Project Summary The vitamin K cycle supports blood coagulation, bone mineralization, and vascular calcium homeostasis. The activity of vitamin-K-dependent proteins (e.g., coagulation factors) is regulated by the γ-carboxylase, GGCX. Epoxidation of vitamin K hydroquinone (KH2) drives catalysis of GGCX and is regenerated by vitamin K epoxide reductase (VKOR) with the assistance of a VKORL paralog and FSP1. VKOR is targeted by warfarin, a widely used oral anticoagulant whose overdose often leads to severe bleeding. Due to poor mechanistic understandings, modulation of the vitamin K cycle to improve anticoagulation therapy has not been explored. In addition, FSP1 regulates ferroptosis by producing KH2, which also affords potent protection against lipid peroxidation. However, the roles of vitamin K reductases in coagulation and ferroptosis, which are linked to numerous cardiovascular disorders, are unclear. Further, the mechanisms of the tightly regulated GGCX catalysis remain elusive. Thus, there is a need to establish a deeper understanding of the entire vitamin K cycle and the biology underlying blood coagulation so new therapeutic strategies can be developed for thromboembolic and cardiovascular diseases. Our long-term goal is to elucidate the physiological process of the entire vitamin K cycle and the underlying mechanisms. The objective of this renewal application is to modulate the redox state of VKOR to regulate anticoagulation, elucidate the relative contributions of VKOR, VKORL and FSP1 to support coagulation and control ferroptosis, and understand the structural mechanisms of GGCX catalysis. Our hypotheses are: (1) shifting VKOR towards a reduced state enhances vitamin K antidoting by increasing VKOR activity and facilitating warfarin release; (2) VKOR/VKORL are primarily responsible for K antidoting and reducing lipid peroxidation in the ER; and (3) GGCX binding of substrates induces conformational changes to tightly regulate the sequential reactions. Our preliminary data obtained 11 crystal structures of VKOR and a VKORL paralog with substrates and antagonists at different redox states. We also discovered that VKOR at reduced state is highly active but poorly inhibited by warfarin, and that K competition at partially oxidized state releases warfarin. We showed that VKOR/VKORL better support carboxylation and K antidoting relative to FSP1. We identified VKOR partner proteins and showed that reduced glutathione maintains VKOR activity. We obtained the first cryo-EM structures of human and conus GGCX that suggests keto-enol tautomerization as an elegant solution that couples epoxidation and carboxylation across the membrane interface. Armed with our expertise in the vitamin K cycle and membrane proteins, we will test our hypotheses with three specific aims: (1) Identify new anticoagulation strategies by employing VKOR redox states; (2) elucidate the interplay of the vitamin K cycle in γ-carboxylation and ferroptosis; and (3) understand the s...