Er 4122shq super high q cavity

Continuous wave X-band electron paramagnetic resonance (EPR) spectra of CYP144A1 proteins were obtained at 10 K using a Bruker ELEXSYS E500 EPR spectrometer equipped with an ER 4122SHQ Super High Q cavity. Temperature control was effected using an Oxford Instruments ESR900 cryostat connected to an ITC 503 temperature controller. Microwave power was 0.5 mW, modulation frequency was 100 KHz and the modulation amplitude was 5 G. EPR spectra were collected for the CYP144A1-FLV and CYP144A1-TRV proteins in the ligand-free state (200 μM) and at the same protein concentration following the addition of the azole inhibitor drug econazole (400 μM).

Continuous wave X-band EPR spectra for the OleT_JE WT and mutant P450s were obtained at 10 K using a Bruker ELEXSYS E500 EPR spectrometer with an ER 4122SHQ Super High Q cavity. Temperature was controlled with an ESR900 cryostat (Oxford Instruments, Abingdon, UK). EPR spectra were collected for WT and OleT_JE mutants at concentrations of 100–200 μm for ligand-free, arachidic acid-bound, and imidazole-bound forms. Arachidic acid was added to dilute OleT_JE protein until the UV-visible spectrum showed no further optical change toward high spin. The enzyme was then concentrated to an appropriate concentration (200 μm for WT, H85Q, and F79A OleT_JE; 150 μm for R245L OleT_JE; and 100 μm for the F79W, F79Y, and R245E mutants) (the latter in its substrate-free form), taking into consideration that some of the mutants are prone to precipitation at high concentrations) using a Vivaspin (30,000 molecular weight cutoff).