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 1 H and 13 C NMR spectra have been assigned. 13 C and 31 P 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.

Trans and cis influences and effects in cobalamins and in their simple models

“…Very recently, Brasch et al have shown that extension of this correlation to a larger number of cobalamins having distances with e.s.d. 's up to 0.01 Å, gave an R 2 value of 0.84 [39,40,43]. However, when the correlation is limited to Co\NB3 distances with e.s.d.…”

“…The fast exchange between the base-on and base-off forms of NOCbl on the NMR time scale explains why the NOCbl signal does not fit the regression line. In alkylcobalamins [43] there is significantly less base-off form, which essentially does not affect the 31 P NMR signal, as for all the other inorganic cobalamins (from H 2 OCbl + to SO 3 Cbl − ), which are in base-on form, without appreciable presence of the base-off form.…”

“…When the trans X ligand varies from H 2 O to NO, an increase of about 6 orders of magnitude in K is observed. [43]. In the literature, K is generally called K base-off .…”

“…This notation will be used from here onwards. The pK base-off increases with the decrease in the electron donating ability of X [43,44]. The plot of pK base-off versus the Co\NB3 axial distances (with e.s.d.…”

Three Redox States of Metallonitrosyls in Aqueous Solution

“…The Co-N α axial bond length to the DMB ligand of NOCbl is greater by 0.13 Å than the distance in the related NO 2 Cbl structure, reflecting the trans-labilizing effect of NO À . Both the axial "base-off" and "base-on" complexes coexist in aqueous solution at mild acidities, with predominance of the latter (deprotonated) form at pHs !7 (163). In an important application of the properties of Co(II)(por)/ Co(III)(por) fragments toward nitrosyl/nitroxyl coordination, a novel amperometric method has been developed, based on the attachment of a Co(II)(por) to a gold electrode and further electrochemical oxidation to Co(III).…”