Table 2. NADH-mediated reduction leads to the release of FMN and concomitant inactivation of the HoxFU moiety.
| Specific activity [U/mg]a | |||
| NADH [µM] | FMN release [%]b | − FMN | + FMN |
| 0 | 16±2 | 294±8 | 356±0 |
| 25 | 29±3 | 297±41 | 371±6 |
| 100 | 62±3 | 138±35 | 356±22 |
| 500 | 96±4 | 59±10 | 246±41 |
a
Purified 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.
b
The 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.