The steady-state kinetic studies leading to establishing the kinetic mechanism were conducted with stopped-flow according to the previously described methodology . The reaction mixture after mixing in stopped-flow contained 100 mM K2HPO4/KH2PO4 buffer pH 6.5, 52.2 nM of AcmB with 50% FAD content, 1% isopropyl alcohol (IPA), and varying concentrations of DCPIP (0.1-0.3 mM) and progesterone (2.5-50 μM). In order to establish the kinetic mechanism, the received data were fitted with nonlinear regression to three two-substrate kinetic models (i.e., sequential ordered bi-bi and random sequential bi-bi and the nonsequential Ping-Pong bi-bi models). The best model was selected based on statistical parameters (i.e., R 2 , χ 2 , AICc, and errors of estimated constants).