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ACCURATE STRUCTURAL DATA DEMYSTIFY B12: HIGH RESOLUTION SOLID-STATE STRUCTURE OF AQUOCOBALAMIN PERCHLORATE AND STRUCTURE ANALYSIS OF THE AQUOCOBALAMIN ION IN SOLUTION

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Author(s)

  • C KRATKY
  • G FARBER
  • K GRUBER
  • K WILSON
  • Z DAUTER
  • H F NOLTING
  • R KONRAT
  • B KRAUTLER

Department/unit(s)

Publication details

JournalJournal of the American Chemical Society
DatePublished - 26 Apr 1995
Issue number16
Volume117
Number of pages17
Pages (from-to)4654-4670
Original languageEnglish

Abstract

Experiments are described to elucidate the structure and solvation of aquocobalamin (vitamin B-12a, If) in the crystal and in aqueous solution. Aquocobalamin (1(+)) is the B-12 derivative in which a water molecule 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 imaging plate detector, yielded the most accurate structural data ever determined for a B-12 molecule. 1(+)ClO(4)(-) crystallizes in the orthorhombic space group P2(l)2(l)2(l), a = 15.042(11) Angstrom, b = 23.715(14) Angstrom, c = 25.104(12) Angstrom, with four 1(+)ClO(4)(-) moieties plus about 100 solvent water molecules per unit cell; 22867 independent 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 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 ''butterfly'' deformation with an ''upward'' fold angle of 18.71(0.07)degrees. The relevance of this observation for the ''upward 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 as from 1(+)ClO(4)(-) dissolved in a 1:1 mixture of water/ethylene glycol. 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 and 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 coordination number of 6 and an average distance of 1.90 Angstrom for both samples. 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 investigations on a B-12 derivative in H2O, including 2D home- and heteronuclear studies on aquocobalamin chloride (1(+)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(III) 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 1(+)ClO(4)(-).

However, significant differences 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-averaged conformation of the hydrogen-bonding c-acetamide side chain.

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