Cofactor F430 Extraction and Purification

To extract and purify cofactor F₄₃₀, Methanosarcina barkeri type strain (DSM 800) was cultured in a 10-L fermenter. Wet cells were sonicated with 1% formic acid on ice and supernatant was recovered by centrifugation (15,000 × g for 30 min). The supernatant was passed though Q sepharose fast-flow column that was pre-equilibrated with 50 mM tris-HCl buffer. F₄₃₀ in the solution was concentrated in a C18 SPE column pre-equilibrated with 1% formic acid and eluted with methanol. The F₄₃₀ fraction was further purified via two stages of liquid chromatography using Agilent 1260 HPLC equipped with a diode array detector and a fraction collector as following. The first HPLC purification was performed with Chromolith Semi Prep RP-18e (10 mm × 100 mm; Merck). Eluents were 100 mM NaClO₄/HClO₄ (pH 2.3) for A and acetonitrile for B. The gradient condition was 0% B at 0 min, 20% B until 2 min, then 27.3% B until 30 min at 1 mL/min of flow rate. The collected F₄₃₀ fraction was desalted using a C18 SPE column under the same condition described above. The second purification was performed with Hypercarb (10 mm × 150 mm; Thermo Scientific). Eluents were HClO₄aq (pH 1) for A and acetonitrile for B. The gradient condition was 0% B at 0 min, 30% B until 3 min, then 52.8% B until 70 min at 1 mL/min of flow rate. The collected F₄₃₀ fraction was desalted using an Oasis HLB SPE column.
One μmol of purified F₄₃₀, 0.2 µmol of methyl- and benzyl-viologen, 100 μmol of Ti(III)-citrate and methyl substrate were dissolved in previously degassed CAPS buffer solution (totally 10 mL) in a 30 mL glass vial and sealed with a butyl rubber stopper and an aluminum cap. The vial was standing at room temperature. Head space gas was periodically sampled for methane measurement using an HP 6890 GC equipped with a flame ionization detector. The compounds were separated with a PoraBOND Q column (25 m, 0.53 mm i.d., 10 μm film thickness; Agilent) using helium as the carrier gas at a flow rate of 8.5 mL/min. Initial oven temperature was 35°C and was heated to 190°C by 20°C/min. Injection was performed in split mode (split ratio 5:1). Concentration was determined by a C₁–C₄ alkane gas standard.