Crystal and Molecular Structure of Vitamin B12 Modified Coenzymes.

Compounds belonging to the vitamin B12 family (X-Cbl, Figure 1), the so-called cobalamins, are octahedral Co(III) complexes co-ordinated in the equatorial positions by the polycyclic corrin nuleus and in the axial positions by a 5,6-dimethybenzi- midazole residue and an X group. X = CN; corresponds to cyanocobalamin or vitamin B12 (CNCbl); X = 5’-deoxyi-5’-adenosyl to coenzyme B12 (adoCbl); X = CH3; methylcobalamin (MeCbl). On the pyrrole rings, there are seven amide side chains a, b, c, d, e, f e g and chain f is bonded to the phosphate group of the axially co-ordinated nucleotide (Figure 1). Since the pioneering work of D. Hodgkin on cyanocobalamin, crystal structures of cobalamins, in contrast with those of the simple models, such as cobaloximes, were not accurate enough for even a basic discussion, having R factor higher that 0.10. 1Only recently have a few structures of cobalamins been reported with accuracy similar to that of high resolution small molecules (R = 0.04-0.06), thanks to the synchrotron radiation.2 In a first series of experiments, high quality of single crystals of cobalamins N3-Cbl·2LiCl, Cl-Cbl·2LiCl, CN-Cbl·2LiC land CN-Cbl·KCl 2b have been obtained and X-ray diffraction data collected at ELETTRA. The structures were solved and refined to R 0.05 at 100 and 275 K. The most part of cobalamins are isomorphous and crystallize in space P212121. A plot of the unit cell edge ratios, c/a versus b/a, shows that they can be grouped in four clusters, having the same arrangement of the molecules in a plane and differing in the way in which these planes are packed. 2, 3 The structures of X-Cbl;2LiCl belong to cluster 4, while that of CN-Cbl;KCl to cluster 1. The structures of vitamin B12 and adoCbl belong to clusters 2 and 3, respectively.
The projections of the crystal structure for clusters 1 and 4 along the crystallographic axis a are depicted in Figures 2 and 3 and show that the molecules form large cavities running along that axis and containing the crystallisation water molecules and the ions.

The cavity of CNCbl.KCl, viewed along the crystallographic axis c is shown in Figure 4, together with the water molecule and ion content.

The cavity consists of channels, containing disordered water molecules, connected to side pockets where the ions and ordered water molecules are located. Similarly shaped cavities characterise the structures of cluster 4, with two cations and two anions located in each pocket. The study of the molecular structures, which have been found scarcely affected by static disorder, has shown that the structures belonging to the same cluster have very similar side chain conformations.2b

The structures of cluster 4 are characterised by two intramolecular H-bonds, one between the chain c and X, the other between the OR8 ribosyl and of the e chain O51 atoms, whereas structures of cluster 1 exhibit only the first type of H-bond.
No intramolecular H-bond is detected in the other clusters. In the structure of cluster 4, presented here, all the amide side chains, except c, are involved in electrostatic interactions with cations through their O atoms and in H-bond with anions through their NH groupings (Figure 5). In CN-Cbl·KCl, belonging to cluster 1, the a, d and e amide chains are not involved in interactions with ions (Figura 6). These structures represent the first structural characterisation of interactions of the corrin side chains with ions, which has been suggested to be important to discriminate MeCbl and adoCbl solution behaviour. 4 In another series of experiments some S-containing cobalamins have been crystallized and their structures solved and refined by using X-ray diffraction data collected at ELETTRA.2c Methylcobalamin, (MeCbl), (X = CH3) is the coenzyme of methionine synthase, which catalyses the conversion of homocysteine to methionine.6 Model chemistry suggested that homocysteinate reacts with MeCbl, to form a Co(I)Cbl intermediate, through the heterolytic Co-C cleavage. A mechanism for the MeCbl function in the enzyme has proposed the Co thiolate coordination trans to the Me group ( thiolate ligation), with consequent weakening of the Co-C bond. However, the reports on this reaction were controversial. 6 With the aim to contribute to this problem, the first structural characterization of the axial fragment in cobalamins, containing S-ligand, is described. The preparation and the crystal structure determination of two cobalamins, (SO3Cbl)(NH4).nH2O (1, X = SO32-) and [(NH2)2CSCbl](PF6).nH2O (2, X= thiourea), based on synchrotron data collected at 100 K, are reported. The refinement gave R= 0.054 for 1 and 0.078 for 2. 2c
The Co-S distances in 1 and 2 are 2.231(1) and 2.300(2) Å, respectively whereas the trans Co-NB3 distance of 2.134(4) Å in 1 is significantly longer than that of 2.032(5) Å in 2. Comparison of axial distances in cobalamins and in the simple model, pyCo(DH)2X (cobaloximes, DH= monoanion of dimethylglyoxime), allows to evaluate the length, not yet experimentally determined, of the Co-NB3 and Co-SR bonds in thiolate cobalamins (RSCbl), to be 2.26 and 2.04 Å, respectively. This result suggests that the RS- trans influence is similar (or slightly larger) than that of thiourea, but significantly smaller than that of sulphite, which in turn is less trans influencing than Me. These findings support the previous suggestion obtained from simple models 6 that the thiolate ligation by homocysteine in MeCbl could be unlikely an important step in the processes involving methionine synthase. Further comparison between the axial fragment in cobalamin with that in cobaloximes shows that the Co-X distances are approximately equal in both series, whereas the Co-py bonds are appreciably shorter than the Co-NB3 ones, when the s-donating ability of X increases. Thus, the difference between the corresponding Co-py and Co-NB3 distances is nearly zero for the weak donor H2O and about 0.2 Å for the good s-donor ado. This seems to supports the greater electron richness of the metal centre in cobalamins with respect to cobaloximes. Further studies are in progress.

References
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  3. K. Gruber, G. Jogl, G. Klintschar and C. Kratky, in Vitamin B12 and B12-Proteins, B. Krautler, D. Arigoni B. T. Golding, Eds. Wiley-VCH, Wienheim, 1998, pag. 335.
    4. N.E. Brasch, A.Zahl and R. van Eldik, Inorg. Chem., 1997, 36, 4891.
  4. C. L. Drennan, M. M. Dixon, D. M. Hoover, J. T. Jarret, C. W. Goulding, R. G. Mattews, M. L. Ludwig, in Vitamin B12 and B12- Proteins", Kräutler, B.; Arigoni, D. and Golding, B. T., Eds., Wiley-VCH publisher, Weinheim, 1998, pag. 133.
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