Equiv Of Zn Research Articles

Lewis Acid Supported Nickel Nitrenoids.

Metalation of the polynucleating ligand F,tbs LH6 (1,3,5-C6 H9 (NC6 H3 -4-F-2-NSiMe2 t Bu)3 ) with two equivalents of Zn(N(SiMe3 )2 )2 affords the dinuclear product (F,tbs LH2 )Zn2 (1), which can be further deprotonated to yield (F,tbs L)Zn2 Li2 (OEt2 )4 (2). Transmetalation of 2 with NiCl2 (py)2 yields the heterometallic, trinuclear cluster (F,tbs L)Zn2 Ni(py) (3). Reduction of 3 with KC8 affords [KC222 ][(F,tbs L)Zn2 Ni] (4) which features a monovalent Ni centre. Addition of 1-adamantyl azide to 4 generates the bridging μ3 -nitrenoid adduct [K(THF)3 ][(F,tbs L)Zn2 Ni(μ3 -NAd)] (5). EPR spectroscopy reveals that the anionic cluster possesses a doublet ground state (S = ). Cyclic voltammetry of 5 reveals two fully reversible redox events. The dianionic nitrenoid [K2 (THF)9 ][(F,tbs L)Zn2 Ni(μ3 -NAd)] (6) was isolated and characterized while the neutral redox isomer was observed to undergo both intra- and intermolecular H-atom abstraction processes. Ni K-edge XAS studies suggest a divalent oxidation state for the Ni centres in both the monoanionic and dianionic [Zn2 Ni] nitrenoid complexes. However, DFT analysis suggests Ni-borne oxidation for 5.

Lewis Acid Supported Nickel Nitrenoids

AbstractMetalation of the polynucleating ligand F,tbsLH6 (1,3,5‐C6H9(NC6H3−4‐F−2‐NSiMe2tBu)3) with two equivalents of Zn(N(SiMe3)2)2 affords the dinuclear product (F,tbsLH2)Zn2 (1), which can be further deprotonated to yield (F,tbsL)Zn2Li2(OEt2)4 (2). Transmetalation of 2 with NiCl2(py)2 yields the heterometallic, trinuclear cluster (F,tbsL)Zn2Ni(py) (3). Reduction of 3 with KC8 affords [KC222][(F,tbsL)Zn2Ni] (4) which features a monovalent Ni centre. Addition of 1‐adamantyl azide to 4 generates the bridging μ3‐nitrenoid adduct [K(THF)3][(F,tbsL)Zn2Ni(μ3‐NAd)] (5). EPR spectroscopy reveals that the anionic cluster possesses a doublet ground state (S = ). Cyclic voltammetry of 5 reveals two fully reversible redox events. The dianionic nitrenoid [K2(THF)9][(F,tbsL)Zn2Ni(μ3‐NAd)] (6) was isolated and characterized while the neutral redox isomer was observed to undergo both intra‐ and intermolecular H‐atom abstraction processes. Ni K‐edge XAS studies suggest a divalent oxidation state for the Ni centres in both the monoanionic and dianionic [Zn2Ni] nitrenoid complexes. However, DFT analysis suggests Ni‐borne oxidation for 5.

A Simple and Effective Method of Determining Lewis Acidity by Using Fluorescence

Summary The strength of a Lewis acid is an important parameter in establishing its utility for practical applications. Here, we report a universal fluorescence-based method for accurately determining the strength of a range of Lewis acids. Utilizing a fluorescent dithienophosphole oxide architecture, we are able to emulate the structural motif of the widely employed Gutmann-Beckett method. This allows us to precisely correlate the optical response to the strength of a Lewis acid. In comparison to commonly used methods that employ 31P NMR chemical shifts or quantum-chemical calculations, our method strongly suggests that using fluorescent Lewis acid-base adducts (FLAs) is more reliable. Moreover, our method provides a direct measure of the Lewis acidity of a given solution, independent of the Lewis base, with direct implications to the overall utility of a Lewis acid in practical applications.

A Noncanonical Metal Center Drives the Activity of the Sediminispirochaeta smaragdinae Metallo-β-lactamase SPS-1.

In an effort to evaluate whether a recently reported putative metallo-β-lactamase (MβL) contains a novel MβL active site, SPS-1 from Sediminispirochaeta smaragdinae was overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not β-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar to that in NDM-1 and other subclass B1 MβLs; however, the Zn1 metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 Å from the position reported in other MβLs. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of β-lactam antibiotics. This study reveals a novel metal binding site in MβLs and suggests that the targeting of metal binding sites in MβLs with inhibitors is now more challenging with the identification of this new MβL.

Probing the Interaction of Aspergillomarasmine A with Metallo-β-lactamases NDM-1, VIM-2, and IMP-7.

Metallo-β-lactamases (MBLs) are a growing threat to the continued efficacy of β-lactam antibiotics. Recently, aspergillomarasmine A (AMA) was identified as an MBL inhibitor, but the mode of inhibition was not fully characterized. Equilibrium dialysis and metal analysis studies revealed that 2 equiv of AMA effectively removes 1 equiv of Zn(II) from MBLs NDM-1, VIM-2, and IMP-7 when the MBL is at micromolar concentrations. Conversely, 1H NMR studies revealed that 2 equiv of AMA remove 2 equiv of Co(II) from Co(II)-substituted NDM-1, VIM-2, and IMP-7 when the MBL/AMA are at millimolar concentrations. Our findings reveal that AMA inhibits the MBLs by removal of the active site metal ions required for β-lactam hydrolysis among the most clinically significant MBLs.

Biochemical and Functional Evaluation of the Intramolecular Disulfide Bonds in the Zinc-Chelating Antimicrobial Protein Human S100A7 (Psoriasin).

Human S100A7 (psoriasin) is a metal-chelating protein expressed by epithelial cells. It is a 22-kDa homodimer with two EF-hand domains per subunit and two transition-metal-binding His3Asp sites at the dimer interface. Each subunit contains two cysteine residues that can exist as free thiols (S100A7red) or as an intramolecular disulfide bond (S100A7ox). Herein, we examine the disulfide bond redox behavior, the Zn(II) binding properties, and the antibacterial activity of S100A7, as well as the effect of Ca(II) ions on these properties. In agreement with prior work [Hein, K. Z., et al. (2013) Proc. Natl. Acad. Sci. U. S. A. 112, 13039-13044], we show that apo S100A7ox is a substrate for the mammalian thioredoxin system; however, negligible reduction of the disulfide bond is observed for Ca(II)- and Zn(II)-bound S100A7ox. Furthermore, metal binding depresses the midpoint potential of the disulfide bond. S100A7ox and S100A7red each coordinate 2 equiv of Zn(II) with subnanomolar affinity in the absence and presence of Ca(II) ions, and the cysteine thiolates in S100A7red do not form a third high-affinity Zn(II) site. These results refute a prior model implicating the Cys thiolates of S100A7red in high-affinity Zn(II) binding [Hein, K. Z., et al. (2013) Proc. Natl. Acad. Sci. U. S. A. 112, 13039-13044]. S100A7ox and the disulfide-null variants show comparable Zn(II)-depletion profiles; however, only S100A7ox exhibits antibacterial activity against select bacterial species. Metal substitution experiments suggest that the disulfide bonds in S100A7 may enhance metal sequestration by the His3Asp sites and thereby confer growth inhibitory properties to S100A7ox.

Phosphorescent Decanuclear Bimetallic Pt6M4 (M = Zn, Fe) Tetrahedral Cages.

Coordination-driven self-assembly delivers discrete, nanoscopic architectures that may preserve or enhance the physicochemical properties of their parent building blocks. Herein, we report the syntheses, characterization, and photophysical properties of two tetrahedral cages, [ZnII4L6](PF6)8 (C1) and [FeII4L6](OTf)8 (C2), where L = PtII(PEt3)2(C≡C-bpy)2 (PEt3 = triethylphosphine; C≡C-bpy = 5-ethynyl-2,2'-bipyridine) and OTf = trifluoromethanesulfonate. C1 and C2 were assembled in isolated yields of 72% and 81% by treating 2 equiv of Zn(NO3)2·6H2O or Fe(OTf)2 with 3 equiv of L, respectively. Both cages were fully characterized by NMR, electrospray ionization mass spectrometry, and single-crystal X-ray diffraction (SCXRD). The local D3 symmetry at each polypyridyl metal node raises the possibility of a number of isomeric cages; however, only the homochiral enantiomers (ΔΔΔΔ and ΛΛΛΛ) are formed based on 1H NMR and SCXRD. C1 exhibits phosphorescence centered at 545 nm with a quantum yield of 10% in N2-degassed acetonitrile at 25 °C. The quantum yield of C2 is significantly lower due to a nonradiative relaxation from 5MC (MC = metal-centered) states introduced by the FeII nodes.

A turn-on fluorescence chemosensor based on a tripodal amine [tris(pyrrolyl-α-methyl)amine]-rhodamine conjugate for the selective detection of zinc ions.

A novel tetradendate ligand derived from a tris(pyrrolyl-α-methyl)amine (H3tpa) and rhodamine-based conjugate (PR) has been designed for use as a sensor, synthesized and characterized spectroscopically. PR {(tris(5-rhodamineiminopyrrol-2-ylmethyl)amine)} serves as a selective colorimetric as well as a fluorescent chemosensor for Zn(2+) in acetonitrile/water (1 : 1, v/v). In the presence of Zn(2+), PR exhibited obvious absorption (558 nm) and emission (577 nm) peaks whose intensity increased along with increasing Zn(2+) concentrations. Titration experiments revealed that a large excess of Zn(2+) was required to saturate the absorption (λmax) and emission intensities. Upon the addition of 1000 equivalents of Zn(2+), the fluorescence intensity of the PR underwent an ∼500-fold increase (Φf = 0.34) with the emission maximum at 580 nm. These kinetics studies demonstrated that the absorption and emission changes were proportional to the Zn(2+) concentration. The color of the solution changed from colorless to a dark pink color. The fluorescence of the PR-Zn(2+) complex can be reversibly restored by using ammonium water or by heating. Competitive ion tests revealed that the intensity of PR-Zn(2+) was not suppressed by excess amounts of other metal ions. The counter anions did not exert obvious influences on the absorption and emission profiles. (1)H-NMR and FT-IR spectroscopic investigations of PR and PR-Zn(2+) revealed that the pyrrole motifs, -C[double bond, length as m-dash]N- groups and spirolactam of rhodamine B are capable of coordinating cation guest species. Because each arm of the tripodal ligand tautomerizes independently, only moderate fluorescence enhancement could be seen until all three -C[double bond, length as m-dash]N- groups were coordinated by zinc, which may be due to the spirolactam ring opening mechanism of the rhodamine unit. Once all three -C[double bond, length as m-dash]N- groups were locked by coordinating with excess of Zn(2+), the isomerization was arrested, and PR exhibited highly enhanced fluorescence. In addition, energy optimized structures of PR were found to be cage-like by Gaussian 09, further supporting that it can access a large excess of Zn(2+). Intriguingly, imaging of HeLa cells by using a confocal microscope revealed that this PR probe could be used for biological applications.

Gd(3+)-Based Magnetic Resonance Imaging Contrast Agent Responsive to Zn(2+).

We report the heteroditopic ligand H5L, which contains a DO3A unit for Gd(3+) complexation connected to an NO2A moiety through a N-propylacetamide linker. The synthesis of the ligand followed a convergent route that involved the preparation of 1,4-bis(tert-butoxycarbonylmethyl)-1,4,7-triazacyclononane following the orthoamide strategy. The luminescence lifetimes of the Tb((5)D4) excited state measured for the TbL complex point to the absence of coordinated water molecules. Density functional theory calculations and (1)H NMR studies indicate that the EuL complex presents a square antiprismatic coordination in aqueous solution, where eight coordination is provided by the seven donor atoms of the DO3A unit and the amide oxygen atom of the N-propylacetamide linker. Addition of Zn(2+) to aqueous solutions of the TbL complex provokes a decrease of the emission intensity as the emission lifetime becomes shorter, which is a consequence of the coordination of a water molecule to the Tb(3+) ion upon Zn(2+) binding to the NO2A moiety. The relaxivity of the GdL complex recorded at 7 T (25 °C) increases by almost 150% in the presence of 1 equiv of Zn(2+), while Ca(2+) and Mg(2+) induced very small relaxivity changes. In vitro magnetic resonance imaging experiments confirmed the ability of GdL to provide response to the presence of Zn(2+).

Zinc Hydroxyacetate and Its Transformation to Nanocrystalline Zinc Oxide

The synthesis of nanocrystalline ZnO by thermal decomposition of zinc hydroxyacetate, Zn(5)(OH)(8)(CH(3)CO(2))(2)·nH(2)O, was investigated. The decomposition process was examined using X-ray diffraction, thermogravimetric analysis, mass spectrometry, electron microscopy, Brunauer-Emmett-Teller surface area analysis, and solid-state NMR spectroscopy. Intermediate Zn(5)(OH)(8)(CH(3)CO(2))(2)·nH(2)O phases form at temperatures up to 110 °C from the starting compound Zn(5)(OH)(8)(CH(3)CO(2))(2)·2H(2)O by partial dehydration. At ∼110 °C, 4 equiv of ZnO and 1 equiv of Zn(CH(3)CO(2))(2) are formed. Further heating causes Zn(CH(3)CO(2))(2) to decompose to acetone, acetic acid, acetic anhydride, and ZnO. Notably, a portion of Zn(CH(3)CO(2))(2) sublimes during the process. Overall, the product of the calcination is equiaxed ZnO nanocrystals of 20-100 nm diameter.

Mechanistic and Spectroscopic Studies of Metallo-β-lactamase NDM-1

In an effort to biochemically characterize metallo-β-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)-NDM-1 fusion proteins with different N-termini (full-length, Δ6, Δ21, and Δ36). All MBP-NDM-1 fusion proteins were soluble; however, only one, MBP-NDM-1Δ36, exhibited high activity and bound 2 equiv of Zn(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1Δ36 variant, which exhibited a k(cat) of 16 s(-1) and a K(m) of 1.1 μM when using nitrocefin as a substrate, bound 2 equiv of Zn(II), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1Δ36 variant indicate the average metal binding site for Zn(II) in this variant consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn-Zn distance of 3.38 Å. This metal binding site is very similar to those of other metallo-β-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1Δ36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-β-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-β-lactamase CcrA.

Characterization of Zn(II)-responsive ribosomal proteins YkgM and L31 in E. coli

RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E. coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes were significantly up-regulated and 146 genes were significantly down-regulated in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA, which encodes for a periplasmic protein known to be involved with Zn(II) import, (2) yodA, which encodes for a periplasmic protein with unknown function, and (3) ykgM, which encodes for a ribosomal protein that is thought to be a paralog of ribosomal protein L31. YodA was over-expressed and purified as a maltose binding protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II). Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand, MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers. Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in MBP-L31 and that the other three cysteines do not bind metal. These results are discussed in light of Zn(II) starvation model that has been postulated for B. subtilis.

Structural and Kinetic Studies on Metallo-β-lactamase IMP-1

In an effort to probe for metal binding to metallo-β-lactamase (MβL) IMP-1, the enzyme was overexpressed, purified, and characterized. The resulting enzyme was shown to bind 2 equiv of Zn(II), exhibit significant catalytic activity, and yield EXAFS results similar to crystallographic data previously reported. Rapid kinetic studies showed that IMP-1 does not stabilize a nitrocefin-derived reaction intermediate; rather, the enzyme follows a simple Michaelis mechanism to hydrolyze nitrocefin. Metal-substituted and metal-reconstituted analogues of IMP-1 were prepared by directly adding metal ion stocks to metal-free enzyme, which was generated by dialysis versus EDTA. UV-vis studies on IMP-1 containing 1 equiv of Co(II) showed a strong ligand-to-metal charge transition at 340 nm, and the intensity of this feature increased when the second equivalent of Co(II) was added to the enzyme. EXAFS fits on IMP-1 containing 1 equiv of Co(II) strongly suggest the presence of a metal-metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equiv of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode.

Reactivity of [Zn2Cp*2] toward Transition-Metal Complexes: Synthesis and Characterization of [Cp*M(ZnCp*)3] (M = Ni, Pd, Pt)

Substitution reactions of labile d10 metal starting complexes with [Zn2Cp*2] (Cp* = pentamethylcyclopentadienyl) are presented. The treatment of [M(cod)2] (M = Ni, Pt; cod =1,5-cyclooctadiene) with stoichiometric amounts of [Zn2Cp*2] results in the formation of [Cp*M(ZnCp*)3] (M = Ni (1), Pt (2)) with the release of 1,3-cyclooctadiene. In addition to Cp* transfer reactions from zinc(I) to the transition-metal centers, the formation of compounds 1 and 2 results via redox chemical pathways: namely, the oxidation of M(0) to M(I) and the reduction of 1 equiv of Zn(I) to Zn(0). The Pd homologue [Cp*Pd(ZnCp*)3] (3) is obtained as a byproduct in the reaction of [Pd(CH3)2(tmeda)] (tmeda = N,N,N′,N′-tetramethylethane-1,2-diamine) with [Zn2Cp*2]. Herein, various side reactions and competing redox chemical processes are involved, including the formation of [Pd(ZnCp*)4(ZnMe)4] as well as [Pd(ZnCp*)4(ZnMe)2(Zn{tmeda})]. Compounds 1–3 have been fully characterized by single-crystal X-ray diffraction, 1H and 13C NMR spectroscopy, IR spectroscopy, and liquid injection field desorption ionization mass spectrometry (LIFDI-MS).

Ratiometric fluorescent Zn 2+ chemosensor constructed by appending a pair of carboxamidoquinoline on 1,2-diaminocyclohexane scaffold

By appending a pair of carboxamidoquinoline pendants onto 1,2-diaminocyclohexane scaffold via N-alkylation, multifunctionalized ACAQ was designed and synthesized as a water soluble fluorescent ratiometric chemosensor for Zn 2+. In 50% aqueous methanol buffer pH 7.4 solution, upon excitation at 316 nm, ACAQ (5 μM) displayed a selective ratiometric fluorescence changes with a shift from 410 to 490 nm in response to the interaction with Zn 2+. After binding with 1 equiv of Zn 2+, ACAQ exhibited a 12-fold enhancement in I 490/I 410 characterized by a clear isoemissive point at 440 nm. The metal sensor binding mode was established by Job’s plot and the combined fluorescence and 1H NMR spectroscopic method. The selectivity of the probe toward biological relevant cations and transition metal ions was proven to be good. In addition, the interference caused by Cu 2+ and Cd 2+ in the quantitation of Zn 2+ can be completely eliminated by the use of diethyldithiocarbamate as the screening agent. Exploitation of ACAQ as the sensing probe, ratiometric determination of Zn 2+ with the limit of detection (LOD) at 28.3 nm can be realized. In addition, the unique responsive properties of the probe toward Fe 3+ and Zn 2+ were used to construct a fluorescent switch. The membrane permeability of ACAQ to living cells and bio-imaging of Zn 2+ were demonstrated.

Converting GLX2-1 into an Active Glyoxalase II

Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) exhibits extensive sequence similarity with GLX2 enzymes but is catalytically inactive with SLG, the GLX2 substrate. In an effort to identify residues essential for GLX2 activity, amino acid residues were altered at positions 219, 246, 248, 325, and 328 in GLX2-1 to be the same as those in catalytically active human GLX2. The resulting enzymes were overexpressed, purified, and characterized using metal analyses, fluorescence spectroscopy, and steady-state kinetics to evaluate how these residues affect metal binding, structure, and catalysis. The R246H/N248Y double mutant exhibited low level S-lactoylglutathione hydrolase activity, while the R246H/N248Y/Q325R/R328K mutant exhibited a 1.5-2-fold increase in k(cat) and a decrease in K(m) as compared to the values exhibited by the double mutant. In contrast, the R246H mutant of GLX2-1 did not exhibit glyoxalase 2 activity. Zn(II)-loaded R246H GLX2-1 enzyme bound 2 equiv of Zn(II), and (1)H NMR spectra of the Co(II)-substituted analogue of this enzyme strongly suggest that the introduced histidine binds to Co(II). EPR studies indicate the presence of significant amounts a dinuclear metal ion-containing center. Therefore, an active GLX2 enzyme requires both the presence of a properly positioned metal center and significant nonmetal, enzyme-substrate contacts, with tyrosine 255 being particularly important.

Bridging Binding Modes of Phosphine‐Stabilized Nitrous Oxide to Zn(C6F5)2

Just say NNO: Reaction of [tBu3PN2O(B(C6H4F)3)] with 1, 1.5, or 2 equivalents of Zn(C6F5)2 affords the species [{tBu3PN2OZn(C6F5)2}2], [{tBu3PN2OZn(C6F5)2}2Zn(C6F5)2], and [tBu3PN2O{Zn(C6F5)2}2] (see structure; red Zn (large sphere), O (small sphere), green N, yellow P) displaying unique binding modes of Zn to the phosphine-stabilized N2O fragment.

NMR structural analysis of the soluble domain of ZiaA-ATPase and the basis of selective interactions with copper metallochaperone Atx1

A Cu(I) metallochaperone, Atx1, interacts with the amino-terminal domain of a Cu(I)-transporting ATPase, PacS(N), but not with a domain of related Zn-transporting ATPase, ZiaA(N) in Synechocystis PCC 6803. This is thought to prevent ZiaA(N) from acquiring Cu(I), which it binds more tightly than Zn. Solution structures of Atx1, PacS(N), and the heterodimer were previously described. Here we report solution structural studies of the ZiaA(N) soluble domain. Apo-ZiaA(N) has a typical ferredoxin-like fold followed by an atypical 34 residues of unstructured polypeptide containing a His(7) motif. ZiaA(N) competes with the metallochromic indicator 4-(2-pyridylazo)resorcinol for 1 equiv of Zn, which can be displaced by thiol-modifying p-mercuriphenylsulfonic acid, establishing that a high-affinity site involves thiols of the CXXC motif within the ferredoxin-like fold. A single equivalent of Zn affects nuclear magnetic resonance signals arising from the CXXC motif as well as all seven His residues. The presence of NMR-line broadening in both sites implies that Zn(1)-ZiaA(N) undergoes exchange phenomena, consistent with CXXC-bound Zn coincidentally sampling various His ligands. These Zn-dependent dynamic changes could either aid metal transfer or alter intramolecular interactions. No formation of Atx1-Cu(I)-ZiaA(N) heterodimers was observed, and in the presence of equimolar ZiaA(N) and PacS(N), only Atx1-Cu(I)-PacS(N) complexes were detected. Residues flanking the CXXC motif of PacS(N) (R(13)-ASS(20)) differ in charge and bulk from those of ZiaA(N) (D(18)-KLK(25)) and make contacts in the Atx1-Cu(I)-PacS(N) complex. Crucially, swapping these residues flanking the CXXC motifs of ZiaA(N) and PacS(N) reciprocally swaps partner choice by Atx1. These few residues of the two ATPases have diverged during evolution to bias Atx1 interactions in favor of PacS(N) rather than ZiaA(N.).

Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from Bacillus anthracis

In an effort to probe the structure, mechanism, and biochemical properties of metallo-beta-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV-vis, (1)H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.

Human Glyoxalase II Contains an Fe(II)Zn(II) Center but Is Active as a Mononuclear Zn(II) Enzyme

Human glyoxalase II (Glx2) was overexpressed in rich medium and in minimal medium containing zinc, iron, or cobalt, and the resulting Glx2 analogues were characterized using metal analyses, steady-state and pre-steady-state kinetics, and NMR and EPR spectroscopies to determine the nature of the metal center in the enzyme. Recombinant human Glx2 tightly binds nearly 1 equiv each of Zn(II) and Fe. In contrast to previous reports, this study demonstrates that an analogue containing 2 equiv of Zn(II) cannot be prepared. EPR studies suggest that most of the iron in recombinant Glx2 is Fe(II). NMR studies show that Fe(II) binds to the consensus Zn(2) site in Glx2 and that this site can also bind Co(II) and Ni(II), suggesting that Zn(II) binds to the consensus Zn(1) site. The NMR studies also reveal the presence of a dinuclear Co(II) center in Co(II)-substituted Glx2. Steady-state and pre-steady-state kinetic studies show that Glx2 containing only 1 equiv of Zn(II) is catalytically active and that the metal ion in the consensus Zn(2) site has little effect on catalytic activity. Taken together, these studies suggest that Glx2 contains a Fe(II)Zn(II) center in vivo but that the catalytic activity is due to Zn(II) in the Zn(1) site.