Protection of aquo/hydroxocobalamin from reduced glutathione by a B12 trafficking chaperone.

We identified a bovine B(12) trafficking chaperone bCblC in Bos taurus that showed 88% amino acid sequence identity with a human homologue. The protein bCblC was purified from E. coli by over-expression of the encoding gene. bCblC bound cyanocobalamin (CNCbl), methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) in the base-off states and eliminated the upper axial ligands forming aquo/hydroxocobalamin (OH(2)/OHCbl) under aerobic conditions. A transition of OH(2)/OHCbl was induced upon binding to bCblC. Interestingly, bCblC-bound OH(2)/OHCbl did not react with reduced glutathione (GSH), while the reaction of free OH(2)/OHCbl with GSH resulted in the formation of glutathionylcobalamin (GSCbl) and glutathione disulfide (GSSG). Furthermore we found that bCblC eliminates the GSH ligand of GSCbl forming OH(2)/ OHCbl. The results demonstrated that bCblC is a B(12) trafficking chaperone that binds cobalamins and protects OH(2)/OHCbl from GSH, which could be oxidized to GSSG by free OH(2)/OHCbl.