Figure 2. Fluorescence emission of flavodoxin and of reconstituted holoprotein.

(a) Spectrum-2 is of 5 μM flavodoxin in buffer, whereas spectrum-1 is of 5 μM reconstituted flavodoxin, which is obtained by mixing apoflavodoxin and FMN at equimolar ratio. On subsequent addition of excess apoflavodoxin to a final concentration of 375 nM, fluorescence emission of both samples changes differently (spectrum-4 and spectrum-3, respectively). This addition reveals that fluorescence of FMN in flavodoxin is fully quenched, whereas in freshly reconstituted holoprotein, it is not. (b) Time-dependent change in fluorescence emission of 5 μM reconstituted flavodoxin (excitation 450 nm, emission 514 nm). One unit of fluorescence corresponds to 0.88 nM FMN. Single-exponential fitting of the data shows that the relaxation time is 4.9±0.2 days.