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X-ray absorption spectroscopy (XAS), including extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) analysis, has been carried out at the ZnKedge of the N-terminal part of the integrase protein of the human immunodeficiency virus, type 2 (HIV-2), and of some zinc coordination compounds. In the presence of excess β-mercaptoethanol, which was present in the NMR structure elucidation of the protein [Eijkelenboomet al.(1997),Curr. Biol.7, 739–746; (2000),J. Biomol. NMR,18, 119–28], the protein spectrum was nearly identical to that recorded in its absence. Comparison of the XANES of the protein with that of model compounds and literature data permits the conclusion that the Zn ion is four-coordinated. The major shell of the EXAFS provides evidence for a mixed (N or O as well as S) coordination sphere, while the minor shells indicate imidazole coordination. Our approach to the analysis of the EXAFS, including quantification of the imidazole by multiple scattering simulations withEXCURV92, was validated on the model compounds. An important result is that with multiple scattering simulations using restraints on the parameters of the imidazole rings the number of imidazoles and their orientation could be determined. The integrase spectra can be fitted with two sulfur ligands at 2.26 Å (Debye–Waller-type factor 0.009 Å2) and two imidazole ligands with the N atoms at 1.99 Å (Debye–Waller-type factor 0.005 Å2). The XAS-derived geometry is fully consistent with that found in the NMR structure determination and, allowing for the volume contraction due to the temperature difference between the experiments, justifies the restraints applied in the structure calculation (Zn—S and Zn—N distances of 2.3 Å and 2.0 Å, respectively).

Two novel tridentate dinucleating ligands containing benzimidazole were prepared, 1,3-bis(2-benzimidazolyl)-2-propanol (Hbbp, 1) and 1,5-bis(2-benzimidazolyl)-3-pentanol (Hbbpen, 2). Their complexing properties toward copper were studied in order to obtain structural and functional models for catechol oxidases. Syntheses and crystal structures of dinuclear Cu(II) complexes derived from these ligands are reported. [Cu(2)bbp(2)](ClO(4))(2).2MeOH, 3, crystallizes in the triclinic space group Ponemacr; with the following unit cell parameters: a = 7.702(3) Å, b = 10.973(6) Å, c = 12.396(6) Å, alpha = 100.59(4) degrees, beta = 99.02(4) degrees, gamma = 98.90(4) degrees, V = 998.7(8) Å(3), and Z = 1. [Cu(2)bbpen(2)](ClO(4))(2).3MeOH, 4, crystallizes in the orthorhombic space group Pccn, with the following unit cell parameters: a = 17.478(9) Å, b = 18.795(8) Å, c = 13.888(6) Å, V = 4562.2(4) Å(3), and Z = 4. Magnetic susceptibility measurements in the temperature ranges 4.6-459 K (3) and 4.6-425 K (4) indicate an antiferromagnetic coupling between the Cu(II) centers of both complexes. In order to determine the structures of the complexes in solution, XAS spectra (EXAFS and XANES) were recorded in the solid state and in solution. The interpretation of these data, including multiple scattering calculations, together with UV-vis titrations, shows that the complexes have the same structure in the crystalline state as well as in methanolic solution. Complex 4 is able to oxidize 3,5-di-tert-butylcatechol (3,5-DTBC) to the quinone (catecholase activity). This reaction was also studied by XAS and UV-vis spectroscopy. These measurements reveal the reduction of Cu(II) to Cu(I) accompanied by a decrease of the coordination number.

Experiments are described to elucidate the structure and solvation of aquocobalamin (vitamin Biza, 1+) in the crystal and in aqueous solution. Aquocobalamin (1+) is the B12 derivative in which a water molecule replaces the axially coordinating organic substituent (methyl or 5'-desoxyadenosyl) at the P-side of the cobalt of the B12 coenzymes. (1) A single-crystal structure analysis of aquocobalamin perchlorate (l'ClO,), using synchrotron radiation in combination with an imaging plate detector, yielded the most accurate structural data ever determined for a B12 molecule. l'C10, crystallizes in the orthorhombic space group P212121, a = 15.042(11) A, b = 23.715(14) A, c = 25.104(12) A, with four 1'CIOi moieties plus about 100 solvent water molecules per unit cell; 22 867 independent and 20 942 significant intensity data were recorded to a nominal resolution of 0.8 A, and refinement against quantities led to a conventional R-value of 0.050 for all 22 867 observations and to a structural model with an average ESD for all carbon-carbon bonds of 0.003 A. In the crystal, the aquocobalamin ion has a very short axial bond between cobalt and the dimethylbenzimidazole (DMB) base of 1.925 (0.002) A, which is rationalized as resulting from the very weak trans axial donor (water). Steric repulsion between the DMB base and the corrin ring induced by this short Co-DMB bond leads to a relatively large "butteffly" deformation with an "upward" fold angle of 18.7 l(0.07)". The relevance of this observation for the "upward conformational deformation" hypothesis for the initiation of Co-C bond homolysis in coenzyme B12 dependent enzymes is discussed. (2) EXAFS spectra were taken from an authentic sample of the aquocobalamin perchlorate crystals used for the X-ray structure analysis, as well as from 1+C104 dissolved in a 1:l mixture of watedethylene glycol. Absorption spectra were recorded at 20 K between 7300 and 8700 eV, and the k3-weighted EXAFS was extracted for a k-value between 2.7 and 14.4 A-l. EXAFS spectra for solid and dissolved l'C10, agree closely, establishing identical cobalt coordination for the aquocobalamin ion in solution and in the solid state. A curve-fitting analysis on the Fourier filtered first-shell data yields a coordination number of 6 and an average distance of 1.90 A for both samples. There is no evidence for a longer Co-N distance. This refutes data published by Sagi and Chance (J. Am. Chem. Soc. 1992, 114, 8061). (3) NMR experiments are described, constituting the first detailed NMR investigations on a Bl2 derivative in H20, including 2D homo- and heteronuclear studies on aquocobalamin chloride (l+Cl-) and assignment of signals due to the exchangeable amide protons of all nitrogens as well a measurements of amide proton exchange rates. The NMR data confirm the occupation of the axial coordination site at the Co(II1) center by water, as well as the occurrence in solution of an intramolecular hydrogen bond to the axially coordinating water molecule, as observed in the crystal structure of l'C10,. However, significant differences of the structure of 1+ in crystals of l'C10, and of 1+ in aqueous solution are indicated from NOE data concerning the time-averaged conformation of the hydrogen-bonding c-acetamide side chain.