Measurements were performed on a Bruker ELEXYS E500 spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). Measurement temperatures ranged from 5 to 30 K, using liquid helium as coolant. The spectrometer was controlled by the Xepr software package (Bruker). EPR samples were frozen and stored in liquid nitrogen. The EPR spectra shown are representative signals from at least two individual experiments. Spin quantification was performed through double integration of the EPR spectra and calculated relative to NrdB∆169Mn. Unless otherwise stated, all spectra were recorded at 10 K, microwave power 1 mW, frequency 9.28 GHz, modulation amplitude 10 G and modulation frequency 100 kHz.
Xepr software package
Manufactured by Bruker
Sourced in Germany
The Xepr software package is a comprehensive software suite designed for the acquisition, processing, and analysis of electron paramagnetic resonance (EPR) data. It provides a user-friendly interface for controlling Bruker EPR spectrometers and performing advanced data analysis tasks.
Automatically generated - may contain errors
Lab products found in correlation
Continuous flow cryostat, by Oxford Instruments (6 mentions)
Itc 503 temperature controller, by Oxford Instruments (6 mentions)
Elexys e500 spectrometer, by Bruker (6 mentions)
Er049x superx microwave bridge, by Bruker (5 mentions)
Shq0601 cavity, by Oxford Instruments (3 mentions)
Elexys 500x band spectrometer, by Bruker (2 mentions)
Emxmicro, by Bruker (1 mentions)
Er4119hs resonator, by Bruker (1 mentions)
Emxmicro spectrometer, by Bruker (1 mentions)
Matlab, by MathWorks (1 mentions)
Itc 503 temperature, by Oxford Instruments (1 mentions)
11 protocols using xepr software package
1
Characterization of Mn-Containing Proteins
2
EPR Characterization of HydA1 Enzyme
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The EPR spectra shown are representative signals from at least two individual experiments. The individual experiments show some preparation dependent differences, but the amplitude of these background signals are negligible compared to the signal intensity of the [2Fe]adt activated HydA1. Measurements were performed on a Bruker ELEXYS E500 spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity (Fig. 2 ) or a Bruker EMX micro equipped with an EMX Premium bridge and an ER4119 HS resonator (Fig. 5 and S13–S15† ), both equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). Measurement temperatures ranged from 10 to 20 K, using liquid helium as coolant, with the following EPR settings unless otherwise stated: microwave power 1 mW, modulation amplitude 1 mT, modulation frequency 100 kHz. The spectrometer was controlled by the Xepr software package (Bruker).
3
Anaerobic Sample Preparation for EPR
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EPR samples were prepared under strict anaerobic conditions. The proteins were reduced with a 10-fold molar excess of sodium dithionite, and the reaction was monitored by UV-visible spectroscopy. The samples were transferred into quartz EPR tubes capped with rubber septa and immediately flash-frozen outside the glovebox. The EPR samples were stored in liquid nitrogen until further usage.
The CW EPR measurements were carried out on a Bruker Elexys 500X-band spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 resonator, equipped with an Oxford Instruments continuous-flow cryostat and an ITC 503 temperature controller (Oxford Instruments). Low temperatures were achieved using liquid helium as the coolant. The spectrometer was controlled by the Xepr software package (Bruker). Standard measuring parameters were 10-G modulation amplitude and 100-kHz modulation frequency. The spectra were averaged over either four or eight scans.
The CW EPR measurements were carried out on a Bruker Elexys 500X-band spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 resonator, equipped with an Oxford Instruments continuous-flow cryostat and an ITC 503 temperature controller (Oxford Instruments). Low temperatures were achieved using liquid helium as the coolant. The spectrometer was controlled by the Xepr software package (Bruker). Standard measuring parameters were 10-G modulation amplitude and 100-kHz modulation frequency. The spectra were averaged over either four or eight scans.
4
EPR and UV-Vis analysis of AaR2
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Measurements were performed
with 214 μM AaR2 on a Bruker ELEXYS E500 spectrometer equipped
with a coldfinger Dewar filled with liquid nitrogen (77 K), a modulation
amplitude of 2 G, and a microwave power of 3.95 mW. The Xepr software
package (Bruker) was used for data acquisition and processing of spectra.
UV–vis spectra were recorded with 11 μM AaR2 at 25 °C
on a PerkinElmer Lambda 35 spectrophotometer.
with 214 μM AaR2 on a Bruker ELEXYS E500 spectrometer equipped
with a coldfinger Dewar filled with liquid nitrogen (77 K), a modulation
amplitude of 2 G, and a microwave power of 3.95 mW. The Xepr software
package (Bruker) was used for data acquisition and processing of spectra.
UV–vis spectra were recorded with 11 μM AaR2 at 25 °C
on a PerkinElmer Lambda 35 spectrophotometer.
5
EPR Spectroscopy with Cryostat Setup
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Measurements were performed on a Bruker ELEXYS E500 spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). The Xepr software package (Bruker) was used for data acquisition and processing of spectra.
6
Electron Paramagnetic Resonance of Photoactive Complex
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All electron paramagnetic resonance spectra were recorded on a Bruker EMX-micro spectrometer equipped with an EMX-Primium bridge and an ER4119HS resonator. Individual solutions were deoxygenated before mixing and the final sample concentrations were 230–250 μM protein, 20–30 μM [Ru(bpy)3]2+, and 4.5 mM [Co(NH3)5Cl]2+. Each sample was ∼100 μL and contained in a flat cell. A dark spectrum was recorded before the sample was exposed to in situ continuous illumination of a 447.5 nm LED (same setup as above) at ambient atmosphere. EPR settings: microwave frequency, 9.85 GHz; microwave power 6.3 mW; modulation frequency 100 kHz; modulation amplitude 0.1 mT. The Xepr software package (Bruker) was used for data acquisition and processing.
7
EPR Spectroscopy of Cryogenic Samples
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Measurements were performed on a Bruker ELEXYS E500 spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments, Oxford, United Kingdom). Measurement temperatures ranged from 5 to 32 K, using liquid helium as coolant. The spectrometer was controlled by the Xepr software package (Bruker).
8
EPR Spectroscopy of Iron-Sulfur Proteins
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EPR samples (generally at 200 μM protein concentration) were prepared under strict anaerobic conditions. The proteins were reduced with 10–20 fold molar equivalents (2–4 mM) of NaDT or oxidized with 10–20 fold molar equivalents (2–4 mM) of thionine acetate for 20–40 minutes. The reduction was followed by UV/Vis, monitoring the disappearance of the [4Fe4S]2+ absorbance around 410 nm. The samples were transferred into quartz EPR tubes, capped with rubber septa before they were removed from the glovebox and immediately flash frozen. The EPR samples were stored in liquid nitrogen.
The low temperature CW EPR measurements were carried out with a Bruker Elexys 500 X-band spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 resonator equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). Low temperature measurements were carried out with liquid helium as coolant. The spectrometer was controlled by the Xepr software package (Bruker). Spectra were recorded with a 10 G modulation amplitude and a 100 kHz modulation frequency. Spectra were averaged over either four or eight scans.
The low temperature CW EPR measurements were carried out with a Bruker Elexys 500 X-band spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 resonator equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). Low temperature measurements were carried out with liquid helium as coolant. The spectrometer was controlled by the Xepr software package (Bruker). Spectra were recorded with a 10 G modulation amplitude and a 100 kHz modulation frequency. Spectra were averaged over either four or eight scans.
9
Determining Zero-Field Splitting in High-Spin Heme
10
EPR Spectroscopy of Metalloenzyme Variants
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X-band EPR measurements were performed
on a Bruker ELEXYS E500 spectrometer equipped with a SuperX EPR049
microwave bridge and a cylindrical TE011 ER 4122SHQE cavity
in connection with an Oxford Instruments continuous flow cryostat.
Measuring temperatures were achieved using liquid helium flow through
an ITC 503 temperature controller (Oxford Instruments). The Xepr software
package (Bruker) was used for data acquisition and processing. EasySpin
software version easyspin-6.0.0-dev.51 was used for spectral simulation
and fitting.39 (link) EPR samples of E252V and
E289D were prepared in 100 mM Tris–HCl, pH 8.0 under a neat
argon atmosphere and either directly flash-frozen (“as prepared”)
or flushed with H2 or CO gas inside the EPR tube for an
hour prior to freezing (“H2- or CO- flushed”).
EPR samples incubated with D2 were prepared in the absence
(4 μL of 1 mM enzyme in 10 mM Tris–HCl, pH 8.0 + 76 μL
100 mM Tris–HCl, pH 8.0) or presence of 95% D2O
(4 μL of 1 mM enzyme in 10 mM Tris–HCl, pH 8.0 + 76 μL
D2O;Figure S6 ). For samples
that were reduced with NaDT, 1 μL of 100 mM stock solution of
NaDT was added into 79 μL of 50 μM enzyme, resulting in
at least 20-fold molar excess of NaDT (Figure S7 ).
on a Bruker ELEXYS E500 spectrometer equipped with a SuperX EPR049
microwave bridge and a cylindrical TE011 ER 4122SHQE cavity
in connection with an Oxford Instruments continuous flow cryostat.
Measuring temperatures were achieved using liquid helium flow through
an ITC 503 temperature controller (Oxford Instruments). The Xepr software
package (Bruker) was used for data acquisition and processing. EasySpin
software version easyspin-6.0.0-dev.51 was used for spectral simulation
and fitting.39 (link) EPR samples of E252V and
E289D were prepared in 100 mM Tris–HCl, pH 8.0 under a neat
argon atmosphere and either directly flash-frozen (“as prepared”)
or flushed with H2 or CO gas inside the EPR tube for an
hour prior to freezing (“H2- or CO- flushed”).
EPR samples incubated with D2 were prepared in the absence
(4 μL of 1 mM enzyme in 10 mM Tris–HCl, pH 8.0 + 76 μL
100 mM Tris–HCl, pH 8.0) or presence of 95% D2O
(4 μL of 1 mM enzyme in 10 mM Tris–HCl, pH 8.0 + 76 μL
D2O;
that were reduced with NaDT, 1 μL of 100 mM stock solution of
NaDT was added into 79 μL of 50 μM enzyme, resulting in
at least 20-fold molar excess of NaDT (
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