For RHR, the FAD reduction was followed at 450 nm at 20°C in the reaction of 9.3 μM of AcmB with 100 μM progesterone using the buffers described above. In each measurement, one glass syringe of the stopped-flow instrument was filled with the solution of the steroid dissolved in EGME (the final concentration of 8%) and buffer solution, while the second one contained the enzyme solution in the buffer.