ACCURATE STRUCTURAL DATA DEMYSTIFY B-12 - HIGH-RESOLUTION SOLID-STATESTRUCTURE OF AQUOCOBALAMIN PERCHLORATE AND STRUCTURE-ANALYSIS OF THE AQUOCOBALAMIN ION IN SOLUTION

Experiments are described to elucidate the structure and solvation of aquocobalamin (vitamin B-12a, If) in the crystal and in aqueous soluti on. Aquocobalamin (1(+)) is the B-12 derivative in which a water molec ule replaces the axially coordinating organic substituent (methyl or 5 '-desoxyadenosyl) at the beta-side of the cobalt of the B-12 coenzymes . (1) A single-crystal structure analysis of aquocobalamin perchlorate (1(+)ClO(4)(-)), using synchrotron radiation in combination with an i maging plate detector, yielded the most accurate structural data ever determined for a B-12 molecule. 1(+)ClO(4)(-) crystallizes in the orth orhombic space group P2(l)2(l)2(l), a = 15.042(11) Angstrom, b = 23.71 5(14) Angstrom, c = 25.104(12) Angstrom, with four 1(+)ClO(4)(-) moiet ies plus about 100 solvent water molecules per unit cell; 22867 indepe ndent and 20942 significant intensity data were recorded to a nominal resolution of 0.8 Angstrom, and refinement against F-2 quantities led to a conventional R-value of 0.050 for all 22867 observations and to a structural model with an average ESD for all carbon-carbon bonds of 0 .003 Angstrom. In the crystal, the aquocobalamin ion has a very short axial bond between cobalt and the dimethylbenzimidazole (DMB) base of 1.925 (0.002) Angstrom, which is rationalized as resulting from the ve ry weak trans axial donor (water). Steric repulsion between the DMB ba se and the corrin ring induced by this short Co-DMB bond leads to a re latively large ''butterfly'' deformation with an ''upward'' fold angle of 18.71(0.07)degrees. The relevance of this observation for the ''up ward conformational deformation'' hypothesis for the initiation of Co- C bond homolysis in coenzyme B-12 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 a s from 1(+)ClO(4)(-) dissolved in a 1:1 mixture of water/ethylene glyc ol. Absorption spectra were recorded at 20 K between 7300 and 8700 eV, and the k(3)-weighted EXAFS was extracted for a k-value between 2.7 a nd 14.4 Angstrom(-1). EXAFS spectra for solid and dissolved 1(+)ClO(4) (-) 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 coordinatio n number of 6 and an average distance of 1.90 Angstrom for both sample s. There is no evidence for a longer Co-N distance. This refutes data published by Sagi and Chance (J. Am. Chem. Sec. 1992, 114, 8061). (3) NMR experiments are described, constituting the first detailed NMR inv estigations on a B-12 derivative in H2O, including 2D home- and hetero nuclear studies on aquocobalamin chloride (1(+)Cl(-)) and assignment o f signals due to the exchangeable amide protons of all nitrogens as we ll a measurements of amide proton exchange rates. The NMR data confirm the occupation of the axial coordination site at the Co(III) center b y water, as well as the occurrence in solution of an intramolecular hy drogen bond to the axially coordinating water molecule, as observed in the crystal structure of 1(+)ClO(4)(-). However, significant differen ces of the structure of 1(+) in crystals of 1(+)ClO(4)(-) and of 1(+) in aqueous solution are indicated from NOE data concerning the time-av eraged conformation of the hydrogen-bonding c-acetamide side chain.