Aerobic decay

To measure the aerobic activity decay, ShVdcCD was first rendered anaerobic by desalting into 20 mM Hepes, 150 mM KCl, pH 7.5 in an anaerobic chamber using a GE Healthcare PD-10 Mini desalting column. prFMN (produced as described above) and MnCl₂ were added in 2 fold excess. The mixture was left for 10 min prior to desalting into the same buffer. A total of 100 µL of active protein was removed from the anaerobic environment in an opaque tube (to ensure no light mediated effects) and the activity was measured every 15 min over a 90 min period using 100 µM PHB in 50 mM NaPi, 50 mM KCl, pH 6, measuring the decrease in absorbance at 247 nm in a Agilent Cary 60 Spectrophotometer using the Kinetics software (Version 5.0.0.999). The protein was constantly exposed to oxygen by leaving the tube open throughout the time course. After 90 minutes the remaining protein was reintroduced to the anaerobic chamber, and a 2-fold excess of prFMN was added. The protein was removed from the chamber, and immediately assayed for activity. The process was performed in duplicate.