A Noncatalytic Cysteine Residue Modulates Cobalamin Reactivity in the Human B12 Processing Enzyme CblC.

Abstract

Human CblC catalyzes the indispensable processing of dietary vitamin B₁₂ by the removal of its β-axial ligand and an either one- or two-electron reduction of its cobalt center to yield cob(II)alamin and cob(I)alamin, respectively. Human CblC possesses five cysteine residues of an unknown function. We hypothesized that Cys149, conserved in mammals, tunes the CblC reactivity. To test this, we recreated an evolutionary early variant of CblC, namely, Cys149Ser, as well as Cys149Ala. Surprisingly, substitution of Cys149 for serine or alanine led to faster observed rates of glutathione-driven dealkylation of MeCbl compared to wild-type CblC. The reaction yielded aquacobalamin and stoichiometric formation of S-methylglutathione as the demethylation products. Determination of end-point oxidized glutathione revealed significantly uncoupled electron transfer in both mutants compared with the wild type. Long incubation times revealed the conversion of aquacobalamin to cob(II)alamin in the presence of oxygen in mutants Cys149Ser and Cys149Ala but not in wild-type CblC, all without an effect on dealkylation rates. This finding is reminiscent of the catalytic behavior of CblC from Caenorhabditis elegans, wherein Cys149 is naturally substituted by Ser, and the reaction mechanism differs from that of human CblC precisely by the unusual stabilization of cob(II)alamin in the presence of oxygen. Thus, Cys149 tunes the catalytic activity of human CblC by minimizing uncoupled electron transfer that forms GSSG. This occurs at the expense of a slower observed rate constant for the demethylation of MeCbl. This adjustment is compatible with diminished needs for intracellular turnover of cobalamins and with life under increased oxygen concentration.