Mechanism of severe WR mutations. Top, in the cellular environment, wild-type (WT) VKOR is largely oxidized (O and PO state), and only a small fraction is in the R state. The fractions of these states are measured by in-cell MS footprinting (Figure 2A). The arrows indicate redox equilibrium, and the short arrow indicates a small propensity for shifting to the R state. Bottom, severe WR mutations (indicated by red star) hinder warfarin binding (dashed lines) and increase the exposure of the VKOR active site to GSH. The increased GSH reduction generates high levels of the R state that is poorly inhibited by warfarin (thin dashed line), resulting in large warfarin-uninhibited activity that manifests as severe WR. In absence of warfarin, the high R state activity compensates for weakened substrate binding, explaining the general lack of VKCFD phenotype.