EXAFS AND X-RAY STUDIES OF B₁₂ MODEL COMPOUNDS. Christoph Kratky, Institut für physikalische Chemie, A-8010 Graz - Austria.

The chemical and structural complexity of the B₁₂ coenzymes (5'-desoxyadenosyl cobalamin and methyl cobalamin) and their biologically relevant reactivities (Co-C bond homolysis and Co-C bond heterolysis, respectively) has been a puzzle ever since the elucidation of their structures by Dorothy Hodgkin's group more than three decades ago. For many years, the focus of structural and chemical research lay on the isolated cofactor or cofactor analogue, with special emphasis on the cobalt center and its coordination environment.

Along these lines, we have determined crystal structures of a number of cobalamins with different [[alpha]]- and [[beta]]-substituents. Using synchrotron radiation in combination with imaging plate detectors for some of these analyses, we were able to collect very accurate and comprehensive data sets, which permitted structure refinement to a level of precision comparable to a well-determined small-molecule crystal structure. From the combined structural data of about 20 cobalamin crystal structures, correlations between several characteristic intra-molecular deformation parameters (upward-folding of the corrin ring, axial Co-N distance, orientation of the dimethylbenzimidazole base) can be established and used to estimate the relative "stiffness" of each of these deformation modes.

In recent years, the B₁₂ field has advanced dramatically as a result of the elucidation of the first crystal structures of proteins binding a B₁₂ cofactor (B₁₂ binding domain of methionine synthase; methylmalonyl CoA mutase). In both proteins, a protein-derived histidine-imidazole occupies the [[alpha]]-axial coodination of the cobalt center, replacing the dimethylbenzimidazole base occupying this position in solutions of the isolated cofactor under physiological conditions. Thus, it appears that some of the above structural correlations refer to a biologically irrelevant cofactor constitution.

For a number of representative compounds, we have also collected X-ray absorption spectra as a basis for the interpretation of EXAFS spectra of cobalamins in ,,non-crystalline" environments (e.g. cobalamins in solution and bound to a protein). Thus, by a comparison of the spectra of Aquocobalamin perchlorate in solution and in crystalline form, we could show that there is no detectable difference in the cobalt environment between the two states and that the conformation observed in the crystal structure is also representative for the aqueous solution.

Very pronounced differences exist in the near-edge region between cobalt(II), cobalt(III) and alkyl (e.g. methyl) cobalamins. For several Sporomusa Ovata proteins carrying a p-cresolyl-cobamide cofactor, strong indications could be derived from the EXAFS and near-edge regions of the X-ray absorption spectrum that the cofactor occurs in the methylated form under oxidative ambient conditions.