Cyanocobalamin

Hydroxocobalamin hydrochloride (HOCbl, ≥ 98%), cyanocobalamin (NCCbl, ≥ 98%), sodium cyanide (≥ 95%) and sodium chlorite (p.a., 80%; further purification as described in reference [15 (link)] did not improve this percentage) were obtained from Sigma-Aldrich (Munich, Germany) and used as received. The buffer solution used was a universal pH buffer, Britton–Robinson, it consists of a mixture of 0.04 M boric acid, 0.04 M phosphoric acid and 0.04 M acetic acid, that has been titrated to pH 7 with 0.2 M sodium hydroxide.
UV–Vis spectra were performed on a Cary 50 UV–Vis spectrophotometer (Varian, Inc., Foster City, CA, USA).
Raman spectra were measured on a Renishaw inVia Raman spectrometer coupled with a Leica microscope at 22 °C. 10 μl of each sample was dropped on a microscope slide covered with aluminum foil. The 532 nm laser line with a power of 100 mW was focused on the sample using a 5X objective. Each spectrum is represented as an average of 4 accumulations and 4 s.
NMR spectra were recorded at 20 °C unless otherwise stated, after diluting the sample (at concentrations indicated in text and Figure legends) with D₂O, on a 500 MHz Bruker instrument. The solvent used was Britton–Robinson universal buffer at pH 7 prepared in D₂O. A water-suppression pulse sequence was used for these measurements.
High-resolution mass spectra (HRMS) were recorded on an LTQ ORBITRAP XL mass spectrometer (ThermoScientific) using positive electrospray ionization. The instrument was externally calibrated. The samples were prepared at room temperature (22 °C) and then inserted into the instrument immediately. The following conditions were used: source voltage, 3.2 kV; sheath and auxiliary gas flow, 8 and 5 arbitrary units, respectively; vaporizer temperature 50 °C, capillary temperature 275 °C, analyzer temperature 26 °C; capillary voltage, 28 V; tube lens voltage, + 110 V. The number of microscans was set to three.
For DFT calculations, the Gaussian09 software package [21 ] was employed following the methodology previously described for Cbl complexes [17 (link)]. The Cbl models were truncated, with the lateral substituents on the corrin as well as the methyl groups on the benzimidazole replaced by hydrogen. Gas-phase geometries and frequency analyses were computed with the aid of the B3PW91 [22 (link), 23 (link)] functional at the def2-SV(P) [24 (link)] double-zeta basis set level. Long-range interactions were accounted by the use of Grimme’s D3 dispersion correction [24 (link)]. Population analyses, NMR [25 (link)] and TD-DFT derived [25 (link)] UV–Vis spectra were computed in the C-PCM solvent continuum adapted for aqueous environment [26 (link)]. In terms of methodology choice for DFT calculations, the methodology employed here was selected for its ability to best mimic trends in UV–Vis spectra as described in our previous study on hydroperoxocobalamin, thus also allowing consistency between the two sets of data [27 (link)]. DFT-derived spectral data were obtained using Chemcraft [28 ]; for the Raman simulations, 298 K and an excitation wavelength of 22,000 cm⁻¹ were assumed.

For uptake measurements cultures were pre-starved for 14 days as described earlier, unless otherwise indicated. Prior to uptake experiments, Anabaena cultures were washed three times with YBG11-Co medium. Uptake was measured in 13 ml of cultures of OD_750nm = 0.2. 20 µl of ⁵⁷Co-cyanocobalamin (stock solution of 0.5 µCi, MP Biomedicals GmbH, Eschwege, Germany) was mixed with 13.6 µl of cyanocobalamin (stock solution 50 nM, Sigma-Aldrich, St. Louis, Missouri, USA) to adjust the final concentration to 92 pM. Cells were incubated in 50-ml falcon tubes in a water bath at 30°C under constant shaking in the dark. 0.5, 4, 8 and 16 minutes after the addition of cyanocobalamin, 2 ml of cell culture were filtered on a hydrophilic membrane (25 mm, 0.45 µm pore size, Merck, Darmstadt, Germany). After filtration, cells were washed three times with 1 ml of washing buffer (1 mM cyanocobalamin, 2 mM NaHCO₃, 20 µM EDTA). Filter digestion and dissolution was previously described [64 (link)]. 10 ml of Aquasafe 300+ scintillation liquid (Zinsser Analytic, Frankfurt, Germany) was added and the ⁵⁷Co-cyanocobalamin determination was done with a Hidex 300 SL liquid scintillation counter (Hidex Deutschland Vertrieb GmbH, Mainz, Germany).