New insights into the solution behavior of cobalamins. Studies of the base-off form of coenzyme B12 using modern two-dimensional NMR methods

Recently developed two-dimensional (2D) NMR methods are used to assign completely the IH and I3C NMR spectra of the base-off form of coenzyme BI2 (5'-deoxyadenosylcobalamin, M, = 1580). The NMR data, including coupling constants, chemical shifts, 2D proton-carbon multiple-bond peak intensities, and 2D NOE peak intensities, are compared with those of the base-on form of coenzyme B12 and with the structure recently determined by neutron diffraction. The NOE signal intensities between protons of the corrin and adenosyl indicate a dynamic equilibrium between at least two states: in one state, the orientation of the adenosyl with respect to the corrin is similar to that observed in the crystal structure, and in the other state, the adenosyl is rotated ca. 50 counterclockwise (when viewed from the adenosyl) about the Co-C bond. Furthermore, we find no evidence for a change in corrin ring pucker on dissociation of the 5,6-dimethylbenzimidazole (DMBz). For the base-off species, the DMBz is repositioned upside down with the B7, B1 1, and R1 protons in the vicinity of the C20 methyl group. Previously unexplainable shift trends in the base-on to base-off conversion are more readily rationalized by use of the new data since, in earlier studies, errors were made both in assigning some of the signals of the base-on form and in following the shift of some of the signals with changes in pH. The protonated nucleotide loop in the base-off form has I3C chemical shifts similar to those of the protonated nucleotide, ribazole-3'-phosphate. The changes in chemical shifts and coupling constants in the ribose moiety of the loop suggest a change in conformation on formation of the base-off form. The primary effects on the side-chain signals of the base-on to base-off conversion are for the signals of the e-propionamide side chain. Relatively little effect is found for the A ring side-chain signals, and intermediate effects are found for the B and D ring side chains. Thus, from the base-off I3C data, there appears to be no reason for concluding that the eastern half of the corrin (rings B and C) is more flexible. 5'-Deoxyadenosylcobalamin (coenzyme BI2, M, = 1580) is a cofactor in over a dozen enzymatic reactions, two of which are known to be important in humans.' It is now generally believed that an essential early step in the catalytic cycle is the homolysis of the bond between Co and the A15 carbon of deoxyadenosyl (Chart I).2-4 The coenzyme is relatively stable to thermolytic homolysis, and it is clear that Co-C bond cleavage is induced by or electronic effects or a combination of both. In a recent review, several mechanisms were discussed in terms of their possible importance in the triggering of Co-C bond homolysi~.~ Four of these included, in some regard, interactions between the 5'-deoxyadenosyl moiety and the corrin ring. In two putative mechanisms, interactions were affected by a repositioning of the 5,6-dimethylbenzimidazole (DMBz) ligand. In this regard, UV data have been interpreted to indicate that, during catalysis, the cobalt-to-DMBz bond is br~ken.~ In addition, analysis of I3C NMR chemical shifts has led to speculation that the DMBz experiences steric compression with side chains of the corrin ring6*' and that the eastern half of the molecule (rings B and C) is more flexible than the western half.*