NMR spectroscopy and molecular modelling studies of nitrosylcobalamin: further evidence that the deprotonated, base-off form is important for nitrosylcobalamin in solution

Abstract

The structure of nitrosylcobalamin (NOCbl) in solution has been studied by NMR spectroscopy and the ¹H and ¹³C NMR spectra have been assigned. ¹³C and ³¹P NMR chemical shifts, the UV-vis spectrum of NOCbl and the observed pK_base-off value of ∼5.1 for NOCbl provide evidence that a significant fraction of NOCbl is present in the base-off, 5,6-dimethylbenzimidazole (DMB) deprotonated, form in solution. NOE-restrained molecular mechanics modelling of base-on NOCbl gave annealed structures with minor conformational differences in the flexible side chains and the nucleotide loop position compared with the X-ray structure. A molecular dynamics simulation at 300 K showed that DMB remains in close proximity to the α face of the corrin in the base-off form of NOCbl. Simulated annealing calculations produced two major conformations of base-off NOCbl. In the first, the DMB is perpendicular to the corrin and its B3 nitrogen is about 3.1 Å away from and pointing directly at the metal ion; in the second the DMB is parallel to and tucked beneath the D ring of the corrin.