Hystar

The LC equipment (Agilent 1200 series, Bremen, Germany) consists of a binary pump, an auto-sampler with 100 µL loop and a UV–Vis detector with a light-pipe flow cell. The UV detector was used at 280 nm to measure the polyphenols. The 5 µm diphenyl column having 250 × 3 mm inner diameter (Varian, Darmstadt, Germany) was used for separation. This was connected to the microTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization source. The internal calibration was achieved by using 0.1 M sodium formate solution at 0.10 mL/min, which was injected through the six-port valve. The calibration was achieved by using the enhanced quadratic mode. Water/formic acid (1000:0.05 v/v) and methanol were used as solvent A and B respectively. The flow rate of the solvents was adjusted to 500 µL/min. A linear gradient was used from 10% B to 80% B in 70 min and a further 10 min was assigned for the gradient to equilibrate from 80% B to 10% B for the next run. 3 µL of the filtered extract was injected into the system. The software used in this system was Bruker Hystar.

UV–visible absorbance measurements were made with a Jasco V550 spectrometer. The extinction coefficient for the E. coli [4Fe–4S] FNR (ε_{406 nm} = 16,200 M⁻¹ cm⁻¹ [28 (link)]) was used to calculate the amount of [4Fe–4S] cluster present in FnrP samples. CD spectra were measured with a Jasco J810 spectropolarimeter. For liquid chromatography–mass spectrometry (LC–MS) an aliquot of FnrP (100 μL, 46 μM [4Fe–4S]) was combined with varying aliquots of aerobic (229 μM O₂, 20 °C) or anaerobic assay buffer (200 μl final volume), and allowed to react for 15 min. Samples were diluted to ~2 μM final concentration, with an aqueous mixture of 1 % (v/v) acetonitrile, 0.3 % (v/v) formic acid, sealed, removed from the anaerobic cabinet and analyzed by an LC–MS instrument consisting of an Ultimate 3000 UHLPC system (Dionex, Leeds, UK), a ProSwift RP-1S column (4.6 × 50 mm) (Thermo Scientific), and a Bruker microQTOF-QIII mass spectrometer, running Hystar (Bruker Daltonics, Coventry, UK), as previously described [9 (link)].