The Y139F VKORC1 mutant fully reduces KO to KH₂to drive carboxylation. (A) Cycling between oxidized (KO) and reduced (KH₂) vitamin K drives VKD protein carboxylation by the γ-glutamyl carboxylase. VKORC1 fully reduces KO to the quinone intermediate (K) and then to KH₂; an unknown warfarin-resistant quinone reductase can also perform the second reaction. (B) Purified human VKORC1 bearing the Y139F mutation was tested for KO reduction to KH₂ by performing all steps under nitrogen to block KH₂ oxidation. (C) Microsomes containing only the carboxylase (Carb) or also expressing wt or Y139F VKORC1 were tested for KO or K supported carboxylation, as assessed by [¹⁴C]-CO₂ incorporation into the peptide FLEEL. (D) The microsomes in panel C were also tested for KH₂-supported carboxylation. (E) Insect cells containing tagged (Y139F_flag) and untagged Y139F or only the individual forms were subjected to immunopurification with anti-FLAG antibody, followed by western analysis using antibody against VKORC1, as before.²²  Mock indicates uninfected cells.