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Chloro-cob(II)alamin formation enhances the thiol oxidase activity of the B 12 -trafficking protein CblC

CblC is a chaperone that catalyzes removal of the β −axial ligand of cobalamin (or B 12 ), generating cob(II)alamin in an early step in the cofactor trafficking pathway. Cob(II)alamin is subsequently partitioned to support cellular needs for the synthesis of the active cobalamin cofactor derivatives. In addition to the β −ligand transferase activity, the Caenorhabdiitis elegans CblC (ceCblC) and the clinical R161G/Q variants of the human protein exhibit robust thiol oxidase activity, converting glutathione to glutathione disulfide while concomitantly reducing O 2 to H 2 O 2 . The chemical efficiency of the thiol oxidase side reaction during ceCblC-catalyzed dealkylation of alkyl-cobalamins is noteworthy in that it effectively scrubs ambient oxygen from the reaction mixture, leading to air stabilization of the highly reactive cob(I)alamin product. In this study, we report that the enhanced thiol oxidase activity of ceCblC requires the presence of KCl, which explains how the wasteful thiol oxidase activity is potentially curtailed inside cells where the chloride concentration is low. We have captured an unusual chloro-cob(II)alamin intermediate that is formed in the presence of potassium chloride, a common component of the reaction buffer, and have characterized it by EPR, MCD and computational analyses. The ability to form a chloro-cob(II)alamin intermediate could represent an evolutionary vestige in ceCblC, A chloro-cob(II)alamin intermediate is observed in the futile oxidation cycle catalyzed by ceCblC, a B 12 -trafficking protein. Characterization of this reaction by EPR, MCD spectroscopy and by computational analyses unexpectedly revealed its dependence on chloride ion and led to the characterization of a Cl-cob(II)alamin intermediate.