Xepr software

All EPR spectra were recorded using a Bruker ELEXSYS-II X/L spectrometer (Rheinstetten, Germany) with either R4123SHQE X-band or ER540R23 L-band resonators. EPR spectra were analyzed using Xepr software (Bruker BioSpin). Hydrogen peroxide, methanol (MS grade), DPPH (2,2-Diphenyl-1-picrylhydrazyl), 3CP (3-Carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-oxyl), and 3CxP were purchased from Sigma-Aldrich (Steinheim, Germany). Spin trap DEPMPO (5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide) was purchased from Focus Biomolecules (Plymouth Meeting, USA). AccuGENE deionized 18 MΩ water was from Lonza (Bornem, Belgium). Iron(II) sulfate heptahydrate (97%) was purchased from Merck (Darmstadt, Germany).

Samples with a volume of 20 µL were filled in glass capillaries (HIRSCHMANN^® ringcaps^®, inner diameter 1.02 mm, Eberstadt, Germany) and sealed with Hemato-Seal™ capillary sealant (Fischer-brand™, Schwerte, Germany). RS spectra were recorded on an Elexsys 500 spectrometer (Bruker, Karlsruhe, Germany) equipped with the Rapid-Scan Accessory (Bruker, Karlsruhe, Germany) [40 (link)] at X-band frequency (9.426 GHz). Sinusoidal rapid magnetic-field scans at a frequency of 20 kHz with a scan width of 20 mT were applied using a 1D field experiment. The center field was set to 336.4 mT, the attenuation to 20 dB (2 mW power), and the background correction function TwoTone included in the XEpr software (Bruker) was chosen. The measurement time was set to 60 s resulting in averaging of 1,202,643 scans (63,297 onboard averages, 19 off-board averages). Recorded spectra were processed with Matlab R2019b (The Mathworks, Inc., Natick, MA, USA) and the toolbox EasySpin 6.00 [41 (link)]. Processing included baseline correction, adjusting the field position of the spectra to correct for small deviations in the microwave frequency between different samples, smoothing of data with a Savitzky–Golay filter function, and the normalization of the intensity.

Continuous wave X-band electron paramagnetic resonance (EPR) spectroscopy was used to characterize the IseG glycyl radical. A 240 μL reaction mixture containing 20 mM HEPES, pH 7.5, 0.1 M KCl, 20 μM IseG, 80 μM reconstituted MBP-IseH, 1 mM SAM, 100 μM Ti(III) citrate and 5% glycerol was incubated at RT for 10 min in the glovebox. A control sample omitting Ti(III) citrate was also prepared. All samples were loaded into EPR tubes with 4 mm o.d. and 8″ length (Wilmad Lab-Glass, 734-LPV-7), sealed with a rubber stopper, removed from the glovebox and frozen in liquid nitrogen prior to EPR analysis. The perpendicular mode X-band EPR spectra were recorded using a Bruker E500 EPR spectrometer. Data acquisition was performed with Xepr software (Bruker). The EPR spectra represent an average of 30 scans and were recorded under the following conditions: temperature, 90 K; center field, 3370 Gauss; range, 200 Gauss; microwave power, 10 μW; microwave frequency, 9.44 MHz; modulation amplitude, 0.5 mT; modulation frequency, 100 kHz; time constant, 20.48 ms; conversion time, 30 ms; scan time, 92.16 s; and receiver gain, 43 dB. The experimental spectra for the glycyl radical were modeled with Bruker Xepr spin fit to obtain g values, hyperfine coupling constants, and line widths. Double integration of the simulated spectra was used to measure spin concentration.

The time-dependent kinetics of tyrosyl radical destruction in human R2 ribonucleotide reductase protein (hR2) by 3-AP and HL² were measured by EPR spectroscopy at 30 K, on a Bruker Elexsys II E540 EPR spectrometer with an Oxford Instruments ER 4112HV helium cryostat. The experimental conditions were: microwave power 3.2 mW, modulation amplitude 5 G, modulation frequency 100 kHz, and conversion time 0.0293 s. Spectra were recorded and analysed using the Bruker Xepr software. The concentration of the tyrosyl radical was determined by double integration of EPR spectra recorded at non-saturating microwave power levels and compared with the copper standard [43 (link)]. Purified, recombinant, iron-reconstituted hR2 [44 (link)] was obtained from the Department of Biochemistry and Biophysics, Stockholm University, Sweden. The samples containing 20 μM hR2 in 50 mM Hepes buffer, pH 7.60/100 mM KCl/5% glycerol, and 20 μM 3-AP or HL² in 1% (v/v) DMSO/H₂O, and 2 mM dithiothreiotol were incubated for indicated times and quickly frozen in cold isopentane. The same sample was used for repeated incubations at room temperature. The experiments were performed in duplicates.

EPR spectra of mixtures of nitroxides and sodium ascorbate in potassium phosphate buffer (0.1 M, $pH$ 7.4, containing 1 mM DTPA) were recorded at 21 °C using an Elexsys E500 spectrometer (Bruker, Wissembourg, France) operating in X-band (9.8 GHz) and equipped with a high sensitivity SHQ cavity. A 4-bore AquaX quartz cell (Bruker) inserted into the cavity was connected to a Bio-Logic MPS-51 stop-flow apparatus with three syringes (Bio-Logic, Claix, France) controlled by the Bio-Logic MPS software. Field-time 2D acquisitions were performed with the following acquisition parameters: modulation frequency, 100 kHz; modulation amplitude, 0.10 mT for compounds 1 and 2, 0.30 mT for compound 3, 0.15 mT for compound 4, and 0.14 mT for compound 5; time constant, 20.48 ms; conversion time, 20.49 ms; center field, 350.3 mT; sweep width, 5.0 mT; sweep time, 10.49 s; microwave power, 10 mW. Data acquisition and processing were performed using Bruker Xepr software. The initial second-order reaction rate constant of the reaction of nitroxides with ascorbate, k₀ values was derived from experimental results as previously described [57 (link),58 (link)].