coli RNA polymerase core enzyme. Autoregulation of sigX was also found in vivo using a sigX'-lacZ gene fusion. RsiX inhibited SigX activity in vivo and in vitro and stabilized the SigX protein. RsiX was localized in the membrane fraction. When RsiX is present, SigX is found in the membrane fraction; in the absence of RsiX, some SigX is detectable in the cytoplasm. We conclude that SigX is a sigma factor that belongs to the ECF (extracytoplasmic function) sigma 70-factor family. It is not known which promoters are recognized by SigX in B. subtilis. SigX may be involved in the regulation of iron metabolism, as evidenced by its activity in E. coli.

Cloning and sequencing of the Vibrio parahaemolyticus fur gene.

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction-based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.

Electrospray ionization mass spectrometry analysis of the apo- and metal-substituted forms of the Fur protein

Fur has been purified and reconstituted with Co 2+ and Mn 2+. The ESI-MS spectra of the apoprotein as well as Mn-Fur and Co-Fur under acidic denaturating conditions showed the existence of two species of molecular mass 16 660±3 and 16 792±3 Da, which correspond, respectively, to the N-terminal methionine `excised' or `non-excised' forms of the monomer. This result proves the absence of any other post-translational modification or modification due to metal incorporation. On the other hand, under soft conditions, ESI spectra provided for the first time direct evidence for dimeric metal-containing forms in solution.