Mechanistic Studies on the Reaction of Nitrocobalamin with Glutathione: Kinetic Evidence for Formation of an Aquacobalamin Intermediate

The essential but also toxic gaseous signaling molecule nitric oxide is scavenged by the reduced vitamin B12 complex cob(II)alamin. The resulting complex, nitroxylcobalamin [NO--Cbl(III)], is rapidly oxidized to nitrocobalamin (NO2Cbl) in the presence of oxygen; however, it is unlikely that nitrocobalamin is itself stable in biological systems. Kinetic studies on the reaction between NO2Cbl and the important intracellular antioxidant, glutathione (GSH), are reported. In this study, a reaction pathway is proposed in which the -axial ligand of NO2Cbl is first substituted by water to give aquacobalamin (H2OCbl+), which then reacts further with GSH to form glutathionylcobalamin (GSCbl). Independent measurements of the four associated rate constants k1, k-1, k2, and k-2 support the proposed mechanism. These findings provide insight into the fundamental mechanism of ligand substitution reactions of cob(III)alamins with inorganic ligands at the -axial site.