Elexsys e580 epr spectrometer

The spectroscopic continuous wave (CW) EPR measurements were performed with a RADIOPAN SE/X-2547 (9 GHz) spectrometer equipped with a RCX661A TM110 resonator and an Oxford CF935 cryostat allowing measurements in a temperature range of 4.2–300 K. The modulation amplitude was 0.05 mT, the microwave power was 11.38 mW (without saturation effects), and the microwave frequency was recalculated for each measured point to match exactly 9 GHz. The number of points per spectra: 1024, accumulations: 2, time per one point: 120. The number of spins was estimated by a direct comparison method with DPPH standard which was earlier calibrated with a primary standard—copper sulphate pentahydrate monocrystals. The EPR relaxation measurements were conducted with a Bruker ELEXSYS E580 EPR Spectrometer equipped with an EN4118X-MD4 resonator in a temperature range of 5–100 K. Field-sweep echo-detected (FSED) spectra were obtained from X-band pulse experiments. The shot repetition time was set at 186 µs, π/2 pulse was set to 20 ns for T_m, T₂, and FSED measurements, and 34 ns for inversion magnetization experiments to obtain the maximum magnetization inversion.

For PELDOR measurements, 15–20 μl of cf-TMD0-His S₂₈C or S₁₃₈C sample containing 15–20% (v/v) deuterated glycerol was transferred into 1.6 mm outer diameter quartz EPR tubes (Suprasil, Wilmad LabGlass) and quick-frozen in liquid nitrogen. Pulsed EPR data were recorded on an ELEXSYS E580 EPR spectrometer (Bruker), which is equipped with a PELDOR unit (E580-400U, Bruker), a continuous-flow helium cryostat (CF935, Oxford Instruments), and a temperature control system (ITC 502, Oxford Instruments). Measurements were performed at Q-band frequencies (33.4 GHz) using an ELEXSYS SuperQ-FT accessory unit and a Bruker AmpQ 10 W amplifier in a Bruker EN5107D2 cavity at 50 K. The dead-time free four-pulse sequence with phase-cycled π/2-pulse was used for PELDOR measurements^{36 (link)}. A 20 ns pump pulse was used, which was placed at the maximum of the echo-detected field swept spectrum. The observer pulse lengths were set to 32 ns (π/2 and π), which were set 70 MHz lower. The deuterium modulations were averaged by increasing the first interpulse delay by 16 ns for 8 steps. The normalized primary PELDOR data V(t)/V(0) were processed to remove background contribution, and the resulting form factors F(t)/F(0) were fitted with a model-free Tikhonov regularization to distance distributions with DeerAnalysis2016 software package^{37 (link)}.

Conventional EPR spectra were obtained at room temperature with a Bruker ESP 380E spectrometer operating at a modulation amplitude of 0.01 mT, with the output microwave (MW) power of 100 mW, the MW attenuation set up to 25 dB and the sweep and constant times of 60 s and 46 ms, respectively. A Bruker ER 4118 X-MD-5 dielectric resonator was used. In pulsed EPR studies, an X-band Bruker ELEXSYS E580 EPR spectrometer was used equipped with a split-ring Bruker ER 4118 X-MS-3 resonator and an Oxford Instruments CF-935 cryostat.
The three-pulse ESEEM sequence (π/2)-τ-(π/2)-t-(π/2)-τ-echo was employed, with excitation at the maximum of the echo-detected EPR spectrum. The pulse lengths were 16 ns and the time delay τ was 204 ns, and the time delay t was scanned from 300 ns to 10 μs, with a 12 or 16 ns time step. The resonator was cooled with a stream of cold nitrogen gas. The temperature was controlled with a nitrogen flow stabilized by a Bruker ER4131VT temperature controller. The sample temperature was kept near 80 K.

The reduction potentials of the T1 and T2Cu centers in Br^2DNiR were determined by titration with sodium dithionite. To facilitate communication between the electrode and the protein, redox mediators were used. The electrochemical potential of the solution was measured using a Thermo Orion oxygen reduction potential electrode at 25 °C. A factor of +207 mV was used to correct values to the standard hydrogen electrode. During titration against dithionite, samples were withdrawn for electron paramagnetic resonance (EPR) analysis. They were placed in 4-mm OD Suprasil quartz EPR tubes (Wilmad LabGlass) under anaerobic conditions and immediately frozen in liquid N₂. To determine the redox potentials of the two copper centers, EPR spectroscopy was used. Continuous wave X-band EPR spectra (∼9.4 GHz) were recorded using a Bruker ELEXSYS E580 EPR spectrometer (Bruker GmbH) using a super-high-Q resonator. Temperature was maintained using an Oxford Instruments ESR900 helium flow cryostat coupled to an ITC 503 controller from the same manufacturer. EPR experiments were carried out at 20 K and employed 0.5-mW microwave power, 100-kHz modulation frequency, and 5-G (0.5 mT) modulation amplitude. Redox potentials of the copper centers were determined by fitting data to the Nernst equation.

Conventional EPR spectra were obtained at room temperature with an X-band EPR benchtop SPINSCAN-X spectrometer (ADANI, Minsk, Belorussia), operating at modulation amplitude of 0.01 mT, with the output microwave (MW) power of 100 mW, the MW attenuation set to −25 dB, and with sweep and constant times of 60 s and 46 ms, respectively.
In pulsed EPR studies, an X-band Bruker ELEXSYS E580 EPR spectrometer was used equipped with a split-ring Bruker ER 4118 X-MS-3 resonator and an Oxford Instruments CF-935 cryostat. A three-pulse ESEEM sequence (π/2)-τ-(π/2)-t-(π/2)-τ-echo was employed, with excitation at the maximum of the echo-detected EPR spectrum. The pulse lengths were 16 ns, time delay τ was 204 ns, the time delay t was scanned from 300 ns to 10 µs, with a 12 or 16 ns time step. The resonator was cooled with a stream of cold nitrogen gas. The temperature was controlled by a nitrogen flow stabilized by a Bruker ER4131VT temperature controller, the sample temperature was kept near 80 K.