Cobalamins and Cobinamides Inhibit Nitric Oxide Synthase Enzymatic Activity.

Cobalamins (Cbl) serve as important co-factors for methionine synthase and methylmalonyl-coA mutase. They also can block the activities of nitric oxide (NO), as the cobalt binds NO and quenches its bioactivity. NO plays very important roles in normal physiology and in various pathologic processes. NO is synthesized by NO synthases (NOS), enzymes that convert L-arginine to L-citrulline and NO. We postulated that various corrin derivatives (e.g., cobalamins or cobinamides) would inhibit the enzymatic activity of NO synthase (NOS) by directly interacting with NOS. We used purified, recombinant neuronal NOS (NOS1) and inducible NOS (NOS2) in in vitro assays measuring conversion of 14-C-L-arginine to 14-C-L-citrulline. Results showed that hydroxocobalamin (OH-Cbl), cobinamide (Cbi), and dicyanocobinamide (CN₂Cbi) were potent NOS1 and NOS2 inhibitors, while cyanocobalamin had much less activity. In general, NOS1 was inhibited more actively than was NOS2, but both were inhibited over 80% by the agents. Methylcobalamin and adenosylcobalamin had very little ability to inhibit NOS activity, but light illumination of these two compounds (a process that liberates the methyl and adenosyl groups from cobalamin, generating OH-Cbl) “activated” them so that they had inhibitory activity comparable to native OH-Cbl

We examined the interactions of NOS2 with cobalamins and cobinamides by spectroscopy. Under reduced conditions, oxygen binds to the NOS heme iron, a step that is obligatory for the NO synthesis catalytic cycle. Carbon monoxide (CO, which serves as an oxygen mimic) causes a marked change in the NOS spectrum under reduced conditions. CN₂Cbi markedly diminished the CO-induced spectral changes in NOS2, indicating that CN2-Cbi likely blocks binding of CO to the NOS2 oxygenase domain under reduced conditions. This suggests that CN₂Cbi-mediated inhibition of NOS activity is due to blocking of oxygen binding to heme, a step required for the NOS catalytic cycle. When we incubated OH-Cbl or CN₂Cbi with cells of the mouse macrophage cell line J774 activated with interferon-gamma and endotoxin to produce NO, there was a dose-dependent inhibition of NO production. While certain cobalamins and cobinamides have been previously noted to bind and quench/scavenge the actions of preformed NO, we show here for the first time that cobalamins and cobinamides can potently inhibit the enzymatic function of NOS and synthesis of NO. These agents (or their derivatives) might serve as NOS inhibitors for treatment of various conditions. Also, native tissue cobalamins and cobinamides might act as important regulators of NOS activity in normal and pathological conditions in vivo.