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In humans, cobalamin or vitamin B(12) is delivered to two target enzymes via a complex intracellular trafficking pathway comprising transporters and chaperones. CblC (or MMACHC) is a processing chaperone that catalyzes an early step in this trafficking pathway. CblC removes the upper axial ligand of cobalamin derivatives, forming an intermediate in the pathway that is subsequently converted to the active cofactor derivatives. Mutations in the cblC gene lead to methylmalonic aciduria and homocystinuria. Here, we report that nitrosylcobalamin (NOCbl), which was developed as an antiproliferative reagent, and is purported to cause cell death by virtue of releasing nitric oxide, is highly unstable in air and is rapidly oxidized to nitrocobalamin (NO(2)Cbl). We demonstrate that CblC catalyzes the GSH-dependent denitration of NO(2)Cbl forming 5-coordinate cob(II)alamin, which had one of two fates. It could be oxidized to aquo-cob(III)alamin or enter a futile thiol oxidase cycle forming GSH disulfide. Arg-161 in the active site of CblC suppressed the NO(2)Cbl-dependent thiol oxidase activity, whereas the disease-associated R161G variant stabilized cob(II)alamin and promoted futile cycling. We also report that CblC exhibits nitrite reductase activity, converting cob(I)alamin and nitrite to NOCbl. Finally, the denitration activity of CblC supported cell proliferation in the presence of NO(2)Cbl, which can serve as a cobalamin source. The newly described nitrite reductase and denitration activities of CblC extend its catalytic versatility, adding to its known decyanation and dealkylation activities. In summary, upon exposure to air, NOCbl is rapidly converted to NO(2)Cbl, which is a substrate for the B(12) trafficking enzyme CblC.