Elexsys 2 spectrometer

X-band (9.40-GHz) EPR spectra were collected on a sample containing 170 μM Nitrosomonas sp. AL212 cyt P460 in 200 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 8.0 with 25% (v/v) glycerol. The measurements were obtained using a Bruker Elexsys-II spectrometer equipped with a liquid He cryostat maintained at 10.0 K. EPR data were simulated using SpinCount.^{17 (link)}

For UV/Vis spectroscopy, 30 μM CcP was prepared in 100 mM KPi, pH 6.0, and hydrogen peroxide was added to a final concentration of 1 mM. Stock solutions were diluted in 100 mM KPi, pH 6.0 to between 1–4 μM for recording optical spectra on an Agilent 8453 Spectrophotometer. For continuous wave EPR spectrosopy, CcP was prepared at ~0.5 mM to obtain stronger signals. Prior to data collection, samples were diluted into a buffer of 100 mM KPi, pH 6.0, 2 mM hydrogen peroxide, and 30% glycerol. Samples were loaded into EPR tubes, flash frozen minutes after addition of hydrogen peroxide, and measured with a Bruker EleXSys II spectrometer at 9 GHz with 1.5 Gauss modulation amplitude, 100 kHz modulation frequency and 25 – 30 dB microwave attenuation.
The L-tyrosine EPR standard was prepared as described^{65 (link)}. L-tyrosine was dissolved in a sodium borate buffer, pH 10, degassed via three freeze-thaw cycles and flame sealed in a quartz EPR tube. While frozen in liquid nitrogen in a finger dewar, the sample was irradiated by a 600 W mercury lamp for 3 – 4 minutes in order to generate the free tyrosyl radical.