Using Transient State Kinetics to Contextualize the Catalytic Strategy of Human Ferroptosis Suppressor Protein 1

Human ferroptosis suppressor protein 1 (HsFSP1) is an NAD(P)H:quinone oxidoreductase with broad substrate specificity that has been widely implicated in aiding malignant neoplastic cell survival. FSP1 is myristoylated and associated with membranes, where it regenerates the reduced forms of quinones using electrons from NADPH. The quinol products intercept reactive oxygen species and ameliorate lipid peroxidation, preventing ferroptosis, a form of regulated cell death. While FSP1 enzymes have been reported to have 6-OH-FAD as an active cofactor, aerobic titration of the enzyme with NADPH in the presence and absence of ubiquinone (UQ) reveals that this is more likely an artifact and that the native form of HsFSP1 has unmodified FAD as the cofactor. Moreover, HsFSP1 suppresses the reaction of the reduced FAD with molecular oxygen three-fold which, from a kinetic standpoint, severely limits the opportunity for cofactor modification. The isolated form of the enzyme has NADP⁺ bound and the rate of release of this product limits the observed rate of reduction by NAD(P)H molecules. The reduction of substrate quinones occurs rapidly (≥2000 s^–1), dictating that the rate of turnover is wholly defined by the rate of release of NADP⁺ from the HsFSP1·NADP⁺ complex. Given that HsFSP1 does not distinguish ubiquinone from ubiquinol by significant differences in binding affinity, this pronounced catalytic commitment to quinone reduction serves to overcome presumed kinetic limitations imposed by the abundance of ubiquinol relative to ubiquinone in the membrane. This characteristic also maintains the enzyme ostensibly fully in the oxidized state under turnover conditions, preventing significant futile reduction of dioxygen.