Functional characterization and metal ion specificity of the metal-citrate complex transporter from Streptomyces coelicolor

Secondary transporters of citrate in complex with metal ions belong to the bacterial CitMHS family, about which little is known. The transport of metal-citrate complexes in Streptomyces coelicolor has been investigated. The best cofactor for citrate uptake in Streptomyces coelicolor is Fe(3+), but uptake was also noted for Ca(2+), Pb(2+), Ba(2+), and Mn(2+). Uptake was not observed with the Mg(2+), Ni(2+), or Co(2+) cofactor. The transportation of iron- and calcium-citrate makes these systems unique among the CitMHS family members reported to date. No complementary uptake akin to that observed for the CitH (Ca(2+), Ba(2+), Sr(2+)) and CitM (Mg(2+), Ni(2+), Mn(2+), Co(2+), Zn(2+)) systems of Bacillus subtilis was noted. Competitive experiments using EGTA confirmed that metal-citrate complex formation promoted citrate uptake. Uptake of free citrate was not observed. The open reading frame postulated as being responsible for the metal-citrate transport observed in Streptomyces coelicolor was cloned and overexpressed in Escherichia coli strains with the primary Fe(3+)-citrate transport system (fecABCDE) removed. Functional expression was successful, with uptake of Ca(2+)-citrate, Fe(3+)-citrate, and Pb(2+)-citrate observed. No free-citrate transport was observed in IPTG (isopropyl-beta-d-thiogalactopyranoside)-induced or -uninduced E. coli. Metabolism of the Fe(3+)-citrate and Ca(2+)-citrate complexes, but not the Pb(2+)-citrate complex, was observed. Rationalization is based on the difference in metal-complex coordination upon binding of the metal by citrate.     

Functional characterization and Me ion specificity of a Ca-citrate transporter from Enterococcus faecalis

Secondary transporters of the bacterial CitMHS family transport citrate in complex with a metal ion. Different members of the family are specific for the metal ion in the complex and have been shown to transport Mg(2+)-citrate, Ca(2+)-citrate or Fe(3+)-citrate. The Fe(3+)-citrate transporter of Streptococcus mutans clusters on the phylogenetic tree on a separate branch with a group of transporters found in the phylum Firmicutes which are believed to be involved in anaerobic citrate degradation. We have cloned and characterized the transporter from Enterococcus faecalis EfCitH in this cluster. The gene was functionally expressed in Escherichia coli and studied using right-side-out membrane vesicles. The transporter catalyzes proton-motive-force-driven uptake of the Ca(2+)-citrate complex with an affinity constant of 3.5 microm. Homologous exchange is catalyzed with a higher efficiency than efflux down a concentration gradient. Analysis of the metal ion specificity of EfCitH activity in right-side-out membrane vesicles revealed a specificity that was highly similar to that of the Bacillus subtilis Ca(2+)-citrate transporter in the same family. In spite of the high sequence identity with the S. mutans Fe(3+)-citrate transporter, no transport activity with Fe(3+) (or Fe(2+)) could be detected. The transporter of E. faecalis catalyzes translocation of citrate in complex with Ca(2+), Sr(2+), Mn(2+), Cd(2+) and Pb(2+) and not with Mg(2+), Zn(2+), Ni(2+) and Co(2+). The specificity appears to correlate with the size of the metal ion in the complex.     

A study of stabilization of gluconeogenic activity in rat liver slices by calcium and manganese ions

1. The effect of some bivalent cations on gluconeogenesis by the rat liver-slice preparation has been investigated. 2. Ca(2+) and Mn(2+) stimulated glucose production from a range of substrates but not from glycerol. Mg(2+) had no effect on the rate of glucose production. 3. Ca(2+) were required to maintain phosphoenolpyruvate carboxylase activity in the slice preparation. 4. Ca(2+) and Mn(2+), but not Mg(2+), retarded the release of lysosomal enzymes from the slice into the incubation medium. 5. It is proposed that Ca(2+) and Mn(2+) stimulate glucose production by stabilizing the lysosome system in the liver-slice preparation. 6. The value of the liver-slice preparation as a means of measuring hepatic gluconeogenesis is discussed.     

Effect of metallic cations on the viability of phenol-treated Escherchia coli

Five metallic cations (Fe(3+), Cr(3+), Ca(2+), Mg(2+), Mn(2+); concentration range, 1.85 x 10(-4) to 37 x 10(-4)m) were incorporated individually as chlorides into nutrient broth and agar media used for the recovery of phenol-treated Escherichia coli. The effects observed varied with the concentration and the ionic species. In nutrient agar, Fe(3+) and Cr(3+) were generally beneficial but were toxic at 37 x 10(-4)m. Of the divalent ions tested, Ca(2+) and Mg(2+) usually gave higher counts in nutrient broth, except at a concentration of 9.25 x 10(-4)m, whereas the effect of Mn(2+) was rather variable. Two possible explanations are suggested to explain these effects. Toxic materials may be removed from the media by the precipitates formed on the addition of Fe(3+) or Cr(3+), or, in the case of the divalent ions, the integrity of the bacterial cell membranes may be maintained.     

Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants.

Two different Cd(2+) uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn(2+) uptake system which also takes up Cd(2+) and is induced by Mn(2+) starvation. The calculated K(m) and V(max) are 0.26 microM and 3.6 micromol g of dry cell(-1) min(-1), respectively. Unlike Mn(2+) uptake, which is facilitated by citrate and related tricarboxylic acids, Cd(2+) uptake is weakly inhibited by citrate. Cd(2+) and Mn(2+) are competitive inhibitors of each other, and the affinity of the system for Cd(2+) is higher than that for Mn(2+). The other Cd(2+) uptake system is expressed in Mn(2+)-sufficient cells, and no K(m) can be calculated for it because uptake is nonsaturable. Mn(2+) does not compete for transport through this system, nor does any other tested cation, i.e., Zn(2+), Cu(2+), Co(2+), Mg(2+), Ca(2+), Fe(2+), or Ni(2+). Both systems require energy, since uncouplers completely inhibit their activities. Two Mn(2+)-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn(2+) for growth as the parental strain. Mn(2+) starvation-induced Cd(2+) uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn(2+) or Cd(2+) accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn(2+) and Cd(2+) uptake system.     

Biochemical analysis of the interaction of calcium with toposome: a major protein component of the sea urchin egg and embryo

We have investigated the biochemical and functional properties of toposome, a major protein component of sea urchin eggs and embryos. Atomic force microscopy was utilized to demonstrate that a Ca(2+)-driven change in secondary structure facilitated toposome binding to a lipid bilayer. Thermal denaturation studies showed that toposome was dependent upon calcium in a manner paralleling the effect of this cation on secondary and tertiary structure. The calcium-induced, secondary, and tertiary structural changes had no effect on the chymotryptic cleavage pattern. However, the digestion pattern of toposome bound to phosphatidyl serine liposomes did vary as a function of calcium concentration. We also investigated the interaction of this protein with various metal ions. Calcium, Mg(2+), Ba(2+), Cd(2+), Mn(2+), and Fe(3+) all bound to toposome. In addition, Cd(2+) and Mn(2+) displaced Ca(2+), prebound to toposome, while Mg(2+), Ba(2+), and Fe(3+) had no effect. Collectively, these results further enhance our understanding of the role of Ca(2+) in modulating the biological activity of toposome.     

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

Characterization of Na/Ca exchange in plasmalemmal vesicles from zona fasciculata cells of the bovine adrenal gland

The presence of an Na/Ca exchange system in fasciculata cells of the bovine adrenal gland was tested using isolated plasmalemmal vesicles. In the presence of an outwardly Na(+) gradient, Ca(2+) uptake was about 2-fold higher than in K(+) condition. Li(+) did not substitute for Na(+) and 5 mM Ni(2+) inhibited Ca(2+) uptake. Ca(2+) efflux from Ca(2+)-loaded vesicles was Na(+)-stimulated and Ni(2+)-inhibited. The saturable part of Na(+)-dependent Ca(2+) uptake displayed Michaelis-Menten kinetics. The relationship of Na(+)-dependent Ca(2+) uptake versus intravesicular Na(+) concentration was sigmoid (apparent K(0.5) approximately 24 mM; Hill number approximately 3) and Na(+) acted on V(max) without significant effect on K(m). Na(+)-stimulated Ca(2+) uptake was temperature-dependent (apparent Q(10) approximately 2.2). The inhibition properties of several divalent cations (Cd(2+), Sr(2+), Ni(2+), Ba(2+), Mn(2+), Mg(2+)) were tested and were similar to those observed in kidney basolateral membrane. The above results indicate the presence of an Na/Ca exchanger located on plasma membrane of zona fasciculata cells of bovine adrenal gland. This exchanger displays similarities with that of renal basolateral cell membrane.     

Metal ion requirements for structure and catalysis of an RNA ligase ribozyme

The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5'-nucleotide via a Watson-Crick base pair, forming a 3',5'-phosphodiester bond between its 5'-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. The ligase requires at least five Mg(2+) for activity and has a [Mg(2+)](1/2) of 70-100 mM. It has an unusual specificity for Mg(2+); there is only marginal activity in Mn(2+) and no detectable activity in Ca(2+), Sr(2+), Ba(2+), Zn(2+), Co(2+), Cd(2+), Pb(2+), Co(NH(3))(6)(3+), or spermine. All tested cations other than Mg(2+), including Mn(2+), inhibit the ribozyme. Hill analysis in the presence of inhibitory cations suggested that Ca(2+) and Co(NH(3))(6)(3+) inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca(2+) or Co(NH(3))(6)(3+), as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca(2+), ribozyme that has been prefolded in Ca(2+) can carry out the self-ligation very quickly upon the addition of Mg(2+). Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min(-1) at pH 9.0.     

Zinc ion acts as a cofactor for serine/threonine kinase MST3 and has a distinct role in autophosphorylation of MST3

We examined the metal ion cofactor preference for MST3 (mammalian Ste20-like kinase 3) of the Ste20 serine/threonine kinase family. Four metal ions (Mg(+2), Mn(+2), Zn(2+), and Co(2+)) activate endogenous, exogenous, and baculovirus-expressed recombinant MST3 within the physiological concentration range. In contrast, Fe(+2) and Ca(+2) do not function as MST3 cofactors. Mn(2+), Co(2+), and Mg(2+)-dependent autophosphorylation of MST3 is mainly on threonine residue while Zn(2+)-stimulated MST3 autophosphorylation is on both serine and threonine residues. The distinct autophosphorylation pattern on MST3 suggests that MST3 may exert various types of kinase reactions depending on the type of metal ion cofactor used. To our knowledge, this is the first report showing Zn(2+) as the metal ion cofactor of a recombinant serine/threonine kinase.     

Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron

Recent studies have shown that overexpression of the transmembrane protein Zrt- and Irt-like protein 14 (Zip14) stimulates the cellular uptake of zinc and nontransferrin-bound iron (NTBI). Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of (55)Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe(2+)) over ferric ion (Fe(3+)). Zip14-mediated (55)Fe(2+) uptake was saturable (K(0.5) ≈ 2 μM), temperature-dependent (apparent activation energy, E(a) = 15 kcal/mol), pH-sensitive, Ca(2+)-dependent, and inhibited by Co(2+), Mn(2+), and Zn(2+). HCO(3)(-) stimulated (55)Fe(2+) transport. These properties are in close agreement with those of NTBI uptake in the perfused rat liver and in isolated hepatocytes reported in the literature. Zip14 also mediated the uptake of (109)Cd(2+), (54)Mn(2+), and (65)Zn(2+) but not (64)Cu (I or II). (65)Zn(2+) uptake also was saturable (K(0.5) ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca(2+) dependence of Zn(2+) transport differed from those of Fe(2+) transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.     

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ～1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.     

A Novel Family of Magnesium Transport Genes in Arabidopsis

Magnesium (Mg(2+)) is the most abundant divalent cation in plant cells and plays a critical role in many physiological processes. We describe the identification of a 10-member Arabidopsis gene family (AtMGT) encoding putative Mg(2+) transport proteins. Most members of the AtMGT family are expressed in a range of Arabidopsis tissues. One member of this family, AtMGT1, functionally complemented a bacterial mutant lacking Mg(2+) transport capability. A second member, AtMGT10, complemented a yeast mutant defective in Mg(2+) uptake and increased the cellular Mg(2+) content of starved cells threefold during a 60-min uptake period. (63)Ni tracer studies in bacteria showed that AtMGT1 has highest affinity for Mg(2+) but may also be capable of transporting several other divalent cations, including Ni(2+), Co(2+), Fe(2+), Mn(2+), and Cu(2+). However, the concentrations required for transport of these other cations are beyond normal physiological ranges. Both AtMGT1 and AtMGT10 are highly sensitive to Al(3+) inhibition, providing potential molecular targets for Al(3+) toxicity in plants. Using green fluorescence protein as a reporter, we localized AtMGT1 protein to the plasma membrane in Arabidopsis plants. We suggest that the AtMGT gene family encodes a Mg(2+) transport system in higher plants.     

Complementary metal ion specificity of the metal-citrate transporters CitM and CitH of Bacillus subtilis

Citrate uptake in Bacillus subtilis is stimulated by a wide range of divalent metal ions. The metal ions were separated into two groups based on the expression pattern of the uptake system. The two groups correlated with the metal ion specificity of two homologous B. subtilis secondary citrate transporters, CitM and CitH, upon expression in Escherichia coli. CitM transported citrate in complex with Mg(2+), Ni(2+), Mn(2+), Co(2+), and Zn(2+) but not in complex with Ca(2+), Ba(2+), and Sr(2+). CitH transported citrate in complex with Ca(2+), Ba(2+), and Sr(2+) but not in complex with Mg(2+), Ni(2+), Mn(2+), Co(2+), and Zn(2+). Both transporters did not transport free citrate. Nevertheless, free citrate uptake could be demonstrated in B. subtilis, indicating the expression of at least a third citrate transporter, whose identity is not known. For both the CitM and CitH transporters it was demonstrated that the metal ion promoted citrate uptake and, vice versa, that citrate promoted uptake of the metal ion, indicating that the complex is the transported species. The results indicate that CitM and CitH are secondary transporters that transport complexes of divalent metal ions and citrate but with a complementary metal ion specificity. The potential physiological function of the two transporters is discussed.     

Divalent metal ions tune the self-splicing reaction of the yeast mitochondrial group II intron <Emphasis Type="Italic">Sc</Emphasis>.ai5γ

Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg(2+) to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis-Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5'-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca(2+), Mn(2+), Ni(2+), Zn(2+), Cd(2+), Pb(2+), and [Co(NH(3))(6)](3+) on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5gamma. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg(2+). For example, the presence of only 5% Ca(2+) relative to Mg(2+) results in a decrease in the maximal turnover rate k (cat) by 50%. Ca(2+) thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg(2+) in the folded state, the latter being indicative of at least one specific Ca(2+) binding pocket interfering directly with catalysis. Similar results are obtained with Mn(2+), Cd(2+), and [Co(NH(3))(6)](3+). Ni(2+) is a much more powerful inhibitor and the presence of either Zn(2+) or Pb(2+) leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg(2+) and raises the question of biological relevance at least in the case of Ca(2+).     

Fluorescence study of the interaction of Suwannee River fulvic acid with metal ions and Al3+-metal ion competition

In this study emission and synchronous-scan fluorescence spectroscopy have been used to investigate the interaction of the class A (oxygen seeking 'hard acid') metal Al(3+), with Suwannee River fulvic acid (SRFA), as well as competition between Al(3+) and several other metal ions (Ca(2+), Mg(2+), Cu(2+), Pd(2+), La(3+), Tb(3+) and Fe(3+)) for binding sites on SRFA. Of the four metal ions possessing very similar (and relatively low) ionic indices (Ca(2+), Mg(2+), Cu(2+) and Pd(2+)) only the latter two paramagnetic ions significantly quenched SRFA fluorescence emission intensity. Of the four metal ions possessing very similar (and relatively low) covalent indices (Ca(2+), Mg(2+), La(3+) and Tb(3+)) only the last paramagnetic ion (Tb(3+)) significantly quenched SRFA fluorescence. None of these metals was able to significantly compete with SRFA-bound Al(3+).Fe(3+), which differs substantially from all of the other metals examined in this study in that it possesses a relatively high ionic index (but not as high as Al(3+)) and a relatively low covalent index (but not as low as Al(3+)), was able not only to quench SRFA fluorescence but also to compete (at least to some extent) with SRFA-bound Al(3+). Synchronous-scan fluorescence SRFA spectra taken in the absence and presence of Fe(3+) and/or Al(3+) support the view that these two metal ions can compete for sites on SRFA. The results of these fluorescence experiments further confirm the Al(3+), and metal ions that have electronic properties somewhat similar to Al(3+) (such as Fe(3+)) are somewhat unique in their ability to interact strongly with binding sites on fulvic acids.