Investigating the interaction mechanism between zinc and Saccharomyces cerevisiae using combined SEM-EDX and XAFS

The interaction mechanism between zinc and the intact yeast cells of Saccharomyces cerevisiae was investigated by using the scanning electron microscopy with energy-dispersive X-ray analysis, as well as X-ray absorption fine structure spectroscopy (XAFS). Displacement of H+, K+, Mg2+, and Na+ during zinc uptake confirmed the existence of both covalent interactions and ionic interactions between Zn2+ and the microbe. Ion exchange mechanism played a role in zinc uptake. The local environment of Zn accumulated in the intact yeast cells was determined by XAFS, which suggests that the nearest neighboring atom of the bound zinc ion on the biomass is oxygen atom. The adsorbed zinc ion on the intact cells of S. cerevisiae is a tetrahedron structure, with the Zn-O bond length of 1.97 A, and the coordination number is only 3.2 of Zn-O structure in the first shell.     

Zinc- and pH-dependent conformational transition in a putative interdomain linker region of the influenza virus matrix protein M1

The matrix protein M1 of influenza A virus forms a shell beneath the viral envelope and sustains the virion architecture by interacting with other viral components. A structural change of M1 upon acidification of the virion interior in an early stage of virus infection is considered to be a key step to virus uncoating. We examined the structure of a 28-mer peptide (M1Lnk) representing a putative linker region between the N- and C-terminal domains of M1 by using circular dichroism, Raman, and absorption spectroscopy. M1Lnk assumes an alpha-helical structure in a mildly hydrophobic environment irrespective of pH, being consistent with the X-ray crystal structures of an N-terminal fragment of M1 at pH 7 and 4. In the presence of Zn(2+), on the other hand, M1Lnk takes a partially unfolded conformation at neutral pH with a tetrahedral coordination of two Cys residues and two His residues to a Zn(2+) ion in the central part of the peptide. Upon acidification, the peptide releases the Zn(2+) ion and refolds into the alpha-helix-rich structure with a midpoint of transition at pH 5.9. The pH-dependent conformational transition of M1Lnk strongly suggests that the interdomain linker region of M1 also undergoes a pH-dependent unfolding-refolding transition in the presence of Zn(2+). A small but significant portion of the M1 protein is bound to Zn(2+) in the virion, and the Zn(2+)-bound M1 molecule may play a special role in virus uncoating by changing the disposition of the N- and C-terminal domains upon acidification of the virion interior.     

The composition and structure of bovine peritubular dentin: mapping by time of flight secondary ion mass spectroscopy

The dentin layer of the tooth is a complex mineralized tissue traversed by a closely packed system of tubules. Each tubule is surrounded by highly mineralized tissue referred to as peritubular dentin (PTD). The remaining mineralized collagen network between the tubules is the intertubular dentin (ITD). A TOF-SIMS analysis of the PTD constituents has been used to compare the PTD to the ITD. The PTD differs from the ITD not only in the degree of mineralization but also in the amount and nature of the mineral elements and amino acids. The organic matrix of the PTD consists of a unique collagen free assembly of proteins rich in glutamic acid, where the ITD organic matrix is collagen-rich and Asp-rich. The apparent concentration of organic fragment ions observed in the PTD in the TOF-SIMS negative ion mode was much higher than expected. The PTD was found to be rich in Ca2+, Na+, Mg2+, and K+. The amount of Mg2+ and K+ in the PTD was significantly reduced after deproteination, while Ca2+ and Na+ were still accumulated in the PTD. This implies that Mg2+ and K+ are mainly associated with the organic matrix rather than with the mineral of the PTD.     

Molecular dynamics characterization of the active cavity of carboxypeptidase A and some of its inhibitor adducts.

Molecular dynamics (MD) calculations have been performed on carboxypeptidase A and on its adducts with inhibitors, such as d-phenylalanine (dPhe) and acetate. The catalytically essential zinc ion present in the protein was explicitly included in all the simulations. The simulation was carried out over a sphere of 15 A centered on the zinc ion. The crystallographic water molecules were explicitly taken into account; then the protein was solvated with a 18 A sphere of water molecules. MD calculations were carried out for 45-60 ps. There is no large deviation from the available X-ray structures of native and the dPhe adduct for the MD structures. Average MD structures were calculated starting from the X-ray structure of the dPhe adduct, and, from a structure obtained by docking the inhibitor in the native structure. Comparison between these two structures and with that of the native protein shows that some of the key variations produced by inhibitor binding are reproduced by MD calculations. Addition of acetate induces structural changes relevant for the understanding of the interaction network in the active cavity. The structural variations induced by different inhibitors are examined. The effects of these interactions on the catalytic mechanism and on the binding of substrate are discussed.     

Enhancement of enzyme activity through three-phase partitioning: crystal structure of a modified serine proteinase at 1.5 A resolution.

Three-phase partitioning is fast developing as a novel bioseparation strategy with a wide range of applications including enzyme stability and enhancement of its catalytic activity. Despite all this, the enzyme behaviour in this process still remains unknown. A serine proteinase, proteinase K, was subjected to three-phase partitioning (TPP). A 3 ml volume of proteinase K solution (3 mg/ml in 0.05 M acetate buffer, pH 6.0) was brought to 30% (w/v) ammonium sulphate saturation by addition of saturated ammonium sulphate. tert-Butanol (6 ml) was added to this solution and the mixture was incubated at 25 degrees C for 1 h. The precipitated protein in the mid-layer was dissolved in 3 ml of 0.05 M acetate buffer, pH 6.0. The specific activity of the processed enzyme was estimated and was found to be 210% of the original enzyme activity. In order to understand the basis of this remarkable enhancement of the enzyme activity, the structure of the TPP-treated enzyme was determined by X-ray diffraction at 1.5 A resolution. The overall structure of the TPP-treated enzyme is similar to the original structure in an aqueous environment. The hydrogen bonding system of the catalytic triad is intact. However, the water structure in the substrate binding site has undergone a rearrangement as some of the water molecules are either displaced or completely absent. Two acetate ions were identified in the structure. One is located in the active site and seems to mimic the role of water in the enzyme activity and stability. The other is located at the surface of the molecule and is involved in stabilizing the local structure of the enzyme. The most striking observation in respect of the present structure pertains to a relatively higher overall temperature factor (B = 19.7 A(2)) than the value of 9.3 A(2) in the original enzyme. As a result of a higher B-factor, a number of residues, particularly their side chains, were found to adopt more than one conformation. It appears that the protein exists in an excited state which might be helping the enzyme to function more rapidly than the original enzyme in aqueous media. Summarily, the basis of increased enzymatic activity could be attributed to (i) the presence of an acetate ion at the active site and (ii) its excited state as reflected by an overall higher B-factor.     

Structural basis for Ni(2+) transport and assembly of the urease active site by the metallochaperone UreE from Bacillus pasteurii

Bacillus pasteurii UreE (BpUreE) is a putative chaperone assisting the insertion of Ni(2+) ions in the active site of urease. The x-ray structure of the protein has been determined for two crystal forms, at 1.7 and 1.85 A resolution, using SIRAS phases derived from a Hg(2+)-derivative. BpUreE is composed of distinct N- and C-terminal domains, connected by a short flexible linker. The structure reveals the topology of an elongated homodimer, formed by interaction of the two C-terminal domains through hydrophobic interactions. A single Zn(2+) ion bound to four conserved His-100 residues, one from each monomer, connects two dimers resulting in a tetrameric BpUreE known to be formed in concentrated solutions. The Zn(2+) ion can be replaced by Ni(2+) as shown by anomalous difference maps obtained on a crystal of BpUreE soaked in a solution containing NiCl(2). A large hydrophobic patch surrounding the metal ion site is surface-exposed in the biologically relevant dimer. The BpUreE structure represents the first for this class of proteins and suggests a possible role for UreE in the urease nickel-center assembly.     

Crystal structure of the superantigen staphylococcal enterotoxin type A.

Staphylococcal enterotoxins are prototype superantigens characterized by their ability to bind to major histocompatibility complex (MHC) class II molecules and subsequently activate a large fraction of T-lymphocytes. The crystal structure of staphylococcal enterotoxin type A (SEA), a 27 kDa monomeric protein, was determined to 1.9 A resolution with an R-factor of 19.9% by multiple isomorphous replacement. SEA is a two domain protein composed of a beta-barrel and a beta-grasp motif demonstrating the same general structure as staphylococcal enterotoxins SEB and TSST-1. Unique for SEA, however, is a Zn2+ coordination site involved in MHC class II binding. Four amino acids including Ser1, His187, His225 and Asp227 were found to be involved in direct coordination of the metal ion. SEA is the first Zn2+ binding enterotoxin that has been structurally determined.     

Kinetic analysis of carboxy ester hydrolysis by a dinuclear Zn(II) complex

A dinuclear Zn(II) complex with hexaaza macrocyclic ligand bearing two 2-hydroxypropyl pendants, 3,6,9,16,19,22-hexaaza-6,19-bis(2-hydroxypropyl)-tricyclo [22,2,2,2(11,14)]triaconta-11,13,24,26,27,29-hexane (L) was synthesized and studied as a catalyst of the cleavage of 4-nitrophenyl acetate (NA). X-ray diffraction analysis of [Zn(2)LCl(2)]Cl(2)(.)6H(2)O revealed that Zn(II) adopts a trigonal-bipyramidal geometry. The complexation constants of L with Zn(II) have been determined at 298 K by means of potentiometric titration. [Zn(2)H(-2)L](2+) is the dominant species in aqueous solution around pH 8. The Zn(2)L-promoted hydrolysis of NA showed a second-order rate constant of 0.33 M(-1)s(-1) at pH 9.0, and the main promoter species are concluded to be the deprotonated species [Zn(2)H(-2)L](2+).     

Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein.

It has been reported that the fusion protein with the protein transduction domain (PTD) peptide of HIV-1 Tat protein can be internalized through the cell membrane of intact cells, although the exact mechanism is unknown. In this report, we investigated whether this new method could be used for the molecular analysis of exocytosis via HPC-1/syntaxin 1A, which plays an important role in transmitter release. When applied to PC12 cells, Tat PTD fusion proteins were rapidly internalized into most cells. In order to show that the internalized protein remained biologically active, the H3 domain of HPC-1/syntaxin 1A was fused to Tat PTD (Tat-H3). Transmitter release in PC12 cells was suppressed by Tat-H3 treatment. These results indicate that the Tat fusion protein is a useful tool for analyzing the process of transmitter release.     

Rat submaxillary gland serine protease, tonin. Structure solution and refinement at 1.8 A resolution

Tonin is a mammalian serine protease that is capable of generating the vasoconstrictive agent, angiotensin II, directly from its precursor protein, angiotensinogen, a process that normally requires two enzymes, renin and angiotensin-converting enzyme. The X-ray crystallographic structure determination and refinement of tonin at 1.8 A resolution and the analysis of the resulting model are reported. The initial phases were obtained by the method of molecular replacement using as the search model the structure of bovine trypsin. The refined model of tonin consists of 227 amino acid residues out of the 235 in the complete molecule, 149 water molecules, and one zinc ion. The R-factor (R = sigma Fo - Fc/sigma Fo) is 0.196 for the 14,997 measured data between 8 and 1.8 A resolution with I greater than or equal to sigma (I). It is estimated that the overall root-mean-square error in the coordinates is about 0.3 A. The structure of tonin that has been determined is not in its active conformation, but one that has been perturbed by the binding of Zn2+ in the active site. Zn2+ was included in the buffer to aid the crystallization. Nevertheless, the structure of tonin that is described is for the most part similar to its native form as indicated by the close tertiary structural homology with kallikrein. The differences in the structures of the two enzymes are concentrated in several loop regions; these structural differences are probably responsible for the differences in their reactivities and specificities.     

Structural analysis of N-acetylglucosamine-6-phosphate deacetylase apoenzyme from Escherichia coli

We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis.     

The role of zinc in the stabilization of the dimeric form of bacterial alpha-amylase.

Bacterial alpha-amylase was shown by equilibrium and velocity-sedimentation studies to be a monomer-dimer equilibrium system in 0.10M-NaCl/0.015M-calcium acetate/0.010M-EDTA, pH7.0; an association constant of 2.4 X 10(3)M-1 is obtained. Studies of the binding of Zn2+ to alpha-amylase in 0.10M-NaCl/0.005M-calcium acetate, pH7.0, yielded binding curves that exhibit dependence on the concentration of alpha-amylase (Zn2+-free) used in the equilibrium-dialysis experiments. Results are described very satisfactorily by a reaction scheme in which Zn2+ binds exclusively to the dimer of the above monomer--dimer system with an association constant of 1.0 X 10(6)M-1. The present results refute the earlier scheme for dimer stabilization by Zn2+ in which the metal ion formed a cross-link between two non polymerizing monomer units.     

Zinc(II) complexes with potent cyclin-dependent kinase inhibitors derived from 6-benzylaminopurine: synthesis, characterization, X-ray structures and biological activity

The synthesis, characterization and biological activity of the first zinc(II) complexes with potent inhibitors of cyclin-dependent kinases (CDKs) derived from 6-benzylaminopurine are described. Based on the results following from elemental analyses, infrared, NMR and ES+MS (electrospray mass spectra in the positive ion mode) spectroscopies, conductivity data, thermal analysis and X-ray structures, the tetrahedral Zn(II) complexes of the compositions [Zn(Olo)Cl(2)](n) (1), [Zn(iprOlo)Cl(2)](n) (2), [Zn(BohH(+))Cl(3)] x H(2)O (3) and [Zn(iprOloH(+))Cl(3)] x H(2)O (4) have been prepared, where Olo=2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine (Olomoucine), iprOlo=2-(2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (i-propyl-Olomoucine), Boh=2-(3-hydroxypropylamino)-6-benzylamino-9-isopropylpurine (Bohemine). The 1D-polymeric chain structure for [Zn(Olo)Cl(2)](n) (1) as well as the monomeric one for [Zn(BohH(+))Cl(3)] x H(2)O (3) and [Zn(iprOloH(+))Cl(3)] x H(2)O (4) have been revealed unambiguously by single crystal X-ray analyses. The 1D-polymeric chain of 1 consists of Zn(Olo)Cl(2) monomeric units in which the Zn(II) ion is coordinated by two chlorine atoms and one oxygen atom of the 2-hydroxyethylamino group of Olomoucine. The next monomeric unit is bonded to Zn(II) through the N7 atom of a purine ring. Thus, each of Zn(II) ions is tetrahedrally coordinated and a ZnCl(2)NO chromophore occurs in the complex 1. The complexes 3 and 4 are mononuclear species with a distorted tetrahedral arrangement of donor atoms around the Zn(II) ion with a ZnCl(3)N chromophore. The corresponding CDK inhibitor, i.e., both Boh and iprOlo, is coordinated to Zn(II) via the N7 atom of the purine ring in 3 and 4. The cytotoxicity of the zinc(II) complexes against human melanoma, sarcoma, leukaemia and carcinoma cell lines has been determined as well as the inhibition of the CDK2/cyclin E kinase. A relationship between the structure and biological activity of the complexes is also discussed.     

Interaction of zinc ions with d(CGCAATTGCG) in a 2.9 Å resolution X-ray structure

We have synthesized and crystallized in the presence of Zn(2+) ions the peptidyl-oligonucleotide adduct CH(3)CO-(Arg)(4)-NH-(CH(2))(6)-NH-p-d(CGCAATTGCG). This is the first structure obtained from a deoxyoligonucleotide crystallized in the presence of zinc ions. Zn ions are clearly visible in the 2.9 A resolution map. On the other hand, the peptide tail is not visible in the crystal structure as determined by X-ray diffraction. The terminal bases C1 and G10 are found in extra-helical positions. Their phosphates are ligands of a Zn(2+) ion, located in a special position of the unit cell. This ion plays an important role in the packing arrangement, since it binds four different DNA molecules. Two other Zn(2+) ions are also important for DNA packing. They interact specifically with the N7 atoms of the terminal G2 and G10 bases, but not with the internal G8. This result supports the hypothesis that transition metals do not interact with the bases of duplex DNA in the B form.     

Comparison of the effects of saccharide binding and crystallization on the zinc transition-metal site of concanavalin A

The Zn site in concanavalin A solution was studied by X-ray absorption fine structure spectroscopy (XAFS) with and without the saccharide methyl alpha-D-glucoside (aMG) bound to the protein. No structural change occurs in the metal-binding site when the saccharide is bound to the protein. There is, however, evidence for structural change remote from the metal site. This is in contrast to the significant changes that we have previously found to occur in the near neighborhood of the Zn atom when an aqueous solution of Zn concanavalin A crystallizes. We propose a structural explanation of these facts based on the known crystal structure of concanavalin A.     

The effect of zinc on the activity and fluorescence of carbonic anhydrase holoenzymes.

The addition of Zn2+ to human carbonic anhydrase B holoenzyme was shown to enhance the protein fluorescence, and this enhancement was correlated with the inhibition of the p-nitrophenyl acetate esterase activity. The affinity for the inhibitory Zn2+ was increased when the ionic inhibitors, acetate or chloride, were added, suggesting that the inhibitory Zn2+-binding site is within the region of the protein that undergoes an anion-induced conformational change. A similar fluorescence enhancement was observed when Zn2+ was added to human carbonic anhydrase C and to bovine carbonic anhydrase, demonstrating that the binding site is not a thiol group. Circular-dichroism studies showed that the C isoenzyme but not the B isoenzyme underwent a major conformational change in the presence of Zn2+. A mechanism for the Zn2+-induced fluorescence enhancement was suggested on the basis of studies with simple compounds.     

Crystal structure of the complex of concanavalin A and tripeptide

The X-ray structure analysis of a cross-linked crystal of concanavalin A soaked with the tripeptide molecule as the probe molecule showed electron density corresponding to full occupation in the binding pocket. The site lies on the surface of concanavalin A and is surrounded by three symmetry-related molecules. The crystal structure of the tripeptide complex was refined at 2.4-Å resolution to an R-factor of 17.5%, (R_free factor of 23.7%), with an RMS deviation in bond distances of 0.01 Å. The model includes all 237 residue of concanavalin A, 1 manganese ion, 1 calcium ion, 161 water molecules, 1 glutaraldehyde molecule, and 1 tripeptide molecule. This X-ray structure analysis also provides an approach to mapping the binding surface of crystalline protein with a probe molecule that is dissolved in a mixture of organic solvent with water or in neat organic solvent but is hardly dissolved in aqueous solution.     

Cysteine-to-serine mutants of the human copper chaperone for superoxide dismutase reveal a copper cluster at a domain III dimer interface

Cysteine-to-serine mutants of a maltose binding protein fusion with the human copper chaperone for superoxide dismutase (hCCS) were studied with respect to (i) their ability to transfer Cu to E,Zn superoxide dismutase (SOD) and (ii) their Zn and Cu binding and X-ray absorption spectroscopic (XAS) properties. Previous work has established that Cu(I) binds to four cysteine residues, two of which, C22 and C25, reside within an Atox1-like N-terminal domain (DI) and two of which, C244 and C246, reside in a short unstructured polypeptide chain at the C-terminus (DIII). The wild-type (WT) protein shows an extended X-ray absorption fine structure (EXAFS) spectrum characteristic of cluster formation, but it is not known how such a cluster is formed. Cys to Ser mutagenesis was used to investigate the Cu binding in more detail. Single Cys to Ser mutations, as represented by C22S and C244S, did little to affect the metal binding ratios of hCCS. Both mutants still showed approximately 2 Cu(I) ions and 1 Zn ion per protein. The double mutants C22/24S and C244/246S, on the other hand, showed Cu binding stoichiometries close to 1:1. The Zn-EXAFS of WT CCS showed a 3-4 histidine ligand environment that is consistent with Zn binding in the SOD-like domain II of CCS. The Zn environment remained unchanged between wild type and all of the mutant CCS proteins. Single Cys to Ser mutations displayed lower activity than WT protein, although close to full activity could be rescued by increasing the CCS:SOD ratios to 8:1 in the assay mixture. The structure of the Cu centers of the single mutants as revealed by EXAFS was also similar to that of WT protein, with clear indications of a Cu cluster. On the other hand, the double mutants showed a greater degree of perturbation. The DI C22/25S mutant was 70% active and formed a cluster with a more intense Cu-Cu interaction. The DIII C244/246S mutant retained only a fraction (16%) of activity and did not form a cluster. The results suggest the formation of a DIII-DIII cluster within a dimeric or tetrameric protein and further suggest that this cluster may be an important element of the copper transfer machinery.     

Structural consequences of the active site substitution Cys181 ==> Ser in metallo-beta-lactamase from Bacteroides fragilis

The metallo-beta-lactamases require divalent cations such as zinc or cadmium for hydrolyzing the amide bond of beta-lactam antibiotics. The crystal structure of the Zn2+ -bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam bond of the substrate. It was previously reported that the replacement of the active site Cys181 by a serine residue severely impaired catalysis while atomic absorption measurements indicated that binding of the two zinc ions remained intact. Contradicting data emerge from recent mass spectrometry results, which show that only a single zinc ion binds to the C181S metallo-beta-lactamase. In the current study, the C181S mutant enzyme was examined at the atomic level by determining the crystal structure at 2.6 A resolution. The overall structure of the mutant enzyme is the same as that of the wild-type enzyme. At the mutation site, the side chain of Ser181 occupies the same position as that of the side chain of Cys181 in the wild-type protein. One zinc ion, Zn1, is present in the crystal structure; however, the site of the second zinc ion, Zn2 is unoccupied. A water molecule is associated with Zn1, reminiscent of the hydroxide seen in the structure of the wild-type enzyme but farther from the metal. The position of the water molecule is off the plane of the carboxylate group of Asp103; therefore, the water molecule may be less nucleophilic than a water molecule which is coplanar with the carboxylate group.     

High resolution X-ray structures of different metal-substituted forms of phosphotriesterase from Pseudomonas diminuta

Phosphotriesterase, isolated from the soil-dwelling bacterium Pseudomonas diminuta, catalyzes the detoxification of organophosphate-based insecticides and chemical warfare agents. The enzyme has attracted significant research attention in light of its possible employment as a bioremediation tool. As naturally isolated, the enzyme is dimeric. Each subunit contains a binuclear zinc center that is situated at the C-terminal portion of a "TIM" barrel motif. The two zincs are separated by approximately 3.4 A and coordinated to the protein via the side chains of His 55, His 57, His 201, His 230, Asp 301, and a carboxylated Lys 169. Both Lys 169 and a water molecule (or hydroxide ion) serve to bridge the two zinc ions together. Interestingly, these metals can be replaced with cadmium or manganese ions without loss of enzymatic activity. Here we describe the three-dimensional structures of the Zn(2+)/Zn(2+)-, Zn(2+)/Cd(2+)-, Cd(2+)/Cd(2+)-, and Mn(2+)/Mn(2+)-substituted forms of phosphotriesterase determined and refined to a nominal resolution of 1.3 A. In each case, the more buried metal ion, referred to as the alpha-metal, is surrounded by ligands in a trigonal bipyramidal ligation sphere. For the more solvent-exposed or beta-metal ion, however, the observed coordination spheres are either octahedral (in the Cd(2+)/Cd(2+)-, Mn(2+)/Mn(2+)-, and the mixed Zn(2+)/Cd(2+)-species) or trigonal bipyramidal (in the Zn(2+)/Zn(2+)-protein). By measuring the anomalous X-ray data from crystals of the Zn(2+)/Cd(2+)-species, it has been possible to determine that the alpha-metal ion is zinc and the beta-site is occupied by cadmium.