Zinc Enzyme Research Articles

A Galvanizing Story—Protein Stability and Zinc Homeostasis

Zinc is an essential component of protein function in all living cells. The intracellular availability of zinc ions must be balanced against their potential toxicity. Maintaining optimal zinc ion levels requires the integration of zinc enzyme expression with zinc transport. Much of the effort to understand bacterial zinc homeostasis, as well as that of other metals, has focused on transcriptional regulation and the biochemical properties of the relevant transcriptional regulators. Less is known about the translational and posttranslational mechanisms of metal homeostasis in bacteria, although these are clearly important (9–11). The significance of protein stability and turnover is emphasized in the results of a study of the zinc-dependent ZntR transcriptional activator, which are presented in this issue (15). These new data provide a way to reconcile the results of previous biochemical and biological studies of ZntR function (6, 13, 18) and provide a better model for understanding zinc homeostasis and one which can be applied to other metals. This study underscores the importance of designing experiments that account for the dynamic nature of cell physiology and its effect on intracellular transition metal utilization. There are many known zinc proteins in Escherichia coli (50), several of which are essential (e.g., RNA polymerase and tRNA synthetases). Growth conditions that change the level of zinc protein expression will require adjustments of intracellular zinc levels to ensure that sufficient metal is available for the generation of active proteins. Access to zinc will depend on the concentration of both extracellular zinc and that recovered from the turnover of other Zncontaining proteins. Zinc ions are transported into the cytoplasm via ZnuABC (14), although other import pathways exist (5). ZnuABC expression is regulated by Zur (14), a Zn-dependent transcriptional repressor homologous to the well-known Fur family of metal-dependent regulators (8). When Zur binds available intracellular zinc, it represses ZnuABC transcription, ultimately leading to cessation of zinc import via this pathway. Excess zinc ions are exported by the zinc-inducible exporter ZntA (1, 16), whose expression is regulated by ZntR (2), a member of the MerR family of transcriptional regulators (7).

Theoretical Study of the Phosphotriesterase Reaction Mechanism

Phosphotriesterase (PTE) is a binuclear zinc enzyme that catalyzes the hydrolysis of extremely toxic organophosphate triesters. In the present work, we have investigated the reaction mechanism of PTE using the hybrid density functional theory method B3LYP. We present a potential energy surface for the reaction and provide characterization of the transition states and intermediates. We used the high resolution crystal structure to construct a model of the active site of PTE, containing the two zinc ions and their first shell ligands. The calculations provide strong support to an associative mechanism for the hydrolysis of phosphotriesters by PTE. No protonation of the leaving group was found to be necessary. In particular, the calculations demonstrate that the nucleophilicity of the bridging hydroxide is sufficient to be utilized in the hydrolysis reaction, a feature that is of importance for a number of other di-zinc enzymes.

The Carboxylate Shift in Zinc Enzymes: A Computational Study

Zinc is the second most abundant transition element in biology and the only metal known to be represented in enzymes from each one of the six classes established by the International Union of Biochemistry. The flexible coordination geometry, the fast ligand exchange, the lack of redox activity, and its role as Lewis acid are just some of the features that make zinc an invaluable element in biological catalysis. In this study, we have analyzed the importance in mononuclear Zn enzymes of an interesting mechanistic phenomenon known as carboxylate shift, which is characterized by a change in the coordination mode of a carboxylate group (mono to bidentate or vice versa) with both ligand entrance or exit from the metal coordination sphere. Using B3LYP calculations, we were able to unveil in detail patterns relating the intrinsic characteristics of a given Zn coordination sphere with the existence or not of a carboxylate-shift mechanism and the additional energy stabilization arising from it. In particular, a specific Zn coordination sphere containing a carboxylate ligand (Asp or Glu), a cysteine, and a histidine has been shown to have the most favorable combination of amino acid residues that ensures a fast ligand exchange.

Carbonic anhydrase inhibitors: Binding of an antiglaucoma glycosyl-sulfanilamide derivative to human isoform II and its consequences for the drug design of enzyme inhibitors incorporating sugar moieties

N-(4-Sulfamoylphenyl)-α- d-glucopyranosylamine, a promising topical antiglaucoma agent, is a potent inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The high resolution X-ray crystal structure of its adduct with the target isoform involved in glaucoma, CA II, is reported here. The sugar sulfanilamide derivative binds to the enzyme in a totally new manner as compared to other CA–inhibitor adducts investigated earlier. The sulfonamide anchor was coordinated to the active site metal ion, and the phenylene ring of the inhibitor filled the channel leading to the active site cavity. The glycosyl moiety responsible for the high water solubility of the compound was oriented towards a hydrophilic region of the active site, where no other inhibitors were observed to be bound up to now. A network of seven hydrogen bonds with four water molecules and the amino acid residues Pro201, Pro202 and Gln92 further stabilize the enzyme–inhibitor adduct. Topiramate, another sugar-based CA inhibitor, binds in a completely different manner to CA II as compared to the sulfonamide investigated here. These findings are useful for the design of potent, sugar-derived enzyme inhibitors.

Zinc and Cobalt(II) Complexes of Tripodal Nitrogen Ligands of the Tris[2-substituted Imidazol-4(5)-yl]phosphane Type. Biomimetic Hydrolysis of an Activated Ester

Novel tris[2-substituted imidazol-4(5)-yl]phosphane (4-TIPR) ligands 2b (R = Ph) and 2c (R = tBu) were prepared as model ligands for the tris(histidine) motif found in many zinc enzymes. They readily form cobalt and zinc nitrato and chloro complexes in protic solvents. The steric requirements of the substituents R in 4(5)-position of the 4-TIPR ligands (R = iPr (2a), Ph (2b), tBu (2c)) is discussed on the basis of their UV/VIS spectra. Zinc complexes of 2a and 2c were studied due to their ability to promote the hydrolysis of the activated ester 4-nitrophenyl pyridine-2-carboxylate (pNpic).

Applications of Tripodal [S(3)] and [Se(3)] L(2)X Donor Ligands to Zinc, Cadmium and Mercury Chemistry: Organometallic and Bioinorganic Perspectives.

The tripodal tris(2-mercapto-1-R-imidazolyl)hydroborato ligand system, [Tm(R)], and its selenium counterpart, [Tse(R)], provide useful platforms for investigating organometallic and bioinorganic aspects of the chemistry of zinc, cadmium and mercury in sulfur-rich and selenium-rich coordination environments. For example, the tridentate [Tm(R)] ligand provides an [S(3)] donor array that is of use for mimicking aspects of zinc enzymes and proteins that have sulfur-rich active sites, such as the Ada DNA repair protein. With respect to mercury, an interesting application of the [Tm(Bu(t) )] ligand is the synthesis of the mercury alkyl compounds [Tm(Bu(t) )]HgR (R = Me, Et) that react with PhSH to yield [Tm(Bu(t) )]HgSPh and RH, a reaction that emulates mercury detoxification by the organomercurial lyase, MerB. In addition to the tridentate [Tm(R)] and [Tse(R)] ligands, applications of the bidentate counterparts, [Bm(R)] and [Bse(R)] are also described.

The activity of the dinuclear cobalt-β-lactamase from Bacillus cereus in catalysing the hydrolysis of β-lactams

Metallo-beta-lactamases are native zinc enzymes that catalyse the hydrolysis of beta-lactam antibiotics, but are also able to function with cobalt(II) and require one or two metal-ions for catalytic activity. The hydrolysis of cefoxitin, cephaloridine and benzylpenicillin catalysed by CoBcII (cobalt-substituted beta-lactamase from Bacillus cereus) has been studied at different pHs and metal-ion concentrations. An enzyme group of pK(a) 6.52+/-0.1 is found to be required in its deprotonated form for metal-ion binding and catalysis. The species that results from the loss of one cobalt ion from the enzyme has no significant catalytic activity and is thought to be the mononuclear CoBcII. It appears that dinuclear CoBcII is the active form of the enzyme necessary for turnover, while the mononuclear CoBcII is only involved in substrate binding. The cobalt-substituted enzyme is a more efficient catalyst than the native enzyme for the hydrolysis of some beta-lactam antibiotics suggesting that the role of the metal-ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.

“Inhibition” of the Enzyme Model TpPh,MeZn–OH by Diketo Compounds

AbstractIn order to gain a structural understanding of zinc enzyme inhibition, the model complex TpPh,MeZn–OH was treated with various diketo compounds. α‐Keto carboxylic acids were attached to the zinc ion as anionic O,O‐chelate ligands, of which benzoylformate was oxidatively decarboxylated in air to form the benzoate complex. Two functionalized β‐diketones did not use their functionality in forming the β‐diketonate complexes. Of the α‐diketones, 2,3‐pentanedione formed the α‐keto enolate complex, while 1‐phenyl‐1,2‐propanedione underwent oxidative C–C coupling resulting in a red dinuclear bis(α‐keto enolato) complex. Of the diaryl‐α‐diketones, benzil did not react, but pyridil underwent hydrolytic cleavage to pyridine‐2‐carbaldehyde and picolinate, of which the latter was bound to the zinc ion as an N,O‐chelate ligand.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Modeling Zinc Enzyme Inhibition with Functional Thiolate Ligands

The blocking of zinc enzymes by thiolate-containing inhibitors was modeled by treating TpPh,MeZn-OH with functional thiols. The latter were chosen such that they contain an additional donor function (COOH, COOR, NH2, NHR, OH) in a position favorable for chelation. Of them, mercapto carboxylic acid esters were incorporated as thiolates. The corresponding mercapto carboxylic acids, however, used only their carboxylate function for coordination. Various mercapto amines, mercapto alcohols, and mercaptophenol were exclusively converted to thiolate ligands. The two modes of inhibitor attachment, terminal or chelating, were observed equally frequently. As a rule, they occur as alternatives for similar ligands. In case of 2-mercaptophenol they coexist in the crystalline state and in solution. Hydrogen bonding, both intra- and intermolecular, seems to be a decisive factor determining the inhibitor attachments. Its persistence in solution is underlined by the observation that TpPh,MeZn-hydroxythiophenolates are methylated about 2 orders of magnitude slower than TpPh,MeZn-SPh itself.

Carbonic Anhydrase Inhibitors: Clash with Ala65 as a Means for Designing Inhibitors with Low Affinity for the Ubiquitous Isozyme II, Exemplified by the Crystal Structure of the Topiramate Sulfamide Analogue

The sulfamide analogue of the antiepileptic drug topiramate is a 210 times less potent inhibitor of isozyme II of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) compared to topiramate but effectively inhibits isozymes CA VA, VB, VII, XIII, and XIV (KI in the range of 21-35 nM). Its weak binding to CA II is due to a clash between one methyl group of the inhibitor and Ala65 and may be exploited for the drug design of compounds with lower affinity for this ubiquitous isozyme, as Ala65 is unique to CA II. As shown by X-ray crystallography, the sulfamide analogue binds to CA II with the deprotonated sulfamide moiety coordinated to Zn(II) and with the organic scaffold making an extended network of hydrogen bonds with Thr199, Gln92, His94, Asn62, and Thr200. Its binding to this isozyme is more similar to that of topiramate and quite different from that of the topiramate cyclic sulfate analogue RWJ-37947.

On the origin of the broad-band selectivity of bovine-lens-leucine-aminopeptidase

Detailed DFT (B3LYP) calculations on the mode of action of bovine lens leucine aminopeptidase (blLAP), a binuclear zinc enzyme that hydrolyzes the N-terminal residue of a peptide chain, provide first explanations for the unusual coordination sphere at Zn(1) as well as the presence of a second water channel. The unusual architecture of the active site promotes the spontaneous generation of a water sluice that continually regenerates a series of species capable of acting as active nucleophiles. We now demonstrate that this sluice mechanism is coupled with substrate docking. Both possibilities lead to a complicated hypersurface in which a multitude of stable intermediates and possible nucleophilic species are generated which are in thermodynamic equilibrium with each other. The hypersurface is characterized by very low barriers for interconversion between the intermediates – all of which possess similar stabilities - and thus provides a first explanation for the broad-band activity of blLAP which is capable of hydrolyzing all amino acids except for proline and arginine. Most steric and electronic constraints in the substrate can be overcome by simply selecting a different pathway on the hypersurface. First calculations indicate that a “classical” twostep hydrolysis over a gem-diol/diolate intermediate has a prohibitively high barrier and is thus a probable dead-end on the hypersurface. Alternatively, a non-classical mechanism in which the nucleophilic attack on the carbonyl bond as well as the C-N bond cleavage occurs simultaneously over a chair-like ring transition structure is postulated.

Measurement of the α-Secondary Kinetic Isotope Effect for the Reaction Catalyzed by Mammalian Protein Farnesyltransferase

Protein farnesytransferase (FTase) catalyzes the transfer of a 15-carbon prenyl group from farnesyl diphosphate (FPP) to the cysteine residue of target proteins and is a member of the newest class of zinc metalloenzymes that catalyze sulfur alkylation. Common substrates of FTase include oncogenic Ras proteins, and therefore inhibitors are under development for the treatment of various cancers. An increased understanding of the salient features of the chemical transition state of FTase may aid in the design of potent inhibitors and enhance our understanding of the mechanism of this class of zinc enzymes. To investigate the transition state of FTase we have used transient kinetics to measure the alpha-secondary 3H kinetic isotope effect at the sensitive C1 position of FPP. The isotope effect for the FTase single turnover reaction using a peptide substrate that is farnesylated rapidly is near unity, indicating that a conformational change, rather than farnesylation, is the rate-limiting step. To look at the chemical step, the kinetic isotope effect was measured as 1.154 +/- 0.006 for a peptide that is farnesylated slowly, and these data suggest that FTase proceeds via a concerted mechanism with dissociative character.

Electronic Structure of Four-Coordinate C3v Nickel(II) Scorpionate Complexes: Investigation by High-Frequency and -Field Electron Paramagnetic Resonance and Electronic Absorption Spectroscopies

A series of complexes of formula TpNiX, where Tp*- = hydrotris(3,5-dimethylpyrazole)borate and X = Cl, Br, I, has been characterized by electronic absorption spectroscopy in the visible and near-infrared (NIR) region and by high-frequency and -field electron paramagnetic resonance (HFEPR) spectroscopy. The crystal structure of TpNiCl has been previously reported; that for TpNiBr is given here: space group = Pmc2(1), a = 13.209(2) A, b = 8.082(2) A, c = 17.639(4) A, alpha = beta = gamma = 90 degrees , Z = 4. TpNiX contains a four-coordinate nickel(II) ion (3d8) with approximate C3v point group symmetry about the metal and a resulting S = 1 high-spin ground state. As a consequence of sizable zero-field splitting (zfs), TpNiX complexes are "EPR silent" with use of conventional EPR; however, HFEPR allows observation of multiple transitions. Analysis of the resonance field versus the frequency dependence of these transitions allows extraction of the full set of spin Hamiltonian parameters. The axial zfs parameter for TpNiX displays pronounced halogen contributions down the series: D = +3.93(2), -11.43(3), -22.81(1) cm(-1), for X = Cl, Br, I, respectively. The magnitude and change in sign of D observed for TpNiX reflects the increasing bromine and iodine spin-orbit contributions facilitated by strong covalent interactions with nickel(II). These spin Hamiltonian parameters are combined with estimates of 3d energy levels based on the visible-NIR spectra to yield ligand-field parameters for these complexes following the angular overlap model (AOM). This description of electronic structure and bonding in a pseudotetrahedral nickel(II) complex can enhance the understanding of similar sites in metalloproteins, both native nickel enzymes and nickel-substituted zinc enzymes.

Fibronectin Increases Matrix Metalloproteinase 9 Expression through Activation of c-Fos via Extracellular-regulated Kinase and Phosphatidylinositol 3-Kinase Pathways in Human Lung Carcinoma Cells

Enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with human lung tumor invasion and/or metastasis. We have demonstrated that fibronectin (FN), a matrix glycoprotein, stimulates human non-small cell lung carcinoma (NSCLC) cell proliferation. The current study examines the effect of FN on MMP-9 expression in NSCLC cells. We show that FN increases MMP-9 protein, mRNA expression, and gelatinolytic activity in NSCLC cells. The integrin alpha5beta1 mediated the effects of FN because alpha5 small interfering RNA blocked FN-stimulated MMP-9 protein expression, and also abrogated FN-induced phosphorylation of ERK and phosphatidylinositol 3-kinase (PI3K) signals. The inhibitor of ERK, PD98095, and of PI3K, wortmannin, but not that of protein kinase A, H89, of Rho kinase, Y-27632, of mTOR, rapamycin, or of JNK, SP600125, prevented FN-induced MMP-9 gelatinolytic activity and gene expression. FN enhanced MMP-9 gene promoter activity; however, there was no response to FN in DNA constructs with an AP-1 site mutation. FN increased AP-1 DNA binding activity, and this was abrogated by cyclic AMP response element decoy oligonucleotides, which also diminished FN-induced MMP-9 promoter activity. FN increased the expression of the AP-1 subunit c-Fos protein, but not in the presence of PD98095 and wortmannin. The AP-1 inhibitor, nordihydroguaiaretic acid, and a c-Fos small interfering RNA eliminated the effect of FN on MMP-9 expression. This study indicates that FN, by binding to the integrin alpha5beta1 receptor, stimulates the expression of MMP-9 through increased AP-1/DNA binding and c-Fos protein expression via ERK and PI3K signaling pathways. The data unveils a novel mechanism by which FN could promote NSCLC cell invasion and metastasis.

Enzyme‐Catalyzed Sulfide Ring Formation in Lantibiotics

Building bridges: The crystal structure of the zinc enzyme NisC was solved recently by X-ray diffraction. This cyclase single-handedly catalyzes the formation of several sulfide bridges in the dehydrated precursor protein NisA of the lantibiotic nisin. The Highlight discusses the impact of these results on the field of lantibiotic research.

Zinc enzyme modelling with zinc complexes of polar pyrazolylborate ligands

The Zn–OH 2 and Zn–OH complexes of the new tris(pyrazolyl)borate ligands with pyridyl and carboxamido substituents were investigated for their reactivity towards hydrolyzeable substrates. Tp 4−Py,MeZn–OH inserted CO 2 and CS 2 in methanol forming the Zn–OCOOMe and Zn–SCSOMe products. In non-aqueous media, both types of complexes with both types of substituents on the Tp ligands effected stoichiometric cleavage of tris( p-nitrophenyl)phosphate and p-nitrophenyl acetate. In solutions containing water and the MOPS buffer, up to eight p-nitrophenyl groups per equivalent of zinc complex could be cleaved from the esters, and the resulting bis( p-nitrophenyl)phosphate was also degraded to mono( p-nitrophenyl)phosphate. This is the first time that pyrazolylborate–zinc complexes have shown catalytic activity in hydrolytic reactions.

Enzyme inhibitor modeling with TpZn complexes of functional hydroxamates and oximates

Salicylhydroxamic acid reacts with the enzyme model Tp Ph,MeZn-OH to form the O, O-chelating hydroxamate complex 1. The hydrogen bonding capacity of zinc enzyme bound hydroxamates is reproduced by cocrystallization of two molecules if 1 with two molecules of methanol and by cocrystallization of one molecule of Tp Ph,MeZn-acetohydroxamate with one molecule of 3-phenyl-5-methylpyrazole. The complex formed from Tp Ph,MeZn-OH and N-tosylproline hydroxamic acid, according to its spectra, contains the hydroxamate as an N, N-chelating ligand. In contrast, the oximate derived from pyruvic aldehyde does not act as a chelating ligand, but is monodentate via the oximate oxygen.

Enzyme Deactivation Due to Metal-Ion Dissociation during Turnover of the Cobalt-β-Lactamase Catalyzed Hydrolysis of β-Lactams

Metallo-beta-lactamases are native zinc enzymes that catalyze the hydrolysis of beta-lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted beta-lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pK(a) = 8.9 +/- 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H(2)O/D(2)O) of 5.2 +/- 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt beta-lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pK(a) 6.32 +/- 0.1 and 7.47 +/- 0.1 being in their deprotonated basic forms and one group of pK(a) 9.48 +/- 0.1 being in its protonated form.

The Variation of Catalytic Efficiency of Bacillus cereus Metallo-β-lactamase with Different Active Site Metal Ions

The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo-beta-lactamase have been investigated. The pH and metal ion dependence of k(cat) and k(cat)/K(m), determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pK(a) 6.3 +/- 0.1 is required in its deprotonated form for metal ion binding and catalysis. The k(cat)/K(m) for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pK(a1) = 8.7 +/- 0.1 required in its deprotonated form and the other of pK(a2) = 9.3 +/- 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, K(i), for CdBcII with l-captopril indicates that pK(a1) = 8.7 +/- 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of k(cat)/K(m) for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pK(a1) = 8.5 +/- 0.1 which needs to be deprotonated and the other of pK(a2) = 9.4 +/- 0.1 which needs to be protonated for catalysis; the pK(a1) was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10(-)(3) M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both k(cat) and k(cat)/K(m) increase with increasing pK(a) of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pK(a) for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.

Binding and Transport of Metal Ions at the Dimer Interface of the Escherichia coli Metal Transporter YiiP

YiiP is a representative member of the cation diffusion facilitator (CDF) family, a class of ubiquitous metal transporters that play an essential role in metal homeostasis. Recently, a pair of Zn2+/Cd2+-selective binding sites has been localized to two highly conserved aspartyl residues (Asp157), each in a 2-fold-symmetry-related transmembrane segment 5 (TM5) of a YiiP homodimer. Here we report the functional and structural interactions between Asp157 and yet another highly conserved Asp49 in the TM2. Calorimetric binding analysis indicated that Asp49 and Asp157 contribute to a common Cd2+ binding site in each subunit. Copper phenanthroline oxidation of YiiP(D49C), YiiP(D157C), and YiiP(D49C/D157C) yielded inter- and intra-subunit cross-links among Cys49 and Cys157, consistent with the spatial proximity of two (Asp49-Asp157) sites at the dimer interface. Hg2+ binding to YiiP(D49C) or YiiP(D49C/D157C) also yielded a Cys49-Hg2+-Cys49 biscysteinate complex across the dimer interface, further establishing the interfacial location of a (Asp49-Asp157)2 bimetal binding center. Two bound Cd2+ ions were found transported cooperatively with a sigmoidal dependence on the Cd2+ concentration (n = 1.4). The binding affinity, transport cooperativity, and rate were modestly reduced by either a D49C or D157C mutation, but greatly diminished when all the bidentate aspartate O-ligands in (Asp49-Asp157)2 were replaced by the monodentate cysteine S-ligands. The functional significance of these findings is discussed based on the unique coordination chemistry of aspartyl residues and a model for the translocation pathway of metal ions at the YiiP dimer interface.