Figure 5. A–D. Spectroscopy of dicyanocobinamide interactions with purified, recombinant NOS2 oxygenase domain, or NO.

Dicyanocobinamide reacts directly with purified, recombinant NOS2 oxygenase domain (A & B): Oxygen reacts directly with the reduced iron of the heme group in NOS. We used carbon monoxide (CO) (that simulates O₂ in binding to the reduced iron) to determine the influence of CN₂-Cbi on oxygen binding to NOS. The reaction of purified NOS2 oxygenase domain (approximately 3 μM final concentration) with CO in a reduced state (with dithionite) markedly changed the UV/vis spectrum, with appearance of the typical CO-bound Fe⁺²-heme peak at 445 nm (A). However, in the presence of CN₂-Cbi, there was no appearance of the 445 nm peak induced by CO in a reduced state using NOS2 oxygenase domain at a final concentration of approximately 4.5 μM (B). Similar findings were noted with OH-Cbl instead of CN₂-Cbi.

Hydroxocobalamin binds NO, but dicyanocobinamide does not (C & D). NO reacts with OH-Cbl (C) as indicated by the change in the UV/Vis spectrum (shift of peak from 352 nm to 354–355 nm, and alteration in the 520 nm region). However, NO does not react with CN₂-Cbi (D) (no change in spectrum).