Cloning and expression of NrdA and NrdB

All chemicals were purchased from Sigma-Aldrich or VWR and used as received unless otherwise stated. Cloning of the N-terminal hexa-histidine tag containing pET-nrdA, pET-nrdB, pET-nrdB∆169 and pET-NrdB∆Grx coding for F. ignava NrdA, NrdB, NrdB∆169 and NrdB∆Grx proteins was done as described in [12 (link)]. Cloning of pET-nrdB∆99 encoding L. blandensis NrdB∆99 (LbNrdB∆99) was done as described in [11 ]. All anaerobic work was performed in an MBRAUN glovebox ([O₂] < 10 ppm). Oxygen detection was done using a standard Clark electrode (Hansatech Instruments), separated from the sample solution by a Teflon membrane, and the signal was recorded using the CalMeter software package by Calmetric. Air-saturated water solutions ([O₂]_25 °C = 253 µM) were used for the calibration of the electrode.