Chlorocob(II)alamin Formation Which Enhances the Thiol Oxidase Activity of the B12-Trafficking Protein CblC.

CblC is a chaperone that catalyzes removal of the ß-axial ligand of cobalamin (or B12), 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 active cobalamin cofactor derivatives. In addition to the ß-ligand transferase activity, the Caenorhabdiitis elegans CblC (ceCblC) and clinical R161G/Q variants of the human protein exhibit robust thiol oxidase activity, converting glutathione to glutathione disulfide while concomitantly reducing O2 to H2O2. The chemical efficiency of the thiol oxidase side reaction during ceCblC-catalyzed dealkylation of alkylcobalamins 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 chlorocob(II)alamin intermediate that is formed in the presence of potassium chloride, a common component of the reaction buffer, and have characterized it by electron paramagnetic resonance, magnetic circular dichroism, and computational analyses. The ability to form a chlorocob(II)alamin intermediate could represent an evolutionary vestige in ceCblC, which is structurally related to bacterial B12-dependent reductive dehalogenases that have been proposed to form halogen cob(II)alamin intermediates in their catalytic cycle.

An Interprotein Co-S Coordination Complex in the B12-Trafficking Pathway.

The CblC and CblD chaperones are involved in early steps in the cobalamin trafficking pathway. Cobalamin derivatives entering the cytoplasm are converted by CblC to a common cob(II)alamin intermediate via glutathione-dependent alkyltransferase or reductive elimination activities. Cob(II)alamin is subsequently converted to one of two biologically active alkylcobalamins by downstream chaperones. The function of CblD has been elusive although it is known to form a complex with CblC under certain conditions. Here, we report that CblD provides a sulfur ligand to cob(II)alamin bound to CblC, forming an interprotein coordination complex that rapidly oxidizes to thiolato-cob(III)alamin. Cysteine scanning mutagenesis and EPR spectroscopy identified Cys-261 on CblD as the sulfur donor. The unusual interprotein Co-S bond was characterized by X-ray absorption spectroscopy and visualized in the crystal structure of the human CblD thiolato-cob(III)alamin complex. Our study provides insights into how cobalamin coordination chemistry could be utilized for cofactor translocation in the trafficking pathway.