Spinach ferredoxin

For in vitro activity assays, 0.01 mL each of purified EtCYP114, EtFd_GA, or spinach ferredoxin (Sigma-Aldrich) and EtFdR (all at 1 mg/mL in buffer 1) were diluted with 1.5 mL of 50 mM phosphate, pH 7.5, 10% glycerol in 4 mL clear glass vials, and KA and NADPH were added to final concentrations of 20 µM and 60 µM, respectively. When H₂O₂ or tBuOOH was used as exogenous oxidant, no NADPH, EtFdR, or Fd was added, instead H₂O₂ was added to a final concentration of 60 µM or tBuOOH to a final concentration of 100 µM. The vial was sealed with a Teflon septum and incubated for 12 h at 25 °C.
For BjFd_GA in vitro activity assays, purified 10 µL EtCYP114 (35 mg/mL), 100 µL BjFd_GA (1 mg/mL) or 20 µL spinach ferredoxin (1 mg/mL; Sigma-Aldrich), and 10 µL spinach FdR (15U/mg; Sigma-Aldrich) were diluted with 0.5 mL of 50 mM phosphate, pH 7.5, 10% glycerol in 4 mL clear glass vials, and KA (5 µg) or 7-ol-KA (15 µg) and excess NADPH were added. The vial was sealed with a Teflon septum and incubated for 12 h at 25 °C.
The assay was acidified with hydrochloric acid to pH 2 and extracted 3 times with 2 mL of a 1:1 mixture of hexanes and ethyl acetate. The resulting organic extract was evaporated to dryness; the residue was methylated with diazomethane and, after reevaporation, dissolved in hexane for GC–MS analysis as previously described (18 (link)).

Oligomycin A, econazole, miconazole, spinach ferredoxin, ferredoxin reductase, and NADPH were purchased from SigmaAldrich (USA). Oligomycin C was purchased from Santa Cruz Biotechnology (USA). Crystal screening kits were obtained from Hampton Research (USA) and Emerald Biosystems (USA). All chemicals were of the highest grade that is commercially available.