Inhibition of Nitric Oxide Synthase (NOS) 1, NOS2, and NOS3 Enzymatic Activities by Vitamin B₁₂ and Related Corrins: Biochemical and Molecular Modeling Studies.

Cobalamins (Cbl) serve as important co-factors for methionine synthase and methylmalonyl-coA mutase. They also can block the activities of NO, as the cobalt in cobalamin 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), inducible NOS (NOS2), and endothelial NOS (NOS3) 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 (CN₂–Cbi) were potent inhibitors of all three isoforms of NOS, while cyanocobalamin had much less activity (Table). In general, NOS1 was most inhibited, with affinities generally NOS1>NOS3>NOS2. Some corrins were extremely potent, with the ID50 of CN₂-Cbi in the nM range. 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. Oxygen binding to the NOS heme iron in the ferrous state is an obligatory step in the NO synthase catalytic cycle. Carbon monoxide (CO, which serves as an oxygen mimic) causes a marked change in the NOS spectrum under reducing conditions. CN₂–Cbi 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 CN₂–Cbi-mediated inhibition of NOS activity is due to blocking of oxygen binding to heme, a step required for the NOS catalytic cycle. Our molecular modeling investigations using manual computer-assisted docking indicate that the corrins can physically access the unusually large heme and substrate binding pocket of NOS. Binding appears to be best in the “base-off” conformation of the lower axial ligand dimethylbenzimidazole, with binding being facilitated by heme ring-corrin ring interactions and possibly by hydrogen bonding to arginine-binding groups in the active site pocket. The total “base-off” structure of CN₂–Cbi and the great potential of OH–Cbl to hydrogen-bond in the pocket help explain their low ID50s for inhibition. The slightly larger active site pocket of NOS1 compared to NOS2 and NOS3 likely explains the lower ID50s for NOS1. 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 all NOS isoforms. These agents (or their derivatives) might serve as NOS inhibitors for treatment of various conditions. Also, native tissue corrins might act as important regulators of NOS activity in normal and pathological conditions in vivo.