NADH-mediated reduction leads to the release of FMN and concomitant inactivation of the HoxFU moiety.

aPurified HoxFU was incubated with various NADH concentrations in the presence (500 µM) and absence of exogenous FMN. After 15 min, benzyl viologen was added and the specific NADH→BV activity was determined. bThe release of FMN from the HoxFU module (without addition of exogenous FMN) was determined by analyzing the HoxFU filtrate from centrifugal filter devices (3 kD cut-off size, 4°C, 7 minutes at 14000× g Millipore, Bedford, MA, USA) by fluorescence spectroscopy.