Elexsys e500 cw x band epr spectrometer

X-band EPR spectra were recorded on a Bruker ELEXSYS E500 CW X-band EPR spectrometer. The temperature of the samples was controlled using an Oxford Instruments ESR900 helium flow cryostat connected to an ITC503 temperature controller. The measurement parameters were: microwave frequency 9.64 GHz, time constant 81.92 ms, conversion time 81.92 ms, and modulation frequency 100 kHz. The microwave power and temperature were varied between measurements and are indicated in the figure legends.
The EPR samples (200 µL) were transferred anaerobically to 4 mm (o.d.) quartz EPR tubes and frozen in liquid nitrogen. All spectra were analyzed with home-written scripts in MATLAB. Spectral simulations were performed using the EasySpin package [37 (link)]. Spin quantification was achieved by comparison with a 1 mM CuSO₄, 10 mM EDTA standard.

A 0.2 ml, 0.2 mM sample of the HydB subunit in 100 mM Tris–HCl, 150 mM NaCl, pH 8 reduced with 10 mM sodium dithionite was transferred to a quartz 4 mm (o.d.) EPR tube and frozen in liquid nitrogen. X-band EPR spectra were recorded on a Bruker ELEXSYS E500 CW X-band EPR spectrometer. The temperature of the sample was controlled using an Oxford Instruments ESR900 helium flow cryostat connected to an ITC503 temperature controller. The measurement parameters were: microwave frequency 9.64 GHz, time constant 81.92 ms, conversion time 81.92 ms, and modulation frequency 100 kHz. The microwave power and temperature were varied between measurements and are indicated in the figure legends. All spectra were analyzed with home-written scripts in MATLAB. Spectral simulations were performed using the EasySpin package (Stoll and Schweiger, 2006 (link)).

Using a Varian Cary 50 Bio spectrophotometer (Varian Inc., Paolo Alto, CA, USA), visible spectra in the 300–800 nm range were captured at 25 °C. For all systems under investigation, magnetic circular dichroism (MCD) spectra were captured at 25 °C using a Jasco J-1500 spectrometer (Jasco Corporation, Ishikawa-machi, Hachioji-shi, Tokyo Japan). The MCD spectra were captured using a Permanent Magnet PM-491 accessory and collected in a magnetic field of +1.6 T in the N/S field direction. All spectroscopic measurements were collected in the pH range of 2.5 to 11. The reactions were carried out in a 0.3 mol L1 KCl solution with a ligand concentration of 6 × 10⁻⁴ mol L⁻¹. 2.0 mL was used for each sample. With the aid of a Mettler Toledo pH meter and modest additions of concentrated KOH and HCl solutions, the pH values were determined. The electron paramagnetic resonance (EPR) spectra were recorded on a Bruker ELEXSYS E500 CW-EPR X-Band spectrometer equipped with an ER 036TM NMR Teslameter and an E 41 FC frequency counter (Bruker AXS GmbH, Karlsruhe, Germany). A microwave power of 10 mW, a modulation frequency of 100 kHz, a modulation amplitude of 10 G, a time constant of 82 ms, and a conversion time of 164 ms were adopted. The samples were sealed in quartz tubes and placed inside a standard EPR quartz tube for measurements.