Esr 900

Optically detected magnetic resonance experiments were carried out using a modified X-band spectrometer (Bruker E300) equipped with a continuous-flow helium cryostat (Oxford ESR 900) and a microwave cavity (Bruker ER4104OR, approximately 9.43 GHz) with optical access. Optical irradiation was performed with a 532 nm continuous-wave laser (Cobolt Samba CW 532 nm DPSSL) from one side-opening of the cavity. PL was detected with a silicon photodiode (Hamamatsu S2281) on the opposite opening, using a 561 nm long-pass filter to reject excitation light. The PL signal was amplified by a current/voltage amplifier (Femto DHPCA-100). For continuous-wave ODMR, PL was recorded by a lock-in detector (Ametek SR 7230) referenced by on–off modulation of microwaves with a frequency of 547 Hz. Microwaves were generated with a microwave signal generator (Anritsu MG3694C), amplified to 3 W (Microsemi) and guided into the cavity. For trODMR, PL was recorded by a digitizer card (GaGe Razor Express 1642 CompuScope) whereby a pulse blaster card (PulseBlasterESR-PRO) triggered the digitizer card and produced microwave pulses for a set length. Microwaves were generated with the same microwave signal generator as in continuous-wave ODMR, whereby they were amplified to 5 W by a travelling wave tube amplifier (Varian VZX 6981 K1ACDK) and guided into the cavity.

To record the CYP125A13 electron paramagnetic resonance (EPR) spectra, a Bruker X-band spectrometer (9.5 GHz) equipped with a continuous supply of helium cryostat ESR 900 and an ITC 4 temperature controller used to achieve a base temperature of 5 K, was used. The microwave frequency and modulation frequency used were 9.647GHz and 100 kHz, respectively. The data were recorded and analyzed at The Korea Basic Science Research Institute, Western Seoul Center. Before the measurement, the oxidized form of 180 μM CYP12A13 in 10 mM potassium phosphate buffer (pH 7.4) was frozen in liquid nitrogen in EPR quartz tubes (Wilmad). The spectral data comprising the g-tensor and line width of the heme centers were acquired by the accumulation of spectra in the Simfonia or Xsophe (Bruker) programs.

EPR spectra were recorded using the Bruker ELEXSYS E500 (Bremen, Germany) with helium cryostat ESR 900 (Abingdon, United Kingdom). Previously calibrated 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used as a standard. Temperature was controlled using the ITC4 503S Oxford thermostat (Abingdon, United Kingdom). Spectra were always recorded with a modulation amplitude at least eight times lower than the line width with a microwave power low enough to avoid saturation. The last strongly over modulated spectrum was recorded at 5 K with 0.64 mW of microwave power and modulation amplitude set to 2 G to enhance the background signals. About 11 mg of dry brown powder were used.