By means of large-angle X-ray scattering zinc(II) and mercury(II) ions in N,N-dimethylthioformamide solution are found to coordinate to four N,N-dimethylthioformamide molecules with Zn-S and Hg-S bond distances of 2.362(5) and 2.527(6) Å, respectively. The intermediate divalent ion in group 12, cadmium, is solvated by six N,N-dimethylthioformamide molecules with a Cd-S bond distance of 2.69(1) Å. Raman and far-infrared spectra have been recorded and assigned for the solvated ions both in solution and in the solid state. The character of the bonds to the metal ion is discussed in order to explain the lower coordination numbers of the zinc and mercury(II) ions.
The zinc and cobalt forms of the prototypic gamma-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and (18)O exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn-Cam or Co-Cam for CO(2) hydration (k(cat)/K(m)) was severalfold greater than HCO(3-) dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn- and Co-Cam, the steady-state parameter k(cat) for CO(2) hydration was pH-dependent with a pK(a) of 6.5-6.8, whereas k(cat)/K(m) was dependent on two ionizations with pK(a) values of 6.7-6.9 and 8.2-8.4. The (18)O exchange assay also identified two ionizable groups in the pH profile of k(cat)/K(m) with apparent pK(a) values of 6.0 and 8.1. The steady-state parameter k(cat) (CO(2) hydration) is buffer-dependent in a saturable manner at pH 8. 2, and the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 x 10(7) M(-1) s(-1). At saturating buffer concentrations and pH 8.5, k(cat) is 2.6-fold higher in H(2)O than in D(2)O, suggesting that an intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k(cat)/K(m) is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes.
The crystal structures of N,N-dimethylthioformamide and N,N-dimethylformamide were determined at 90 ± 2 K from single crystal X-ray diffraction data. Both compounds comprise planar molecules, a consequence of the π-electron delocalization over the N-C-S and N-C-O entities, respectively. In N,N-dimethylthioformamide, almost linear, 175.4(7)Њ, C-H ؒ ؒ ؒ S cooperative hydrogen bonding between the thioformyl groups connects the molecules in helix-shaped chains with an intermolecular C ؒ ؒ ؒ S distance of 378.10(7) pm. The two crystallographically independent molecules in N,N-dimethylformamide form four-membered centrosymmetric rings held together by C-H ؒ ؒ ؒ O intermolecular interactions, two via the formyl protons, C ؒ ؒ ؒ O 329.41(9) pm, and two involving methyl protons, C ؒ ؒ ؒ O 341.41(9) pm. The structures of both liquids were studied at room temperature by large angle X-ray scattering in transmission mode and for N,N-dimethylthioformamide also in reflection geometry. The structure of liquid N,N-dimethylformamide can, despite the stronger hydrogen bond acceptor properties of the oxygen atom, be described without hydrogen bonding. This apparent anomaly with more significant effect of hydrogen bonding in both crystal and liquid forms of N,N-dimethylthioformamide than N,N-dimethylformamide is discussed using results from theoretical calculations on single molecules. Mulliken population analyses indicate a lower positive charge and thus weaker hydrogen-bond donor properties of the formyl than of the thioformyl hydrogen atom. Raman and infrared spectra of the solids and the liquids are used for discussions of the hydrogen bonding effects.
MerR, the metalloregulatory protein of the mercury-resistance operon (mer) has unusually high affinity and specificity for ionic mercury, Hg(II). Prior genetic and biochemical evidence suggested that the protein has a structure consisting of an N-terminal DNA binding domain, a C-terminal Hg(II)-binding domain, and an intervening region involved with communication between these two domains. We have characterized a series of MerR deletion mutants and found that as little as 30% of the protein (residues 80-128) forms a stable dimer and retains high affinity for Hg(II). Biophysical measures indicate that this minimal Hg(II)-binding domain assumes the structural characteristics of the wild-type full-length protein both in the Hg(II) center itself and in an immediately adjacent helical protein domain. Our observations are consistent with the core Hg(II)-binding domain of the MerR dimer being constituted by a pair of antiparallel helices (possibly in a coiled-coil conformation) comprised of residues cysteine 82 through cysteine 117 from each monomer followed by a flexible loop through residue cysteine 126. These antiparallel helices would have a potential Hg(II)-binding site at each end. However, just as in the full-length protein, only one of these potential binding sites in the deleted proteins actually binds Hg(II).
The α subunit of carbon monoxide dehydrogenase from Clostridium thermoaceticum was isolated, treated as described below, and examined by XAS, EPR, and UV−vis spectroscopies. This subunit contains the active site for acetyl-coenzyme A synthesis, the A-cluster, a Ni ion bridged to an Fe4S4 cube. Populations of α subunits contain two major forms of A-clusters, a catalytically active form called Ni - labile and an inactive form called nonlabile. The objective of this study was to elucidate the redox and spectroscopic properties of these A-cluster forms and thereby understand their structural and functional differences. The Ni-labile form could be reduced either by CO and a catalytic amount of native enzyme or by electrochemically reduced triquat in the presence of CO. The Ni2+ component of the Ni-labile form reduced to Ni1+ and bound CO. CO-binding raised E°‘ for the Ni2+/Ni1+ couple, thereby rendering CO and triquat effective reductants. Dithionite did not reduce the Ni-labile form, though its addition to CO/CODH-reduced Ni-labile clusters caused an intracluster electron transfer from the Ni1+ to the [Fe4S4]2+ cluster. Dithionite reduced the [Fe4S4]2+ component of the nonlabile form, as well as the cluster of the Ni-labile form once Ni was removed. Ni may not be bridged to the cube in the nonlabile form. XAS reveals that the Ni in the nonlabile form has a distorted square-planar geometry with two N/O scatters at 1.87 Å and two S scatters at 2.20 Å. The [Fe4S4]2+ portion of Ni-labile A-clusters may maintain the Ni in a geometry conducive to reduction, CO and methyl group binding, and the migratory-insertion step used in catalysis. It may also transfer electrons to and from the redox-active D site during reductive activation.
The crystalline solvates of the divalent group 12 metal ions with the soft sulfur donor N,N-dimethylthioformamide display an unusual variation in coordination number and geometry with two, four, and six ligands attached to the mercury(II), zinc(II), and cadmium(II), ions, respectively. Bis(N,N-dimethylthioformamide)mercury(II) perchlorate precipitates from acetonitrile solution when adding less than 2 equiv of N,N-dimethylthioformamide, while the zinc and cadmium solvates crystallize from saturated N,N-dimethylthioformamide solutions. The disolvate [Hg(SCHN(CH(3))(2))(2)](ClO(4))(2) crystallizes in the monoclinic space group P2(1)/n (No. 14) with a = 6.208(1) Å, b = 15.239(7) Å, c = 8.681(2) Å, beta = 99.30(3) degrees, and Z = 2. Centrosymmetric mercury(II) complexes with strong collinear bonds to two N,N-dimethylthioformamide molecules, Hg-S 2.350(2) Å, are joined by double bridges of perchlorate ions in chains along the a-axis by four weak interactions between the mercury and the perchlorate oxygen atoms, mean Hg-O distance 2.84 Å. Tetrakis(N,N-dimethylthioformamide)zinc trifluoromethanesulfonate, [Zn(SCHN(CH(3))(2))(4)](CF(3)SO(3))(2), crystallizes in the triclinic space group P&onemacr; (No. 2) with a = 10.487(3) Å, b = 12.910(3) Å, c = 13.489(5) Å, alpha = 68.800(4) degrees, beta = 69.260(4) degrees, gamma = 74.06(1) degrees, and Z = 2, with the zinc ions tetrahedrally surrounded by four N,N-dimethylthioformamide ligands, mean Zn-S distance 2.34 Å. Also the cadmium solvate of corresponding composition, [Cd(SCHN(CH(3))(2))(4)(CF(3)SO(3))(2)], crystallizes in the space group P&onemacr;, with a = 8.670(1) Å, b = 9.529(1) Å, c = 10.685(1) Å, alpha = 75.20(1) degrees, beta = 66.97(1) degrees, gamma = 65.31(1) degrees, and Z = 1, although the structure comprises centrosymmetric tetrakis(N,N-dimethylthioformamide)bis(trifluoromethanesulfonato)cadmium(II) complexes in which four Cd-S bonds (mean 2.65 Å) and two weaker Cd-O bonds at 2.470(2) Å to the trifluoromethanesulfonate ions give rise to a pseudo-octahedral coordination around the cadmium ion. When using perchlorate instead as counterion, hexakis(N,N-dimethylthioformamide)cadmium perchlorate, [Cd(SCHN(CH(3))(2))(6)](ClO(4))(2), crystallizes in the space group P2(1)/n with a = 12.757(1) Å, b = 7.4681(6) Å, c = 19.732(2) Å, beta = 96.31(1) degrees, and Z = 2. The mean Cd-S bond distance increases to 2.715 Å in the fully solvated cadmium ion with almost regular octahedral coordination geometry to its six centrosymmetrically related ligands. The effect of the weak internal hydrogen bonding occuring between the hydrogen atom of a -CHS group and the sulfur atom of neighboring N,N-dimethylthioformamide ligands is discussed.
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