Bovine cytochrome c

Methanol (> 99.9% purity) was purchased from Sigma-Aldrich (UK). Ammonium acetate was supplied by Fisher Scientific (Loughborough, UK). Ultrapure water was obtained from a Milli-Q Advantage A10 ultrapure water filtration system (Merck Millipore, Darmstadt, Germany).
Bovine ubiquitin, equine myoglobin and bovine cytochrome c were purchased from Sigma-Aldrich (UK) as lyophilized powders with purities of ≥ 98, 90 and 95% respectively. Proteins were dissolved in 200 mM Ammonium acetate. Myoglobin in Ammonium acetate was desalted twice using Micro Bio-Spin 6 chromatography columns (Bio-Rad Laboratories, Hercules, CA, US).
All samples were diluted to a final protein concentration of 10 μM.

ATR2, ATR2Ncut, PsCPR and RnCPR were expressed in BL21 (AN1067, AN1068, AN1069 and AN1070, respectively). Overnight cultures of these strains were inoculated into 50 ml of TB medium (per litre: 12 g tryptone (Difco), 24 g yeast extract (Diffco), 9.4 g K₂HPO₄, 2.2 g KH₂PO₄ and 4 ml glycerol) supplemented with 50 mg l⁻¹ ampicillin in a baffled shake flask at 25 °C. IPTG (1 mM) was added for induction at 12 h post inoculation. Following further incubation for 12 h, cells were harvested by centrifugation and suspended in 100 mM sodium phosphate buffer (pH 7.3) containing 10% glycerol. The crude extract containing a CPR was obtained by centrifugation following sonication. The protein concentration was measured using a BCA Protein Assay Kit (Pierce). Verification of CPR activity was carried out by a method of P450 reductase activity assay22 (link). The reaction mixture contained 2.1 μg of total protein, 100 μg of bovine cytochrome c (Sigma), 100 μM NADPH and 100 mM potassium phosphate (pH 7.3). The samples were incubated at 25 °C for 5 or 10 min. Degradation ratio of NADPH was measured at 550 nm by comparing before with after incubation.