Metal-binding characteristics of the amino-terminal domain of ZntA: binding of lead is different compared to cadmium and zinc

ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).     

The cysteine-rich amino-terminal domain of ZntA, a Pb(II)/Zn(II)/Cd(II)-translocating ATPase from Escherichia coli, is not essential for its function.

Soft metal-translocating P1-type ATPases have a distinctive amino-terminal domain that contains one to six copies of the conserved metal-binding motif, GXXCXXC. ZntA from Escherichia coli, a Pb(II)-, Zn(II)-, and Cd(II)-transporting ATPase, has an approximately 120 residue amino-terminal domain with one copy of the GXXCXXC motif as well as four additional cysteine residues. The function of this domain was investigated by constructing a mutant of ZntA lacking the first approximately 100 residues. The mutant, DeltaN-ZntA, was able to confer resistance to Pb(II), Zn(II), and Cd(II) salts, in a manner similar to ZntA. The soft metal dependent ATP hydrolysis activity of purified DeltaN-ZntA was characterized. Purified DeltaN-ZntA and ZntA were both inactivated by oxidation. The K(m) for MgATP was unchanged for DeltaN-ZntA relative to ZntA. DeltaN-ZntA displayed the same metal ion specificity as ZntA. Thiolates increased the activities of both ZntA and DeltaN-ZntA. The V(max) values for DeltaN-ZntA were approximately 3-fold lower than for ZntA for all three metal ions. Thus, the amino-terminal domain is not essential for the function of ZntA or for conferring specificity toward particular soft metals. Its function may be to increase the overall catalytic rate by increasing the rate of metal ion binding to the transporter. Residues involved in the ATP-dependent soft metal ion-translocating mechanism as well as those responsible for recognition of specific metal ions must be part of the core structure of the P1-type ATPases.     

Kinetic analysis of metal binding to the amino-terminal domain of ZntA by monitoring metal-thiolate charge-transfer complexes

ZntA, a P(1B)-ATPase transporter from Escherichia coli, mediates resistance specifically to Pb(2+), Zn(2+), and Cd(2+) by active efflux. ZntA has a hydrophilic N-terminal domain that binds one metal ion. This domain, approximately 120 residues long, contains the GXXCXXC motif that has been shown to be the binding site for metal ions such as Cu(+) and Zn(2+) in P(1B)-type ATPases, and an additional cysteine-rich motif, CCCDGAC. We report here that binding of Pb(2+) and Cd(2+) to this domain produces changes in the absorbance spectrum in the 250-400 nm range indicative of metal-thiolate charge-transfer complexes. The spectral changes indicate that only two cysteines are ligands to Cd(2+), but three or more cysteines are involved in binding Pb(2+); this confirms earlier results that the GXXCXXC sequence is not sufficient to bind Pb(2+), which likely involves residues from the CCCDGAC motif. The absorbance changes were used to measure metal binding kinetics of the N-terminal domain using stopped-flow techniques. Binding was described by simple second-order kinetics with a rate constant, k(on), of approximately 10(6)-10(7) M(-)(1) s(-)(1), at 4 degrees C. The activation energy of binding is similar for both Pb(2+) and Cd(2+); however, the entropy change is greater for Pb(2+). The surprisingly large rate constant for metal binding to the N-terminal domain of ZntA, compared to its low turnover rate, indicates that this step is not rate limiting in the overall transport mechanism. These results, in conjunction with earlier studies, suggest that metal binding to the transmembrane site in ZntA or metal release from the transporter is the slow step in the reaction cycle.     

Conservative and nonconservative mutations of the transmembrane CPC motif in ZntA: effect on metal selectivity and activity

ZntA from Escherichia coli belongs to the P1B-ATPase transporter family and mediates resistance to toxic levels of selected divalent metal ions. P1B-type ATPases can be divided into subgroups based on substrate cation selectivity. ZntA has the highest selectivity for Pb2+, followed by Zn2+ and Cd2+; it also shows low levels of activity with Cu2+, Ni2+, and Co2+. It has two high-affinity metal-binding sites, one each in the N-terminus and the transmembrane domains. Ligands to the transmembrane metal site in ZntA include the cysteine residues of the conserved 392CPC394 motif in the sixth transmembrane helix. Pro393 is invariant in all P-type ATPases. For ZntA homologues with different metal ion selectivity, the cysteines are replaced by serine, histidine, and threonine. To test the effect on activity and metal ion selectivity, single alanine, histidine, and serine substitutions at Cys392 or Cys394 in ZntA were characterized, as well as double substitutions of both cysteines by histidine or serine. P393A was also characterized. C392A, C394A, and P393A lost the ability to bind a metal ion with high affinity in the transmembrane domain. Histidine and serine substitutions at Cys392 and Cys394 resulted in loss of binding of Pb2+ at the transmembrane site, indicating that both cysteines of the CPC motif are required for binding Pb2+ with high affinity in ZntA homologues. However, C392H, C392S, C394H, C394S, C392S/C394S, and C392H/C394H could bind other divalent metal ions at the transmembrane site and retained low but measurable activity. Interestingly, these mutants lost the predominant selectivity for Zn2+ and Cd2+ shown by wtZntA. Therefore, conserved residues contribute to metal selectivity by supplying ligands that bind metal ions not only with high affinity, as for Pb2+, but also with the most favorable binding geometry that results in efficient catalysis.     

Metal-binding affinity of the transmembrane site in ZntA: implications for metal selectivity

ZntA, a P1B-type ATPase, confers resistance specifically to Pb2+, Zn2+, and Cd2 in Escherichia coli. Inductively coupled plasma mass spectrometry measurements show that ZntA binds two metal ions with high affinity, one in the N-terminal domain and another in the transmembrane domain. Both sites can bind monovalent and divalent metal ions. Two proteins, deltaN-ZntA, in which the N-terminal domain is deleted, and C59A/C62A-ZntA, in which the N-terminal metal-binding site is disabled by site-specific mutagenesis, can only bind one metal ion. Because C59A/C62A-ZntA can bind a metal ion at the transmembrane site, the N-terminal domain does not block direct access of metal ions to it from the cytosol. A third mutant protein, C392A/C394A-ZntA, in which cysteines from the conserved CPC motif in transmembrane helix 6 are altered, binds metal ions only at the N-terminal site, indicating that both these cysteines form part of the transmembrane site. The metal affinity of the transmembrane site was determined in deltaN-ZntA and C59A/C62A-ZntA by competition titration using a metal ion indicator and by tryptophan fluorescence quenching. The binding affinity for the physiological substrates, Zn2+, Pb2+, and Cd2+, as well as for the extremely poor substrates, Cu2+, Ni2+, and Co2+, range from 10(6)-10(10) M(-1), and does not correlate with the metal selectivity shown by ZntA. Selectivity in ZntA possibly results from differences in metal-binding geometry that produce different structural responses. The affinity of the transmembrane site for metal ions is of similar magnitude to that of the N-terminal site [Liu J. et al. (2005) Biochemistry 44, 5159-5167]; thus, metal transfer between them would be facile.     

Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.     

A zinc(II)/lead(II)/cadmium(II)-inducible operon from the Cyanobacterium anabaena is regulated by AztR, an alpha3N ArsR/SmtB metalloregulator

A novel Zn(II)/Pb(II)/Cd(II)-responsive operon that consists of genes encoding a Zn(II)/Pb(II) CPx-ATPase efflux pump (aztA) and a Zn(II)/Cd(II)/Pb(II)-specific SmtB/ArsR family repressor (aztR) has been identified and characterized from the cyanobacterium Anabaena PCC 7120. In vivo real time quantitative RT-PCR assays reveal that both aztR and aztA expression are induced by divalent metal ions Zn(II), Cd(II), and Pb(II) but not by other divalent [Co(II), Ni(II)] or monovalent metal ions [Cu(I) and Ag(I)]. The introduction of a plasmid containing the azt operon into a Zn(II)/Cd(II)-hypersensitive Escherichia coli strain GG48 functionally restores Zn(II) and Pb(II) resistance with a limited effect on Cd(II) resistance. Gel mobility shift assays and aztR O/P-lacZ induction experiments confirm that AztR is the metal-regulated repressor of this operon. In vitro biochemical and mutagenesis studies indicate that AztR contains a sole metal-binding site, designated the alpha3N site, that binds Zn(II), Cd(II), and Pb(II) with a high affinity. Optical absorption spectra of Co(II)- and Cd(II)-substituted AztR and (113)Cd NMR spectroscopy of (113)Cd(II)-substituted AztR reveal that the sole alpha3N site in AztR is a CadC-like distorted tetrahedral S(3)(N,O) metal site. The first metal-coordination shell in the AztR alpha3N site differs from other alpha3N family members that sense Cd(II)/Pb(II) and those alpha5 repressors that sense Zn(II)/Co(II). Our results reveal that the alpha3N site in AztR mediates derepression of the azt operon in the presence of Zn(II), as well as Cd(II) and Pb(II); this might have provided Anabaena with an evolutionary advantage to adapt to heavy-metal-rich environments, while maintaining homeostasis of an essential metal ion, Zn(II).     

The ATP hydrolytic activity of purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli

ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity. The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized. The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione. The maximal ATPase activity of ZntA was approximately 3 micromol/(mg x min) obtained with the Pb(II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo. These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Synthesis, spectroscopic, structural and complexation studies of a new tetra-naphthylmethylene pendant-armed macrocyclic ligand

A novel emissive tetra-naphthylmethylene pendant-armed macrocyclic ligand and a series of complexes with monovalent and divalent metal ions have been synthesized. Solid compounds have been isolated as mononuclear (Co(II), Cu(II) and Zn(II)) or dinuclear (Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Ag(I)), complexes, depending on the counterions used. The chemical and photophysical properties of the free ligand, the protonation behavior and its metal complexes have been investigated in solution. UV-Vis spectroscopy has revealed a 1:1 binding stoichiometry for Cu(II), Zn(II), Cd(II), Ni(II) and Co(II), and 2:1 molar ratio for Ag(I). In chloroform, the free ligand presents two emission bands related to the monomer naphthalene emission and a red-shifted band attibutable to an exciplex due to a charge transfer from the nitrogen lone electron pair to the excited chromophore. Upon protonation of the free amines or due to metal complexation, the exciplex band disappears. The crystal structure of [Ag₂L(NO₃)₂] is also reported. The structure reveals that both metal ions are into the macrocyclic cavity in a distorted square plane {AgN₃O} environment. Each Ag(I) atom interacts with two neighbouring amine nitrogen atoms, one pyridine nitrogen and one oxygen atom from a monodentate nitrate ion.     

Metal(II) binding ability of a novel N-protected amino acid. A solution-state investigation on binary and ternary complexes with 2,2'-bipyridine

The binary and ternary systems 2,2'-bipyridine (bpy)-M(II)-NO2psglyH2 (M(II) = Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Pb(II); NO2psglyH2 = N-(2-nitrophenylsulfonyl)glycine) were investigated in aqueous solution by means of potentiometry and electron spectroscopy in order to identify the type, number and stability of the complex species as a function of pH and metal-to-ligand molar ratio. The aim is to evaluate the effect of a substituent on the phenyl ring of the N-sulfonyl amino acids on their coordination properties. The prevailing species in the binary systems is the [ML] (M = Co(II), Ni(II), Cu(II), Cd(II), Pb(II)) where the amino acid molecule is in the dianionic form and coordinates the metal ion through both carboxylic oxygen and deprotonated sulfonamidic nitrogen, while in the Mn(II)- and Zn(II)-containing binary system the only complex species found are those with the amino acid in the monoanionic form. In the ternary 2,2'-bipyridine-containing systems the chelating coordination mode of the dianionic amino acid is maintained with M(II) = Co(II), Ni(II), Cu(II), Cd(II), Pb(II) and the addition of the aromatic base also enables the Zn(II) ion to substitute for the sulfonamide nitrogen-bound hydrogen of NO2psglyH2.     

Biosorption of heavy metals by the exopolysaccharide produced by <Emphasis Type="Italic">Paenibacillus jamilae</Emphasis>

The biosorption of several toxic heavy metals (Pb, Cd, Co, Ni, Zn and Cu) by the exopolysaccharide (EPS) produced by Paenibacillus jamilae, a potential biosorbent for metal remediation and recovery was studied. Firstly, the biochemical composition of this bacterial polymer was determined. Glucose was the most abundant neutral sugar, followed by galactose, rhamnose, fucose and mannose. The polymer presented a high content of uronic acids (28.29%), which may serve as binding sites for divalent cations. The presence of carboxylic groups was also detected by infrared spectroscopy. The EPS presented an interesting affinity for Pb in comparison with the other five metals. Lead biosorption (303.03 mg g⁻¹) was tenfold higher (in terms of mg of metal adsorbed per gram of EPS) than the biosorption of the rest of metals. Biosorption kinetics, the effect of pH and the effect of competitive biosorption were determined. Finally, we found that the EPS was able to precipitate Fe(III), but the EPS-metal precipitate did not form with Fe(II), Pb(II), Cd(II), Co(II), Ni(II), Cu(II) and Zn(II).     

Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Adsorption of heavy metal ions from aqueous solutions by activated carbon prepared from apricot stone

Apricot stones were carbonised and activated after treatment with sulphuric acid (1:1) at 200 degrees C for 24 h. The ability of the activated carbon to remove Ni(II), Co(II), Cd(II), Cu(II), Pb(II), Cr(III) and Cr(VI) ions from aqueous solutions by adsorption was investigated. Batch adsorption experiments were conducted to observe the effect of pH (1-6) on the activated carbon. The adsorptions of these metals were found to be dependent on solution pH. Highest adsorption occurred at 1-2 for Cr(VI) and 3-6 for the rest of the metal ions, respectively. Adsorption capacities for the metal ions were obtained in the descending order of Cr(VI) > Cd(II) > Co(II) > Cr(III) > Ni(II) > Cu(II) > Pb(II) for the activated carbon prepared from apricot stone (ASAC).     

Heavy metal contamination in water,soil, and milk of the industrial area adjacent to Swan River,Islamabad, Pakistan

Trace heavy metals such as Cr(III), Ni(II), Cd(II), Zn(II), Pb(II), and Cu(II) are hazardous pollutants and are rich in areas with high anthropogenic activities. Their concentrations were analyzed using atomic absorption spectroscopy, and it was found that their concentrations were several fold higher in downstream Swan River water samples of the Kahuta Industrial Triangle as compared to upstream. Heavy metal soil concentrations taken from the downstream site were 149% for Cr, 131% for Ni, 176% for Cd, 139% for Zn, 224% for Pb, and 182% for Cu when compared to samples from the upstream site. Quantitative analysis concluded that these metals were higher in milk samples collected from downstream as compared to the samples from upstream water-irrigated sites. The order of metal in milk was as Zn > Cr > Cu > Cd > Pb = Ni. Heavy metal contaminations may affect the drinking water quality, food chain, and ecological environment. It was also suggested that the toxicity due to such polluted water, soil, and milk are seriously dangerous to human health in future.     

Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.