Elexsys e500 epr spectrometer

X-band CW-EPR measurements
were performed by using a Bruker E500 ElexSys EPR spectrometer and
ER 4123SHQE X-band cavity resonator with temperature calibration and
control, as described,^{41 (link)} by using the following
acquisition parameters: microwave frequency, 9.5 GHz; microwave power,
0.2 mW; magnetic field modulation, 0.2 mT; modulation frequency, 100
kHz. Four to eight spectra were averaged at each temperature.

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).

All reactions were performed under an atmosphere of argon or nitrogen using standard Schlenk or dry box techniques; solvents were dried over Na metal, K metal or CaH₂. Reagents were of analytical grade, obtained from commercial suppliers and used without further purification. ¹H, ¹¹B, ¹³C and ¹⁹F NMR spectra were obtained with a Bruker AVIII 400 MHz BBFO1 spectrometer at 298 K unless otherwise stated. NMR multiplicities are abbreviated as follows: s=singlet, d=doublet, t=triplet, m=multiplet and br=broad signal. Coupling constants J are given in Hz. Most of signals for the quaternary carbon atoms bonding to boron atom could not be detected, presumably due to coupling with the B atom. Electrospray ionization (ESI) mass spectra were obtained at the Mass Spectrometry Laboratory at the Division of Chemistry and Biological Chemistry, Nanyang Technological University. Melting points were measured with an OpticMelt Stanford Research System. Infrared spectra were measured with the Bruker Alpha-FT-IR Spectrometer with an ECO-ATR module. Continuous wave X-band electron paramagnetic resonance (EPR) spectrum was checked using a Bruker ELEXSYS E500 EPR spectrometer.