EPR spectroscopy was performed on a Bruker Elexsys E580 spectrometer operating at X- and Q-bands using similar equipment and settings as described previously in ref. 26 (link) and 35 (link). CW spectra were recorded at the X-band over the temperature range 20–120 K using a non-saturating microwave power.
Elexsys e580
Manufactured by Bruker
Sourced in Germany
The ELEXSYS E580 is a high-performance electron paramagnetic resonance (EPR) spectrometer produced by Bruker. It is designed to perform advanced EPR experiments and analysis. The core function of the ELEXSYS E580 is to detect and characterize unpaired electrons in a variety of sample types.
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Lab products found in correlation
Cf935, by Oxford Instruments (4 mentions)
Glycerol, by Thermo Fisher Scientific (2 mentions)
Ecz 400s nmr spectrometer, by JEOL (2 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Chloroform 13 c 99 atom, by Merck Group (2 mentions)
En 4118x md4, by Bruker (2 mentions)
Er4102st, by Bruker (1 mentions)
White led light, by Thorlabs (1 mentions)
Helium gas flow cryostat, by Oxford Instruments (1 mentions)
Er4122shqe, by Bruker (1 mentions)
Elexsys e500, by Bruker (1 mentions)
Esr900, by Oxford Instruments (1 mentions)
Nicolet is10 ft ir spectrometer, by Thermo Fisher Scientific (1 mentions)
Evo 50 series, by Zeiss (1 mentions)
Tristar 2, by Micromeritics (1 mentions)
400u second microwave source unit, by Bruker (1 mentions)
Shq cavity, by Bruker (1 mentions)
Toluene d8, by Merck Group (1 mentions)
Cf935 cryostat, by Oxford Instruments (1 mentions)
E2695 separations module, by Waters Corporation (1 mentions)
36 protocols using elexsys e580
1
EPR Spectroscopy of Biological Samples
2
EPR Spectroscopy of Cytochrome bc1 Complexes
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All the measurements were performed using a Bruker ElexSys E580 spectrometer equipped with a super-HighQ (SQHE0511) resonator and ESR900 cryostat unit (Oxford Instruments). The chromatophore membranes were measured using the parameters described by Sarewicz et al. (2018) (link). Briefly, the temperature of the measured samples was 20 K, microwave power of 2 mW and frequency 9.4 GHz, modulation amplitude of 14.36 G, sweep width of 926.8 G, sweep time of 40.96 s, and time constant of 81.92 ms. The number of scans was usually 1–3 depending on the signal-to-noise (S/N) ratio. The registered spectra were processed and analyzed using the Eleana EPR program.
The isolated WT and H198N Cytbc1 complexes were measured as follows. The X-band EPR spectra of low- and high-field transitions of hemes were measured at 10 K, with 2 mW microwave power, frequency of 9.4 GHz, and modulation amplitude of 6 G. The X-band EPR spectra of the 2Fe2S cluster was measured at 30 K with 0.65 mW microwave power and modulation amplitude of 16 G.
The isolated WT and H198N Cytbc1 complexes were measured as follows. The X-band EPR spectra of low- and high-field transitions of hemes were measured at 10 K, with 2 mW microwave power, frequency of 9.4 GHz, and modulation amplitude of 6 G. The X-band EPR spectra of the 2Fe2S cluster was measured at 30 K with 0.65 mW microwave power and modulation amplitude of 16 G.
3
Continuous Wave EPR Experiments
4
Characterization of Copper-Containing Peptides
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Peptides were synthesized, characterized, and purified as previously reported (34 (link)). XAS measurements were recorded at the Cu K-edge absorption edge (8,979 eV) at beamline I20-Scanning at Diamond Light Source (United Kingdom). Briefly and in addition to the main text and figure captions, pulsed EPR experiments [relaxation, ED-FS, ESEEM, ENDOR (44 (link)49 (no link found, link, no link found, no link found, no link found), 53 (link)), DEER, and RIDME (55 62 (link, link, link, no link found, link, link, no link found))] were performed on protonated and deuterated solutions of the peptides with 50% glycerol at X- or Q-band using a Bruker ELEXSYS E580 spectrometer operating at cryogenic temperatures (10 to 50 K). Full experimental details are provided in accompanying SI Appendix .
5
Temperature Dependence of [2Fe-2S] Cluster Relaxation
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The temperature dependence of phase relaxation rate of [2Fe-2S] cluster was measured in isolated complexes using pulse EPR spectroscopy. The measurements were carried out on a Bruker Elexsys-E580 spectrometer at Q-band (33.5 GHz). The electron spin echo decay of each sample was recorded in temperature range from 12 to 24 K in the same manner as described previously (41 (link)). The relaxation rates were determined from fitting a stretched exponential function to the measured electron spin echo curves. The samples were prepared in 50 mm Bicine buffer (pH 8) and 100 mm NaCl under reducing conditions (1 mm sodium ascorbate) in the presence of 20% glycerol as described previously (41 (link)). The measured relaxation rates concern intracomplex interactions and are highly reproducible, falling within a standard error typical of fitting procedure (1–2%), irrespective of protein isolation.
6
Variable-Temperature EPR Spectroscopy
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X (9.8 GHz)- and Q (34 GHz)- band EPR measurements were performed at variable temperature (295–20 K) with the Bruker ELEXSYS-E580 and −E500Q spectrometers from the Center for advanced ESR/EPR techniques (CetRESav). The equipment and magnetic field calibration procedures are described in ref. 61 and at http://cetresav.infim.ro/ . The sample holders were quartz tubes of 2 mm and 3 mm inner diameter for the Q-band and X-band measurements, respectively. The EPR spectra were recorded with 100 kHz modulation frequency and relatively high modulation amplitude of 3 G, at the highest microwave power level for which saturation effects did not occur (10 mW). Multiple scans (up to 15) were performed in order to increase the signal to noise ratio.
7
Pulsed X-Band EPR Spectroscopy
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CW X-Band EPR spectra were recorded on a Bruker Elexsys E500 spectrometer equipped with a SHQ cavity (ν = 9.39 GHz). Low temperature measurements were obtained using an Oxford Instruments ESR900 continuous flow helium cryostat. Pulsed EPR measurements were carried out with a Bruker Elexsys E580 at X-band (ν ≅ 9.70 GHz) equipped with a flexline dielectric ring ENDOR resonator (Bruker EN 4118X-MD4). Temperatures between 4.5 and 100 K were obtained with an Oxford Instruments CF935 continuous flow helium cryostat. Echo detected field swept EPR spectra were recorded by using the Hahn Echo pulse sequence (π/2 – τ – π – τ – echo) with a fixed interpulse delay time τ = 200 ns, tπ/2 = 16 ns and tπ = 32 ns. Both phase memory times were measured by using the Hahn Echo sequence upon increasing the interpulse delay τ starting from τ = 98 ns. Spin-lattice relaxation times were measured using the standard inversion recovery sequence (π – td – π/2 – τ – π – τ – echo), with π/2 = 16 ns. The uncertainty in T1 estimated from replicate measurements was 5–10% depending upon the signal-to-noise ratio at a given temperature-field combination.
8
Pulsed EPR Spectroscopy for Sample Analysis
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Pulsed EPR spectra were recorded with a Bruker ElexSys E580 instrument equipped with either a MD5 or a MD4 resonator and operating at ca. 9.7 GHz and various temperatures. Solution samples of different concentrations (2, 5 and 10 mM in THF) were investigated to check reproducibility and to achieve an acceptable signal-to-noise response in HYSCORE and ENDOR experiments.
9
Measuring Spin-Lattice Relaxation Time in EPR
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All experiments were carried out using a Bruker ELEXSYS E580 spectrometer operating at X-band with a dielectric ring resonator 4118X-MD4 and a Bruker 400U microwave source unit. All measurements were made at 50 K with the sample in a frozen glassy state. The resonator was over-coupled giving a Q factor of approximately 100. The video bandwidth was set to 20 MHz.
Experiments to determine the phase memory time (Tm) were performed by measuring the intensity of a Hahn echo as it decayed with increasing inter-pulse delay. The pulse sequence used was π/2–t1–π, where the π pulse was 32 ns and the initial time delay t1 was 400 ns, in addition two-step phase cycling was employed to eliminate receiver offsets. Timings and delays were used appropriately for each sample. The experiment repetition time was 4 ms and 50 shots were taken at each time point.
Echo decay curves in a deuterated medium are dominated, initially, by ESEEM oscillations and so Tm was estimated by fitting of Eq.(1) to the tail end of the data that is largely free of ESEEM.
Experiments to determine the phase memory time (Tm) were performed by measuring the intensity of a Hahn echo as it decayed with increasing inter-pulse delay. The pulse sequence used was π/2–t1–π, where the π pulse was 32 ns and the initial time delay t1 was 400 ns, in addition two-step phase cycling was employed to eliminate receiver offsets. Timings and delays were used appropriately for each sample. The experiment repetition time was 4 ms and 50 shots were taken at each time point.
Echo decay curves in a deuterated medium are dominated, initially, by ESEEM oscillations and so Tm was estimated by fitting of Eq.
10
Spectroscopic analysis of hemin and SPD_0090
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A buffer of 20 mM Tris-HCl (pH 7.4, containing 100 mM NaCl) was used to prepare 400 μl each of 100 μM hemin and SPD_0090 + hemin solution (SPD_0090: hemin = 1:1). The electron paramagnetic resonance spectra of hemin and SPD_0090 + hemin solution were measured by a Bruker spectrometer (Elexsys E580, Bruker, Germany) with the following parameters: frequency, 9.4 GHz; power, 4 mW; modulation amplitude, 3 G; modulation frequency, 100 kHz; and temperature, 10 K. The obtained data were processed and plotted using the Origin version 9.0 software.
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