Solid-state NMR studies of cobalamins

Abstract

Solid state NMR studies have been performed on two members of the cobalamin family, namely vitamin B12 and methylcobalamin using single-crystal NMR, statis powder NMR, and magic angle spinning NMR techniques.

The chemical shift and quadrupolar coupling tensors of the cobalt centre of vitamin B12 have been determined via single-crystal 59Co NMR at 11.75 T. The chemical shift tensor was found to span (f!) 1173 ± 62 ppm, to have an isotropic shift (5iso) of 4549 ± 75 ppm, and possess a skew (K) of 0.31 ± 0.02. The quadrupolar coupling tensor of vitamin B12 was found to be large with a quadrupolar coupling constant (x) of 27.31 ± 0.08 MHz and an asymmetry (77) of 0.243 ± 0.005.

Selective labelling of the cyano group of vitamin B12 using 15N and 15N, 13C cyanide and preparation. of methyl-13C-cobalamin allowed for the study of these nuclei around the cobalt centre in these two cobalamins. The nitrogen was too far removed to see any effect due to the cobalt nucleus. However, the carbon spectra were greatly affected by both J-coupling and residual dipolar coupling due to the proximity of the directly bonded spin-~ (59Co) nucleus. Simulation of the methyl- 13C-cobalamin was not possible, but that of cyano 15N, 13C-cobalamin yielded a one bond Co-C J-coupling of 165 ± 10 Hz.

The two cobalamins in this study can be forced to adapt a base-off conformation via chemical means. Using 31 P NMR, we have been able to determine if a cobalamin is base-on or base-off from the isotropic chemical shifts ( 60 n = -1.3 ± 1 ppm, 6of 1= 4.6 ± 0.3 ppm), the spans of the chemical shift tensors (n0n= 184 ± 3 ppm, no11= 112.5 ± 7.5 ppm), as well as the chemical shift tensor skews (Kon= 0.35.5 ± 0.0:35, K.01 i= -0. 72 ± 0.01 ). We believe that this trend can be used to determine if an enzyme-bound cobalamin adapts a base-on or base off conformation. We have also studied the 59Co NMR spectra of the base-off cobalamins.

Deuterium NMR studies of methyl-decobalamin and its base-off analogue have revealed information about the dynamics of these two complexes. The activation energy for methyl group rotation of the base-off conformation is significantly less than that for the base-on conformation indicating that base-off cobalamins have a weaker cobalt-carbon bond.