Stabilising pentavalent actinides--visible-near infrared and X-ray absorption spectroscopic studies of the utility of the [(Np3W4O15)(H2O)3(MW9O33)3]18- (M = Sb, Bi) structural type.

We report the interaction between B-type tri-lacunary heteropolyoxotungstate anions and actinyl(V) cations in aqueous solution, yielding a greater understanding of the stability of the O≡An≡O(1+) linear dioxo actinide moiety. Previously we reported that B-α-[BiW(9)O(33)](9-) and B-α-[SbW(9)O(33)](9-) will react with NpO(2)(1+) to yield [(Np(3)W(4)O(15))(H(2)O)(3)(MW(9)O(33))(3)](18-) (M = Bi, or Sb). Single crystal structural characterisation of salts of these complexes revealed a core in which three Np(V) atoms interact with a central W(VI) atom through bridging oxo groups. These bridging oxygen atoms come from one of the two axial oxygens in O≡Np≡O(1+) and represent a highly unusual interaction for a discrete molecular species. In this study visible/near infra-red spectroscopy indicates that [(Np(3)W(4)O(15))(H(2)O)(3)(BiW(9)O(33))(3)](18-) could be readily stabilized in solution at near neutral pH for several months, with (NH(4))(14)Na(4)[(Np(3)W(4)O(15))(H(2)O)(39)BiW(9)O(33))(3)]·62H(2)O crystallising from solution in high yield. At lower pH and [BiW(9)O(33)](9-) : NpO(2)(1+) ratios additional Np(V) species could be observed in solution. Stabilization of [(Np(3)W(4)O(15))(H(2)O)(3)(SbW(9)O(33))(3)](18-) in solution proved more challenging, with several distinctive Np(V) near infra-red transitions observed in solution. Slow complexation kinetics and reduction to Np(IV) was also observed. High [SbW(9)O(33)](9-) : NpO(2)(1+) molar ratios and careful control of solution pH was required to prepare solutions in which [(Np(3)W(4)O(15))(H(2)O)(3)(SbW(9)O(33))(3)](18-) was the only neptunium containing species. In stark contrast to the NpO(2)(1+) chemistry, [BiW(9)O(33)](9-) readily oxidizes PuO(2)(1+) to PuO(2)(2+) yielding further evidence of the decreased stability of Pu(V)vs. Np(V). Np L(II)-edge XAFS measurement revealed very good agreement with single crystal diffraction data for the Np structural environment for [(Np(3)W(4)O(15))(H(2)O)(3)(MW(9)O(33))(3)](18-) (M = Bi, or Sb) in the solid state. There was also good agreement between coordination shells for [(Np(3)W(4)O(15))(H(2)O)(3)(BiW(9)O(33))(3)](18-) in the solid state and in solution, yielding further confirmation of the high stability of this particular cluster.

Investigating the impact of cholesterol on magnetically aligned sphingomyelin/cholesterol multilamellar vesicles using static (31)P NMR.

The effect of cholesterol (5-40mol%) on the magnetic induced orientation of sphingomyelin/cholesterol multilamellar vesicles (MLVs) was examined using static solid state (31)P NMR spectroscopy. The orientation was modeled assuming an ellipsoidal deformation of the vesicles and was monitored as a function of cholesterol concentration and temperature. In addition, the static (31)P chemical shift anisotropy (CSA) was used to assess the motional and dynamical changes occurring in the bilayer are reported. An exploration of the factors determining the magnetic orientation in sphingomyelin/cholesterol bilayers from the gel (s(o)) to liquid crystalline (or liquid-ordered, l(o)) phases is presented and discussed.

Differential binding of Co(II) and Zn(II) to metallo-beta-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.

Engineered mononuclear variants in Bacillus cereus metallo-beta-lactamase BcII are inactive.

Metallo-beta-lactamases (MbetaLs) are zinc enzymes able to hydrolyze almost all beta-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of MbetaLs has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-beta-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms. Despite extensive studies, there is still controversy about the nature of the active BcII species. Here we have designed two mutant enzymes in which each one of the metal binding sites was selectively removed. Both mutants were almost inactive, despite preserving most of the structural features of each metal site. These results reveal that neither site isolated in the MbetaL scaffold is sufficient to render a fully active enzyme. This suggests that only the dinuclear species is active or that the mononuclear variants can be active only if aided by other residues that would be metal ligands in the dinuclear species.

Using 31P MAS NMR to monitor a gel phase thermal disorder transition in sphingomyelin/cholesterol bilayers.

The impact of low cholesterol concentrations on an egg sphingomyelin bilayer is investigated using 31P magic angle spinning (MAS) NMR spectroscopy. The magnitude of the isotropic 31P MAS NMR line width is used to monitor the main gel to liquid crystalline phase transition, along with a unique gel phase pretransition. In addition, the 31P chemical shift anisotropy (CSA) and spin-spin relaxation times (T2), along with the effects of spinning speed, proton decoupling and magnetic field strength, are reported. The variation of this unique gel phase thermal pretransition with the inclusion of 5 through 21 mol% cholesterol is presented and discussed.

The Zn2 position in metallo-beta-lactamases is critical for activity: a study on chimeric metal sites on a conserved protein scaffold.

Metallo-beta-lactamases (MbetaLs) are bacterial Zn(II)-dependent hydrolases that confer broad-spectrum resistance to beta-lactam antibiotics. These enzymes can be subdivided into three subclasses (B1, B2 and B3) that differ in their metal binding sites and their characteristic tertiary structure. To date there are no clinically useful pan-MbetaL inhibitors available, mainly due to the unawareness of key catalytic features common to all MbetaL brands. Here we have designed, expressed and characterized two double mutants of BcII, a di-Zn(II) B1-MbetaL from Bacillus cereus, namely BcII-R121H/C221D (BcII-HD) and BcII-R121H/C221S (BcII-HS). These mutants display modified environments at the so-called Zn2 site or DCH site, reproducing the metal coordination environments of structurally related metallohydrolases. Through a combination of structural and functional studies, we found that BcII-HD is an impaired beta-lactamase even as a di-Zn(II) enzyme, whereas BcII-HS exhibits the ability to exist as mono or di-Zn(II) species in solution, with different catalytic performances. We show that these effects result from an altered position of Zn2, which is incapable of providing a productive interaction with the substrate beta-lactam ring. These results indicate that the position of Zn2 is essential for a productive substrate binding and hydrolysis.

The metallo-beta-lactamase GOB is a mono-Zn(II) enzyme with a novel active site.

Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes able to hydrolyze and inactivate most beta-lactam antibiotics. The large diversity of active site structures and metal content among MbetaLs from different sources has limited the design of a pan-MbetaL inhibitor. Here we report the biochemical and biophysical characterization of a novel MbetaL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MbetaLs. Contrasting all other related MbetaLs, GOB-18 is fully active against a broad range of beta-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MbetaLs.

Isostructural neo-pentoxide derivatives of group 3 and the lanthanide series metals for the production of Ln2O3 nanoparticles.

The synthesis and characterization of a series of neo-pentoxide (OCH2C(CH3)3 or ONep) derivatives of group 3 and the lanthanide (Ln) series' metals were undertaken via an amide/alcohol exchange route. Surprisingly, the products isolated and characterized by single-crystal X-ray diffraction yielded isostructural species for every trivalent cation studied: [Ln(mu-ONep)2(ONep)]4 [Ln=Sc (1), Y (2), La (3), Ce (4), Pr (5), Nd (6), Sm (7), Eu (8), Gd (9), Tb (10), Dy (11), Ho (12), Er (13), Tm (14), Yb (15), Lu (16)]. Compounds 3, 4, 6, and 11 have been previously reported. Within this series of complexes, the Ln metal centers are oriented in a square with each Ln-Ln edge interconnected via two mu-ONep ligands; each metal center also binds one terminal ONep ligand. NMR data of 1-3 indicate that the solid-state structure is retained in solution. FTIR spectroscopy (KBr pellet) revealed the presence of significant Ln---H-C interactions within one set of the bridging ONep ligands in all cases; the stretching frequencies of these C-H bonds appear to increase in magnitude with decrease in metal ion radius. These complexes were used to generate nanoparticles through solution hydrolysis routes, resulting in the formation of lanthanide oxide nanoparticles and rods. The emission properties of these ceramics were preliminarily investigated using UV-vis and PL measurements.

X-ray absorption spectroscopy of the zinc-binding sites in the class B2 metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria.

X-ray absorption spectroscopy was used to investigate the metal-binding sites of ImiS from Aeromonas veronii bv. sobria in catalytically active (1-Zn), product-inhibited (1-Zn plus imipenem), and inactive (2-Zn) forms. The first equivalent of zinc(II) was found to bind to the consensus Zn(2) site. The reaction of 1-Zn ImiS with imipenem leads to a product-bound species, coordinated to Zn via a carboxylate group. The inhibitory binding site of ImiS was examined by a comparison of wild-type ImiS with 1 and 2 equiv of bound zinc. 2-Zn ImiS extended X-ray absorption fine structure data support a binding site that is distant from the active site and contains both one sulfur donor and one histidine ligand. On the basis of the amino acid sequence of ImiS and the crystal structure of CphA [Garau et al. (2005) J. Mol. Biol. 345, 785-795], we propose that the inhibitory binding site is formed by M146, found on the B2-distinct alpha3 helix, and H118, a canonical Zn(1) ligand, proposed to help activate the nucleophilic water. The mutation of M146 to isoleucine abolishes metal inhibition. This is the first characterization of ImiS with the native metal Zn and establishes, for the first time, the location of the inhibitory metal site.

Substrate-assisted cysteine deprotonation in the mechanism of dimethylargininase (DDAH) from Pseudomonas aeruginosa.

The enzyme dimethylargininase (also known as dimethylarginine dimethylaminohydrolase or DDAH; EC 3.5.3.18) catalyzes the hydrolysis of endogenous nitric oxide synthase inhibitors, N(omega)-methyl-l-arginine and N(omega),N(omega)-dimethyl-l-arginine. Understanding the mechanism and regulation of DDAH activity is important for developing ways to control nitric oxide production during angiogenesis and in many cases of vascular endothelial pathobiology. Several possible physiological regulation mechanisms of DDAH depend upon the presence of an active-site cysteine residue, Cys249 in Pseudomonas aeruginosa (Pa) DDAH, which is proposed to serve as a nucleophile in the catalytic mechanism. Through the use of pH-dependent ultraviolet and visible (UV-vis) difference spectroscopy and inactivation kinetics, the pK(a) of the active-site Cys249 in the resting enzyme was found to be unperturbed from pK(a) values of typical noncatalytic cysteine residues. In contrast, the pH dependence of k(cat) values indicates a much lower apparent pK(a) value. UV-vis difference spectroscopy between wild-type and C249S DDAH shows absorbance changes consistent with Cys249 deprotonation to the anionic thiolate upon binding positively charged ligands. The proton from Cys249 is lost either to the solvent or to an unidentified general base. A mutation of the active-site histidine residue, H162G, does not eliminate cysteine nucleophilicity, further arguing against a pre-formed ion pair with Cys249. Finally, UV-vis and X-ray absorption spectroscopy revealed that inhibitory metal ions can bind at these two active-site residues, Cys249 and His162, and also stabilize the anionic form of Cys249. These results support a proposed substrate-assisted mechanism for Pa DDAH in which ligand binding modulates the reactivity of the active-site cysteine.

Sequential binding of cobalt(II) to metallo-beta-lactamase CcrA.

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.

The quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein.

Lactonases from Bacillus species hydrolyze the N-acylhomoserine lactone (AHL) signaling molecules used in quorum-sensing pathways of many Gram-negative bacteria, including Pseudomonas aeruginosa and Erwinia carotovora, both significant pathogens. Because of sequence similarity, these AHL lactonases have been assigned to the metallo-beta-lactamase superfamily of proteins, which includes metalloenzymes of diverse activity, mechanism, and metal content. However, a recent study claims that AHL lactonase from Bacillus sp. 240B1 is not a metalloprotein [Wang, L. H., et al. (2004) J. Biol. Chem. 279, 13645]. Here, the gene for an AHL lactonase from Bacillus thuringiensis is cloned, and the protein is expressed, purified, and found to bind 2 equiv of zinc. The metal-bound form of AHL lactonase catalyzes the hydrolysis of N-hexanoyl-(S)-homoserine lactone but not the (R) enantiomer. Removal of both zinc ions results in loss of activity, and reconstitution with zinc restores activity, indicating the importance of metal ions for catalytic activity. Metal content, sequence alignments, and X-ray absorption spectroscopy of the zinc-containing lactonase all support a proposed dinuclear zinc binding site similar to that found in glyoxalase II.

Comparison of cysteine and penicillamine ligands in a Co(II) maquette.

l-Penicillamine (Pen) has been investigated as a ligand for metalloprotein design by examining the binding of Co(II) to the sequence NH(2)-KL(Pen)EGG.(Pen)IG(Pen)GA(Pen).GGW-CONH(2). For comparison, we have studied Co(II) binding to the analogous sequence with Cys ligands, the ferredoxin maquette ligand IGA that was originally designed to bind a [4Fe-4S] cluster. The Co(II) affinity and UV-vis spectroscopic properties of IGA indicate formation of a pseudotetrahedral tetrathiolate ligated Co(II). In contrast, IGA-Pen showed formation of a pseudotetrahedral complex with Co(II) bound by three Pen ligands and an exogenous H(2)O. EXAFS data on both Co(II) complexes confirms not only the proposed primary coordination spheres but also shows six Co(II)-C(beta) methyl group distances in Co(II)-IGA-Pen. These results demonstrate that ligand sterics in simple peptides can be designed to provide asymmetric coordination spheres such as those commonly observed in natural metalloproteins.

Transient mixed-valence character of Re(I)(4)(CO)(12)(4,4'-bpy)(4)Cl(4).

This study addresses, in detail, the orbital nature and the extent of metal-metal communication in the lowest emitting triplet state of Re(4)(CO)(12)(4,4'-bpy)(4)Cl(4) (where 4,4'-bpy = 4,4'-bipyridine) as well as the symmetry of the lowest (3)MLCT manifold in comparison to that of the ground state. All spectral evidence points to (1). a (3)MLCT excited manifold localized between a single Re(I) corner and an adjacent bridging ligand, (2). a transient mixed-valence state that is completely localized between a single transiently oxidized Re center and the adjacent metals, and (3). a second-order charge transfer from a localized transiently reduced bridging ligand to the adjacent Re(I) center to which it is attached, effectively lowering its oxidation state. The orbital nature of the lowest (3)MLCT manifold is fully corroborated by a molecular orbital diagram derived from quantum chemical modeling studies, while the existence of the localization, localized mixed valency, and second-order charge transfer rely on spectral evidence alone. This work makes use of low-temperature time-resolved infrared (TRIR) techniques as well as a luminescence study. Many of the nuances of the luminescence and TRIR data interpretation are extracted from statistical analysis and quantum chemical modeling studies. The relative concentrations of the dominant conformers that exist for Re(4)(CO)(12)(4,4'-bpy)(4)Cl(4) have also been estimated from Boltzmann statistics.