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CF2HCbl, CF3Cbl , and CF3CH2Cbl have been synthesized and characterized in solution by (1)H NMR and UV-vis spectroscopy, and their X-ray crystal structures have been determined using synchrotron radiation. The structure of CF3CH2Cbl is reported for the first time, whereas those of CF2HCbl and CF3Cbl are re-examined to obtain more precise structural data. Comparison of the structural data obtained with the alkylcobalamin analogues, MeCbl and EtCbl, indicates that the Co-C and Co-NB3 bond lengths are shorter in the fluoroalkylcobalamins. The structural data of the fluoroalkylcobalamins previously reported in the literature had been conflicting in this regard. Thus, a much less dramatic shortening of the two axial bonds was found for CF3Cbl, whereas in the case of CF2HCbl, the Co-NB3 bond length is shorter than in MeCbl. Direct comparison of the structures of CF3CH2Cbl and EtCbl indicates a large distortion of the axial fragment in the former case that can be attributed to steric effects. A number of previously reported correlations of the effect of the β-ligand on the structure and properties of cobalamins are re-examined in light of the present results. Particular emphasis is placed on the axial fragment. This analysis substantially confirms and, with the new data reported here, adjusts and expands the data set for correlations between trans and cis influences of the β-ligand of cobalamins and their structure (Co-X and Co-NB3 distances and corrin fold angle) and properties (UV-vis spectra, NMR spectra, and pK(base-off)).

The interligand interactions in coordination compounds have been principally interpreted in terms of cis and trans influences and effects, which can be defined as the ability of a ligand X to affect the bond of another ligand, cis or trans to X, to the metal. This review analyzes these effects/influences in cobalamins (XCbl) and their simple models cobaloximes, LCo(chel)X. Important properties of these complexes, such as geometry, stability, and reactivity, can be rationalized in terms of steric and electronic factors of the ligands. Experimental evidence of normal and inverse trans influence is described in alkylcobaloximes for the first time. The study of simple B(12) models has complemented that on the more complex cobalamins, with particular emphasis on the properties of the axial L-Co-X moiety. Some of the conclusions reached for the axial fragment of simple models have also been qualitatively detected in cobalamins and have furnished new insight into the as yet unestablished mechanism for the homolytic cleavage of the Co - C bond in the AdoCbl-based enzymes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Protein carbamylation is of great concern both in vivo and in vitro. Here, we report the first structural characterization of a protein carbamylated at the N-terminal proline. The unexpected carbamylation of the α-amino group of the least reactive codified amino acid has been detected in high-resolution electron density maps of a new crystal form of the HIV-1 protease/saquinavir complex. The carbamyl group is found coplanar to the proline ring with a trans conformation. The reaction of N-terminal with cyanate ion derived from the chaotropic agent urea was confirmed by mass spectra analysis on protease single crystals. Implications of carbamylation process in vitro and in vivo are discussed.

The chemistry and biochemistry of the vitamin B(12) compounds (cobalamins, XCbl) are described, with particular emphasis on their structural aspects and their relationships with properties and function. A brief history of B(12), reveals how much the effort of chemists, biochemists and crystallographers have contributed in the past to understand the basic properties of this very complex vitamin. The properties of the two cobalamins, the two important B(12) cofactors Ado- and MeCbl are described, with particular emphasis on how the Co-C bond cleavage is involved in the enzymatic mechanisms. The main structural features of cobalamins are described, with particular reference to the axial fragment. The structure/property relationships in cobalamins are summarized. The recent studies on base-off/base-on equilibrium are emphasized for their relevance to the mode of binding of the cofactor to the protein scaffold. The absorption, transport and cellular uptake of cobalamins and the structure of the B(12) transport proteins, IF and TC, in mammals are reviewed. The B(12) transport in bacteria and the structure of the so far determined proteins are briefly described. The currently accepted mechanisms for the catalytic cycles of the AdoCbl and MeCbl enzymes are reported. The structure and function of B(12) enzymes, particularly the important mammalian enzymes methyltransferase (MetH) and methyl-malonyl-coenzyme A mutase (MMCM), are described and briefly discussed. Since fast proliferating cells require higher amount of vitamin B(12) than that required by normal cells, the study of B(12 )conjugates as targeting agents has recently gained importance. Bioconjugates have been studied as potential agents for delivering radioisotopes and NMR probes or as various cytotoxic agents towards cancer cells in humans and the most recent studies are described. Specifically, functionalized bioconjugates are used as "Trojan horses" to carry into the cell the appropriate antitumour or diagnostic label. Possible future developments of B(12) work are summarized.

Density functional theory has been applied to study the origin of the inverse and normal trans influence in alkylcobalamins. In order to cover the X-ray structural data available for alkylcobalamins with a variety of axial substituents, geometries of 28 related corrin-containing models have been optimized and analyzed. The BP86/6-31G(d) level of theory was applied which showed good reliability in reproducing the axial bond lengths. Comparison of experimental and calculated data allowed to conclude that the inverse trans influence is not a general feature of cobalamins, as it appeared from the experimental data analysis alone. Inverse trans influence is observed for the series of R groups with increasing bulk and electron donating ability. For the series of R groups having similar medium bulk, but differing significantly in the electron donating ability, normal trans influence was found. Finally, it was determined, that the axial bond lengths correlate well but differently in the two series of R groups with the orbital energies of the six molecular orbitals essential in axial interligand bonding.

Two probes consisting of vitamin B(12) (CNCbl) conjugated to Gd chelates by esterification of the ribose 5'-OH moiety, Gd-DTPA-CNCbl (1; DTPA = diethylenetriamine-N,N,N',N'',N''-pentaacetic acid) and Gd-TTHA-CNCbl (2; TTHA = triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), have been synthesised and characterised. The crystal structure of a dimeric form of 1, obtained by crystallisation with an excess of GdCl(3), has been determined. The kinetics of binding to and dissociation from transcobalamin II show that 1 and 2 maintain high-affinity binding to the vitamin B(12) transport protein. Complex 2 is very stable with respect to Gd(3+) release owing to the saturated co-ordination of the Gd(3+) ion by four amino and five carboxylate groups. Hydrolysis of the ester functionality occurs on the time scale of several hours. The lack of saturation and the possible involvement of the ester functionality in co-ordination result in lower stability of 1 towards hydrolysis and in a considerable release of Gd(3+) in vitro. Gd(3+) ions released from 1 are avidly taken up by the K562 tumour cells to an extent corresponding to approximately 10(10) Gd(3+) per cell. The internalisation of toxic Gd(3+) ions causes a marked decrease in cell viability as assessed by Trypan blue and WST-1 tests. On the contrary, the experiments with the more stable 2 did not show any significant cell internalisation of Gd(3+) ions and any influence on cell viability. The results point to new avenues of in situ generation of cytotoxic pathways based on the release of toxic Gd(3+) ions by vitamin B(12) bioconjugates.

The ligand substitution reactions of complexes of the type trans-[(R)Co(Chel)S](+/0) with L, where chel = (DO)(DOH)pn = 2,2'-(1,3-diaminopropanebis(2-methyl-3-butanone)oxime), R = CH(3), L = imidazole, pyrazole, 1,2,4-triazole and 1-methylimidazole, and S = water and MeOH, and chel = (Hdmg)(2) = bis(dimethylglyoximate), R = CH(2)Cl, CH(2)Br, and CH(2)I, L = thiourea and pyridine, and S = water, were studied in detail as a function of temperature and pressure. The reported activation parameters (DeltaH, DeltaS and DeltaV) support the operation of a dissociative interchange (I(d)) mechanism. Complexes of the type trans-[RCo(Hdmg)(2)L] (R = CH(2)Cl, CH(2)Br, and CH(2)I; L = H(2)O and Py) were fully optimized at the B3LYP/LANL2DZp level, and the structural data support the mechanistic assignment based on the reported activation parameters. For the reaction of trans-[(CH(3))(2)Co((DO)(DOH)pn)] with acid, the activation parameters (DeltaH, DeltaS and DeltaV) were found to be 37 +/- 1 kJ mol(-1), -86 +/- 3 J mol(-1) K(-1) and -18.9 +/- 0.7 cm(3) mol(-1), respectively, and support a protonation mechanism.

A highly nanoporous material has been obtained by self-assembly of calixarene and porphyrin building blocks. This supramolecular zeolite-like structure was successively functionalized by diffusion and coordination of metal ions to form a new bifunctionalized nanoporous material containing a porphyrinic pigment together with a metal center.

Time-dependent density functional theory (TDDFT) has been applied to the analysis of the electronic spectra of methylcobalamin (MeCbl) and its derivative in which the trans axial base was replaced by a water molecule (MeCbi[Single Bond]H(2)O). The latter corresponds to the situation encountered in strongly acidic solutions. The study primarily focuses on the accuracy of two functionals, the hybrid B3LYP and the gradient corrected BP86, in dealing with the electronic excitations. The high resolution crystal structure of MeCbl was the source of the initial coordinates. To generate the initial structures, the full MeCbl was simplified by replacing the corrin side chains by H atoms. The vertical excitation energies, together with the corresponding oscillator strengths, were calculated at the optimized BP86 and B3LYP structures of the ground electronic state of the complexes. The NBO analysis shows that the B3LYP functional gives a bonding description of the ground state as a more polarized covalent bond compared to that given by BP86. The latter functional has more covalent bonding and is thus more appropriate for modeling the axial bonding properties. To validate the accuracy of the present TDDFT analysis, the computed excitations were directly compared to the absorption spectra of MeCbl. In order to obtain a reliable agreement between experiment and theory, the two-parameter scaling technique was introduced, which compensates differently the low-energy and high-energy excitations. Electronic excitations strongly depend on the choice of the functional. Transitions involving corrin pi-->pi(*) excitations are better described by the B3LYP functional while transitions associated with metal-to-ligand (dpi-->pi(*)d) excitations are better described by BP86. These differences can be associated with the different bonding descriptions obtained by B3LYP and BP86.

Cobalamin (Cbl, vitamin B12) is an essential micronutrient that is synthesized only by bacteria. Mammals have developed a complex system for internalization of this vitamin from the diet. Three binding proteins (haptocorrin, intrinsic factor, transcobalamin (TC)) and several specific cell surface receptors are involved in the process of intestinal absorption, plasma transport and cellular uptake. The recent literature on the binding proteins is briefly reviewed. A structural study is presented addressing a unique feature of TC among the three proteins, i.e., the displacement of the weak Co(III)-ligand H2O at the upper (or beta) axial side of H2O-Cbl by a histidine side chain. We have investigated crystallographically the beta-ligand exchange on Cbl bound to TC by crystallization of bovine holo-TC in the presence of either cyanide or sulfite. The resulting electron density maps show that the histidine side chain has been displaced by an exogenous ligand CN(-) or SO(3)(-2)to a lower extent than expected based on their higher affinity for Co and excess concentration with respect to histidine. This may reflect either reduced affinities of CN(-) and SO(3)(-2)or the advantageous binding of the protein-integrated His-residue when competing for the beta-site of Cbl bound to TC. The loop hosting the histidine residue appears more flexible after disruption of the coordination bond His-Cbl but no other differences are observed in the overall structure of holo-TC. These structural results are discussed in relation to a possible physiological role of histidine substitution for H2O and regarding the role of beta-conjugated Cbl-analogues recently proposed for targeted delivery of imaging agents.

Four tetraphosphorylated TiiiiPO cavitands 1 encapsulate the pyridinium heads of a tetrakis(N-methylpyridinium)porphyrin iodide to form a 4:1:4 (host)4/guest4+/4I- complex. The single-crystal X-ray diffraction analysis shows the arrangement of the four cavities bound to the CH3N+ groups of the porphyrin moiety and the four iodide anions nested between the phenethyl substituents of the hosts. 1H NMR investigations show that the structure is preserved in chloroform solution and underscore the effect of the counteranions.

An efficient procedure for highly chemo- and stereoselective cyclization of (S)-allylalanine derivatives is reported (diastereomeric ratios up to 96:4) where the reaction course can be completely controlled by switching from gamma-lactones to cyclic carbamates simply with the proper choice of the amino acid protecting groups. Both processes are stereoconvergent and afford the (S,S)-products in high yields, short reaction times, and mild reaction conditions.

Studies comparing the binding of genuine cobalamin (vitamin B12) to that of its natural or synthetic analogues have long established increasing ligand specificity in the order haptocorrin, transcobalamin and intrinsic factor, the high-affinity binding proteins involved in cobalamin transport in mammals. In the present study, ligand specificity was investigated from a structural point of view, for which comparative models of intrinsic factor and haptocorrin are produced based on the crystal structure of the homologous transcobalamin and validated by results of published binding assays. Many interactions between cobalamin and its binding site in the interface of the two domains are conserved among the transporters. A structural comparison suggests that the determinant of specificity regarding cobalamin ligands with modified nucleotide moiety resides in the beta-hairpin motif beta3-turn-beta4 of the smaller C-terminal domain. In haptocorrin, it provides hydrophobic contacts to the benzimidazole moiety through the apolar regions of Arg357, Trp359 and Tyr362. Together, these large side chains may compensate for the missing nucleotide upon cobinamide binding. Intrinsic factor possesses only the tryptophan residue and transcobalamin only the tyrosine residue, consistent with their low affinity for cobinamide. Relative affinity constants for other analogues are rationalized similarly by analysis of steric and electrostatic interactions with the three transporters. The structures also indicate that the C-terminal domain is the first site of cobalamin-binding since part of the beta-hairpin motif is trapped between the nucleotide moiety and the N-terminal domain in the final holo-proteins.

Pax-8 is a member of the Pax family of transcription factors and is essential in the development of thyroid follicular cells. Pax-8 has two DNA-binding domains: the paired domain and the homeo domain. In this study, a preliminary X-ray diffraction analysis of the mammalian Pax-8 paired domain in complex with the C-site of the thyroglobulin promoter was achieved. The Pax-8 paired domain was crystallized by the hanging-drop vapor-diffusion method in complex with both a blunt-ended 26 bp DNA fragment and with a sticky-ended 24 bp DNA fragment with two additional overhanging bases. Crystallization experiments make clear that the growth of transparent crystals with large dimensions and regular shape is particularly influenced by ionic strength. The crystals of Pax-8 complex with blunt-ended and sticky-ended DNA, diffracted synchrotron radiation to 6.0 and 8.0 A resolution and belongs both to the C centered monoclinic system with cell dimensions: a = 89.88 A, b = 80.05 A, c = 67.73 A, and beta = 124.3 degrees and a = 256.56, b = 69.07, c = 99.32 A, and beta = 98.1 degrees , respectively. Fluorescence experiments suggest that the crystalline disorder, deduced by the poor diffraction, can be attributed to the low homogeneity of the protein-DNA sample. The theoretical comparative model of the Pax-8 paired domain complexed with the C-site of the thyroglobulin promoter shows the probable presence of some specific protein-DNA interactions already observed in other Pax proteins and the important role of the cysteine residues of PAI subdomain in the redox control of the DNA recognition.

The two peptides, rich in C(alpha)-tetrasubstituted amino acids, Ac-[Aib-L-(alphaMe)Val-Aib](2)-L-His-NH(2) (1) and Ac-[Aib-L-(alphaMe)Val-Aib](2)-O-tBu (2 a) are prevalently helical. They present the unique property of changing their conformation from the alpha- to the 3(10)-helix as a function of the polarity of the solvent: alpha in more polar solvents, 3(10) in less polar ones. Conclusive evidence of this reversible change of conformation is reported on the basis of the circular dichroism (CD) spectra and a detailed two-dimensional NMR analysis in two solvents (trifluoroethanol and methanol) refined with molecular dynamics calculations. The X-ray diffractometric analysis of the crystals of both peptides reveals that they assume a prevalent 3(10)-helix conformation in the solid state. This conformation is practically superimposable on that obtained from the NMR analysis of 1 in methanol. The NMR results further validate the reported CD signature of the 3(10)-helix and the use of the CD technique for its assessment.

Structural and 1H NMR data have been obtained for cobaloximes with the bulkiest substituted pyridines reported so far. We have isolated in noncoordinating solvents the complexes CH3Co(DH)2L (methylcobaloxime, where DH = the monoanion of dimethylglyoxime) with L = sterically hindered N-donor ligands: quinoline, 4-CH3quinoline, 2,4-(CH3)2pyridine, and 2-R-pyridine (R = CH3, OCH3, CH2CH3, CH=CH2). We have found that the Co-N(ax) bond is very long in the structurally characterized complexes. In particular, CH3Co(DH)2(4-CH3quinoline) has a longer Co-N(ax) bond (2.193(3) A) than any reported for methylcobaloximes. The main cause of the long bonds is unambiguously identified as the steric bulk of L by the fairly linear relationship found for Co-N(ax) distance vs CCA (calculated cone angle, CCA, a computed measure of bulk) over an extensive series of methylcobaloximes. The linear relationship improves if L basicity (quantified by pKa) is taken into account. In anhydrous CDCl3 at 25 degrees C, all complexes except the 2-aminopyridine adduct exhibit 1H NMR spectra consistent with partial dissociation of L to form the methylcobaloxime dimer. 1H NMR experiments at -20 degrees C allowed us to assess qualitatively the relative binding ability of L as follows: 2,4-(CH3)2pyridine > 4-CH3quinoline approximately = quinoline approximately = 2-CH3pyridine > 2-CH3Opyridine > 2-CH3CH2pyridine > 2-CH2=CHpyridine. The broadness of the 1H NMR signals at 25 degrees C suggests a similar order for the ligand exchange rate. The lack of dissociation by 2-aminopyridine is attributed to an intramolecular hydrogen bond between the NH2 group and an oxime O atom. The weaker than expected binding of 2-vinylpyridine relative to the Co-N(ax) bond length is attributed to rotation of the 2-vinyl group required for this bulky ligand to bind to the metal center, a conclusion supported by pronounced changes in 2-vinylpyridine signals upon coordination.

Cobalamin (Cbl, vitamin B(12)) serves for two essential cofactors in mammals. The pathway for its intestinal absorption, plasma transport, and cellular uptake uses cell surface receptors and three Cbl-transporting proteins, haptocorrin, intrinsic factor, and transcobalamin (TC). We present the structure determination of a member of the mammalian Cbl-transporter family. The crystal structures of recombinant human and bovine holo-TCs reveal a two-domain architecture, with an N-terminal alpha(6)-alpha(6) barrel and a smaller C-terminal domain. One Cbl molecule in base-on conformation is buried inside the domain interface. Structural data combined with previous binding assays indicate a domain motion in the first step of Cbl binding. In a second step, the weakly coordinated ligand H(2)O at the upper axial side of added H(2)O-Cbl is displaced by a histidine residue of the alpha(6)-alpha(6) barrel. Analysis of amino acid conservation on TC's surface in orthologous proteins suggests the location of the TC-receptor-recognition site in an extended region on the alpha(6)-alpha(6) barrel. The TC structure allows for the mapping of sites of amino acid variation due to polymorphisms of the human TC gene. Structural information is used to predict the overall fold of haptocorrin and intrinsic factor and permits a rational approach to the design of new Cbl-based bioconjugates for diagnostic or therapeutic drug delivery.

The interactions of the tetracationic meso-tetrakis(N-methyl-4-pyridyl)porphyrin (H(2)TMPyP) and its metallo derivatives (MTMPyP) (where M=copper(II), zinc(II), and gold(III) with the octa-anionic form (at neutral pH) of 5,11,17,23-tetrasulfonato-25,26,27,28-tetrakis(hydroxycarbonylmethoxy)calix[4]arene (C(4)TsTc) lead to a series of complex species whose stoichiometry and porphyrin sequence can be easily tuned. Crystallographic, spectroscopic, and diffusion NMR studies converge towards a common picture in which a central 1:4 porphyrin/calixarene unit serves as a template for the formation of more complex species. These species arise by successive, stepwise addition of single porphyrin molecules above and below the plane of the 1:4 central core to ultimately give a 7:4 complex. Noticeably, the stoichiometry of the various complex species corresponds to the actual concentration ratio of porphyrins and calixarenes in solution allowing the stoichiometry of these species to be easily tuned. This behavior and the remarkable stability of these species allow homo-porphyrin and hetero-(metallo)porphyrin species to be formed with control of not only the stoichiometry but also the sequence of the porphyrin array. The flexibility and ease of this approach permit, in principle, the design and synthesis of porphyrin arrays for predetermined purposes. For example, we have shown that it is very easy to design and obtain mixed porphyrin species in which a foreseen photoinduced electron-transfer is indeed observed.

The de novo protein DF1 is a minimal model for diiron and dimanganese metalloproteins, such as soluble methane monooxygenase. DF1 is a homodimeric four-helix bundle whose dinuclear center is formed by two bridging Glu side chains, two chelating Glu side chains, and two monodentate His ligands. Here, we report the di-Mn(II) and di-Co(II) derivatives of variants of this protein. Together with previously solved structures, 23 crystallographically independent four-helix bundle structures of DF1 variants have been determined, which differ in the bound metal ions and size of the active site cavity. For the di-Mn(II) derivatives, as the size of the cavity increases, the number and polarity of exogenous ligands increases. This collection of structures was analyzed to determine the relationship between protein conformation and the geometry of the active site. The primary mode of backbone movement involves a coordinated tilting and sliding of the first helix in the helix-loop-helix motif. Sliding depends on crystal-packing forces, the steric bulk of a critical residue that determines the dimensions of the active site access cavity, and the intermetal distance. Additionally, a torsional motion of the bridging carboxylates modulates the intermetal distance. This analysis provides a critical evaluation of how conformation, flexibility, and active site accessibility affect the geometry and ligand-binding properties of a metal center. The geometric parameters defining the DF structures were compared to natural diiron proteins; DF proteins have a restricted active site cavity, which may have implications for substrate recognition and chemical stability.

The electronic structure of hydroxocobalamin (OHCbl) has been calculated by a density functional method, using the orthogonalized linear combination of the atomic orbitals method (OLCAO). The X-ray crystal structure has been determined from synchrotron X-ray diffraction data and the geometry determined was used in the calculations. Comparison with the recently reported electronic structures of cyanocobalamin (CNCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) shows that Mulliken charges (Q*) and bond orders (BO) vary only on the axial fragment.

Protein design provides an attractive approach to test the essential features required for folding and function. Previously, we described the design and structural characterization in solution of mimochromes, a series of miniaturized metalloproteins, patterned after the F-helix of the hemoglobin beta-chain. Mimochromes consist of two medium-sized helical peptides, covalently linked to the deuteroporphyrin. CD and NMR characterization of the prototype, mimochrome I, revealed that the overall structure conforms well to the design. However, formation of Delta and Lambda diastereomers was observed. To overcome the problem of diastereomer formation, we re-designed mimochrome I, by engineering intramolecular, interchain interactions. The resulting model was mimochrome IV: the solution structural characterization showed the presence of the Lambda isomer as a unique form. To examine the extent to which the stereochemical stability and uniqueness of mimochrome IV was retained in the solid state, the crystal structure of Co(III)-mimochrome IV was solved by X-ray diffraction, and compared to the solution structure of the same derivative. Co(III)-mimochrome IV structures, both in solution and in the solid state, are characterized by the following common features: a bis-His axial coordination, a Lambda configuration around the metal ion, and a predominant helical conformation of the peptide chains. However, in the crystal structure, intrachain Glu1-Arg9 ion pairs are preferred over the designed, and experimentally found in solution, interchain interactions. This ion pairing switch may be related to strong packing interactions.

The kinetics of axial water substitution by azoles (pyrazole and 1,2,4-triazole) in three different cobaloximes, viz.trans-[Co(Hdmg)(2)(R)H(2)O] where Hdmg = dimethylglyoximate, R = PhCH(2), Et and CF(3)CH(2), were studied as a function of azole concentration, temperature and pressure in aqueous solution. The second order rate constants for the substitution of water in trans-[Co(Hdmg)(2)(R)H(2)O] for R = Et at pH 6.0, 25 degrees C and I= 0.1 M (NaClO(4)), were found to be 1309 and 1200 M(-1) s(-1) for pyrazole (Pz) and 1,2,4-triazole (Tz), respectively, and those obtained for R = PhCH(2) were found to be 755 and 691 M(-1) s(-1), respectively. The second order rate constants in the case of R = CF(3)CH(2) were found to be 0.358 and 0.348 M(-1) s(-1) for Pz and Tz, respectively. The relative order of reactivity for the different alkyls being Et > PhCH(2) > CF(3)CH(2). The activation parameters (DeltaH([not equal]), DeltaS([not equal]) and DeltaV([not equal])) obtained for these reactions were found to be in the range of 65-87 kJ mol(-1), 24-47 J mol(-1) K(-1) and 2.5-7.7 cm(3) mol(-1), respectively. These data suggest that an I(d) substitution mechanism operates where the azoles participate in the transition state.

The reaction of a cis beta folded organocobalt derivative with a salen-type ligand, 1, isolated as racemic compound of Delta and Lambda enantiomers, with enantiomerically pure alpha-l amino acids is reported. The reaction between racemic 1 and l-tyrosine afforded a mixture of the two diastereoisomers Delta-2 and Lambda-2, which could be separated by fractional crystallization owing to the lower solubility of Delta-2. The absolute configuration of the two diastereomers was unequivocally assigned from the X-ray structure, using the known absolute configuration of the asymmetric carbon of the amino acid as internal reference. The reaction of racemic 1 with trans-4-hydroxy-l-proline afforded only the diastereoisomer with a Delta configuration of the tetradentate ligand, as proved by X-ray diffractometric analysis. For both l-tyrosine and trans-4-hydroxy-l-proline, the amino acid initially coordinates both to the Delta and to the Lambda enantiomers of 1, leading to an about equimolar mixture of diastereoisomers. In the case of l-tyrosine the diastereoisomers have about the same energy, so that the successive isomerization is negligible. In the case of trans-4-hydroxy-l-proline, Delta-3 is much more stable than Lambda-3, and the isomerization reaction Lambda-3 --> Delta-3 goes practically to completion.

The electronic structure of adenosylcobalamin (B12 coenzyme, AdoCbl) has been calculated by a density functional method, using the orthogonalized linear combination of the atomic orbital method (OLCAO). Since a fixed accurately determined geometry was needed in such calculations, the crystal structure of adenosylcobalamin has been redone and refined to R = 0.065, using synchrotron diffraction data. Comparison with the recently reported electronic structures of cyano- (CNCbl) and methylcobalamin (MeCbl) shows that the net charges and bond orders vary only on the axial donors. The values in the three cobalamins suggest that the Co-C bond in MeCbl has a strength similar to that in AdoCbl, but it is significantly weaker that that in CNCbl. Present results are compared with those previously reported for the analogous corrin derivatives; i.e., simplified cobalamins with the side chains a-f replaced by H atoms. Despite a qualitative agreement, a discrepancy in the calculated HOMO-LUMO gap is found.

The kinetics of axial water substitution by cysteine in six different cobaloximes, viz.trans-RCo(Hdmg)(2)H(2)O, where Hdmg = dimethylglyoximate, R = cyclo-C(5)H(9) (c-P), CH(3)CH(2) (Et), CH(3) (Me), C(6)H(5)CH(2) (Bz), C(6)H(5) (Ph) and CF(3)CH(2), were studied as a function of cysteine concentration, temperature and pressure. It was found that cysteine substitutes the coordinated H(2)O molecule trans to the alkyl group with second order rate constants that follow the order of reactivity: c-P > Et > Bz > Me > Ph > CF(3)CH(2). Rate and activation parameters (Deltan H(++), Delta S(++) and Delta V(++)) enable the formulation of a reaction mechanism that can account for the substitution behaviour of the investigated alkylcobaloximes. In particular, a gradual mechanistic changeover from I(d) to I is observed along the series of R groups from c-P to CF(3)CH(2).

The reduction of [Co(III)(tmsalen)py(2)](+)ClO(4)(-), where tmsalen = 4,4',7,7'-tetramethylsalen, with NaBH(4)/PdCl(2) in alkaline methanolic solution, followed by the oxidative addition of CH(2)ClI, leads to the expected trans organometallic dimeric species 1, [CH(2)ClCo(tmsalen)](2), provided that the product is recovered from the reaction mixture immediately after the completion of the reaction. If 1 is left for longer time in contact with the reaction mixture, the intramolecular reaction of the axial chloromethyl group with the equatorial chelate leads to the formation of the monocationic complex 2, containing a seven-membered ring. In this complex the novel tetradentate ligand coordinates Co in a cis fashion, the other two positions being occupied by one py and one water molecule. The resulting complex is chiral, even if the reaction product is a racemic compound. The unidentate ligands of 2 have been exchanged quantitatively for N-MeIm, and the resulting complex 3 still maintains the beta cis geometry. Therefore, 2 may be considered the precursor of a new class of organocobalt derivatives with a folded tetradentate ligand and two adjacent exchangeable sites. On the basis of the geometry of the tetradentate Schiff bases in complexes, where they adopt a planar geometry, it was suggested that there is a significant electron density delocalization involving the metal center over the two chemically equivalent moieties of Co(chel). Comparison of the geometry of the planar salicylaldiminate (sal) moiety with that of the cyclized methoxy-iminate one (imi) in 2 and 3 strongly supports that the delocalization, still present in sal, is essentially either lost or strongly reduced in imi.

Diffraction data from two non-isomorphous crystals (forms 1 and 2) of an artificial protein with a four-helix bundle motif, di-Co(II)-DF1-L13A, have been collected using synchrotron radiation. The phase of form 1 has been assigned using the group and minimal non-isomorphic supergroup relation between the space group of the previously determined di-Mn(II)-DF1-L13G structure and the space group of this form. This unconventional method of solving the phase problem has also been tested with form 2 using a reverse relation. The structure of the latter form has been solved using the group and maximal non-isomorphic subgroup relation with the space group of form 2 of the analogous dimanganese protein. This application has shown that this phasing method can be used for solving the protein structures of polymorphic crystals as an alternative to the molecular-replacement method.

Results of the accurate crystal structure determination of NO(2)Cbl.2LiCl (1), NO(2)Cbl.NaCl (2), NCSCbl (3) and NCSeCbl (4), based on synchrotron diffraction data collected at 100 K, are described. The nitro group in (1) was found to be disordered with two orientations that differ by a rotation of approximately 60 degrees about the Co-NO(2) bond, whereas in (2) the nitro group has only one orientation. The first X-ray structural determination of a cobalamin with a Co-Se bond is reported. Comparison of the axial distances indicates that SeCN has a bond length of 2.384 (3) A and that the trans influence on the Co-N bond is only slightly greater than that of SCN. The crystals of the thiocyanate cobalamin contain both the S- and N-bonded coordination isomers in a 3:2 ratio. The structural features of the Co-S bond in cobalamins are discussed. The crystal chemistry of cobalamins is discussed in terms of packing of roughly spherical molecules. The unit-cell parameters can be used to group the cobalamins' crystal structures in different arrays intermediate between distorted hexagonal close packing and primitive hexagonal arrangements. The structural features of cobalamins, and of cobaloximes that have the same axial fragment as the cobalamins, are reviewed and discussed in terms of the cis influence of the equatorial ligand.

The guest-induced synthesis of a molecular box from methylaquacobaloxime and 1,4-phenylenebisboronic acid, with pyrazine (pz) as guest, is described. The resulting supramolecular species was characterized by X-ray structural analysis, 1H and 13C NMR spectroscopy, and low-resolution electrospray ionization (ES) mass spectrometry. The assembly was monitored by a time dependent 1H NMR experiment, which showed that the guest thermodynamically drives the assembly of the host. The effectiveness of pyrazine in this role arises from its having both the correct geometry and a sufficiently low pKa value. Several other ditopic ligands were tested, but none led to formation of an analogous product. However, a second supramolecular species was formed in the case of ethylenediamine (en). X-ray structural analysis and 1H and 13C spectroscopy revealed that this is closely related to the first, with a phenyl side removed and the pz bridge substituted by the en bridge.

The complex mer-[Co(III)(L(1)Npy)(2)](+) (1') where the L(1)Npy(-) is the tridentate 3-[(2-pyridyl)methylimino]butan-2-one oximate ligand, gives alkyl-cobalt derivatives after reduction with NaBH(4)/Pd(2+) to the Co(I) and alkylation. The formation of the cobalt-carbon bond is accompanied by the reduction to the amino form of one or both imino ligands (depending on the experimental conditions) initially present in 1'. In one series of experiments, complexes of the type fac-[RCo(III)(L(1)Npy)(H-L(1)NHpy)](+) (R = Me, i-Pr, CH(2)Cl, CH(2)Br, CH(2)CF(3), and Bz) were obtained, in which only one of the two ligands was reduced to the amino form (H-L(1)NHpy). The saturation of one azomethine group causes the products to assume a fac configuration and induces the formation of one asymmetric carbon and one asymmetric nitrogen center in the chelating system. When an excess of reducing agent is used, both azomethine groups may be saturated, causing the introduction of one pair of chiral carbons and one pair of chiral nitrogens. Two isomers of the methyl derivative [MeCo(III)(L(1)NHpy)(H-L(1)NHpy)](+) were isolated. The X-ray analysis reveals that these isomers differ from one another in configuration of the C and N chiral centers. Possible reaction mechanisms leading to these different types of complexes are proposed.

The three-dimensional structures of the native cytochrome c(2) from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 A and in the reduced state at 1.95 A resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c(2) with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3(10)-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c(2) with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles phi and psi of approximately -140 and -130 degrees, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 A resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c(2) of the rare six-residue insertion in the helix 3(10) conformation that increases Met loop flexibility.

De novo design of proteins provides an attractive approach to uncover the essential features required for their functions. Previously, we described the design and crystal structure determination of a di-Zn(II) complex of "due-ferri-1" (DF1), a protein patterned after the diiron-dimanganese class of redox-active proteins [Lombardi, A.; Summa, C.; Geremia, S.; Randaccio, L.; Pavone, V.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6298-6305]. The overall structure of DF1, which contains a carboxylate-bridged dinuclear metal site, agrees well with the intended design. However, access to this dimetal site is blocked by a pair of hydrophobic leucine residues (L13 and L13'), which prevent facile entry of metal ions and small molecules. We have now taken the next step in the eventual construction of a catalytically active metalloenzyme by engineering an active site cavity into DF1 through the replacement of these two leucine residues with smaller residues. The crystal structure of the dimanganous form of L13A-DF1 indeed shows a substrate access channel to the dimetal center. In the crystal structure, water molecules and a ligating dimethyl sulfoxide molecule, which forms a monatomic bridge between the metal ions, occupy the cavity. Furthermore, the diferric form of a derivative of L13A-DF1, DF2, is shown to bind azide, acetate, and small aromatic molecules.

Transcobalamin is a cobalamin-binding protein in mammalian plasma that facilitates the cellular uptake of vitamin B(12). Human transcobalamin was crystallized using polyethylene glycol and ethanol as precipitants. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.04, b = 145.27, c = 164.96 A. A complete data set to 3.2 A resolution was collected from a single crystal using synchrotron radiation. Estimation of the crystal packing (V(M) = 3.2 A(3) Da(-1)) and self-rotation function analysis suggest the presence of two molecules in the asymmetric unit related by non-crystallographic twofold symmetry.

Self-aggregation of water-soluble pyridinium-substituted porphyrins with functionalized calixarenes leads to well-defined pH-tunable stoichiometries. Two assemblies with different porphyrin:calixarene ratios that crystallized at pH 2 and 6 have been structurally characterized. The Job plots of Soret band broadenings indicate that these structures are also preserved in solution., (Copyright © 2001 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)

The reaction of CH(3)Co(DH)(2)H(2)O with 4-pyridinyl boronic acid in methanol or water affords the dinuclear complexes [MeCo(DH)(DB(OR)(4-Py))](2), with R = Me (2) or H (3), respectively, through reaction of boron with the oxime oxygens of the alkylcobaloxime and coordination of the pyridinyl N to cobalt. The reaction is strongly pH dependent, and the formation of the complexes requires a neutral medium. The complexes have been fully characterized by (1)H and (13)C NMR spectroscopy, ESI-MS spectrometry, and elemental analysis. The X-ray structure shows that in 2, the pyridinyl groups are facing each other and nearly perpendicular both to the plane of the Co B Co1 B1 atoms and to the mean equatorial plane, so that the complex may be considered a molecular box. A dimeric arrangement has already been found in the related [MeCo(DH)(DB(OMe)(3-Py))](2) (1) complex, which forms a distorted molecular rectangle [Dreos, R.; Nardin, G.; Randaccio, L.; Tauzher, G.; Vuano, S. Inorg. Chem. 1997, 36, 2463]. The dimerization is possible in both cases, as the conformational freedom of the B bridge compensates for the different position (3- or 4-) of the pyridinyl N donor.

Treatment of R = -CH(2)X (X = halogen) derivatives of the type [RCo(III)(LNH-py)(HLNH-py)](+), where HLNH-py = 2-(2-pyridyl-ethyl)amino-3-butanone oxime and LNH-py its conjugated base with diluted NaOH, afforded a new complex containing a three-membered ring by a pathway involving the intramolecular nucleophilic addition of an equatorial nitrogen donor to the axial carbon. The X-ray analysis reveals a highly distorted structure. The C-Co-N angle is acute (42.8 degrees ) with the distortion of the coordination sphere concentrated in the Co-C axial and Co-N bonds, which move away from the pseudo-octahedral positions in the CH(2)X parent complex to form the C-N bond of the metallocycle. Kinetic studies of the formation of this novel complex starting from [(XCH(2)Co(III)(LNH-py)(HLNH-py)](+) (X = Cl,Br,I) showed that the metallocycle formation rates increase in the order Cl < Br < I. Kinetic data are consistent with a mechanism involving an intermediate species resulting from the deprotonation of an amine equatorial nitrogen in a rapid preequilibrium, followed by the slow step of the ring closure.

Cytochrome c(2) from Rhodopseudomonas palustris has been crystallized in two different crystal forms: a monoclinic form I at pH 4.4 from both reduced and oxidized protein solution and a trigonal form II at pH 9.0 from reduced protein solution. Complete 1. 7 and 1.4 A resolution data sets were collected from the oxidized form I and from the form II, respectively. The preliminary structures show an important change in the iron coordination environment in the trigonal form obtained at basic pH arising from the substitution of the Met ligand by an ammonia molecule.

The accurate crystal structure determinations of MeCbl (1), CNCbl.2LiCl (2), and CNCbl.KCl (3), based on synchrotron diffraction data collected at 100 K and using high-quality single crystals, are reported. Refinements gave R1 indices of 0.0834 (1), 0.0434 (2), and 0.0773 (3). The influence of the water of crystallization and ion content on the crystal packing of these and other cobalamins (XCbl) is discussed, and a relationship between the crystal packing and the corrin side chain conformations is presented. An analysis of the bond lengths within the corrin moiety, based on 13 accurate structures with several X groups, shows that the trend of the C-C and C-N distances can be interpreted in terms of electronic and steric factors. The variation in structural, NMR and IR spectroscopic, and electrochemical properties are compared with those of cobaloximes, the B12 model, when X is varied. This comparison indicates that the pi-back-donation from metal to the CN axial ligand and the transmission of the trans influence of the X ligand are more effective in cobalamins than in cobaloximes. These findings are consistent with a significantly greater availability of electron charge on Co in cobalamins, and, hence, a semiquantitative evaluation of the electronic difference between the cobalt centers in the two systems is allowed.

De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 A rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the Glu-Xxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 A. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation.

The dicopper(II) complex with the ligand N,N,N',N',N"-pentakis[(1-methyl-2-benzimidazolyl)methyl]dipropylenetriamine (LB5) has been synthesized and structurally characterized. The small size and the quality of the single crystal required that data be collected using synchrotron radiation at 276 K. [Cu(2)(LB5)(H(2)O)(2)][ClO(4)](4): platelet shaped, P&onemacr;, a = 11.028 Å, b = 17.915 Å, c = 20.745 Å, alpha = 107.44 degrees, beta = 101.56 degrees, gamma = 104.89 degrees, V = 3603.7 Å(3), Z = 2; number of unique data, I >/= 2sigma(I) = 3447; number of refined parameters = 428; R = 0.12. The ligand binds the two coppers nonsymmetrically; Cu1 is coordinated through five N donors and Cu2 through the remaining three N donors, while two water molecules complete the coordination sphere. Cu1 has distorted TBP geometry, while Cu2 has distorted SP geometry. Voltammetric experiments show quasireversible reductions at the two copper centers, with redox potential higher for the CuN(3) center (0.40 V) and lower for the CuN(5) center (0.17 V). The complex binds azide in the terminal mode at the CuN(3) center with affinity lower than that exhibited by related dinuclear polyaminobenzimidazole complexes where this ligand is bound in the bridging mode. The catechol oxidase activity of [Cu(2)(LB5)](4+) has been examined in comparison with that exhibited by [Cu(2)(L-55)](4+) (L-55 = alpha,alpha'-bis{bis[(1-methyl-2-benzimidazolyl)methyl]amino}-m-xylene) and [Cu(2)(L-66)](4+) (L-66 = alpha,alpha'-bis{bis[2-(1-methyl-2-benzimidazolyl)ethyl]amino}-m-xylene) by studying the catalytic oxidation of 3,5-di-tert-butylcatechol in methanol/aqueous buffer pH 5.1. Kinetic experiments show that [Cu(2)(L-55)](4+) is the most efficient catalyst (rate constant 140 M(-1) s(-1)), followed by [Cu(2)(LB5)](4+) (60 M(-1) s(-1)), in this oxidation, while [Cu(2)(L-66)](4+) undergoes an extremely fast stoichiometric phase followed by a slow and substrate-concentration-independent catalytic phase. The catalytic activity of [Cu(2)(L-66)](4+), however, is strongly promoted by hydrogen peroxide, because this oxidant allows a fast reoxidation of the dicopper(I) complex during turnover. The activity of [Cu(2)(LB5)](4+) is also promoted by hydrogen peroxide, while that of [Cu(2)(L-55)](4+) is little affected. The phenol monooxygenase activity of [Cu(2)(LB5)](2+) has been compared with that of [Cu(2)(L-55)](2+) and [Cu(2)(L-66)](2+) by studying the ortho hydroxylation of methyl 4-hydroxybenzoate to give methyl 3,4-dihydroxybenzoate. The LB5 complex is much more selective than the other complexes since its reaction produces only catechol, while the main product obtained with the other complexes is an addition product containing a phenol residue condensed at ring position 2 of the catechol.