Circular Dichroism Spectroscopy

CD spectra were recorded on a JASCO J-815 spectropolarimeter in the range of 200–280 nm using a 1 mm cuvette. Data points were recorded every 1 nm and averaged over three scans. The protein concentration was kept at 5 µM in 10 mM phosphate buffer pH 7.5 and the spectra was recorded at 20 °C. Holo protein measurements were carried out in excess CoPPIX (20 μM). Thermal denaturation was performed by heating samples from 4 to 90 °C at a rate of 1°C min⁻¹, monitoring the loss of signal at 222 nm.