Glutathione increases the binding affinity of a bovine B12 trafficking chaperone bCblC for vitamin B12

Abstract

Intracellular B12 metabolism involves a B12 trafficking chaperone CblC that is well conserved in mammals including human. The protein CblC is known to bind cyanocobalamin (CNCbl, vitamin B12) inducing the base-off transition and convert it into an intermediate that can be used in enzyme cofactor synthesis. The binding affinity of human CblC for CNCbl was determined to be Kd=≈6–16μM, which is relatively low considering sub-micromolar B12 concentrations (0.03–0.7μM) in normal cells. In the current study, we discovered that the base-off transition of CNCbl upon binding to bCblC, a bovine homolog of human CblC, is facilitated in the presence of reduced form of glutathione (GSH). In addition, GSH dramatically increases the binding affinity for CNCbl lowering the Kd from 27.1±0.2–0.24±0.09μM. The effect of GSH is due to conformational change of bCblC upon binding with GSH, which was indicated by limited proteolysis and urea-induced equilibrium denaturation of the protein. The results of this study suggest that GSH positively modulates bCblC by increasing the binding affinity for CNCbl, which would enhance functional efficiency of the protein.