Low-temperature EPR spectra were acquired on a Bruker X-band EMX plus 10/12 spectrometer equipped with an Oxford Instruments ESR 910 continuous helium-flow cryostat. A cylindrical resonator (ER4119hs TE011) was used for EPR data collection. The purified DnfA and the sample reduced by 1 mM dithionite in anaerobic chamber (“reduced” state) were assayed. For samples in mixed-valence state, DnfA was mixed with NADH, FAD, and NH2OH, and the reaction was allowed to incubate at 30 °C for 30 min before EPR measurement. About 10% glycerol was added to the protein samples as cryoprotective agent, and each sample was placed into quartz EPR tubes (Wilmad; 707-SQ-250 M, 3 mm inner diameter, 4 mm outer diameter). The EPR tubes were frozen in liquid N2 for subsequent EPR measurement. For each sample, multiple scans were accumulated to obtain a good S/N ratio. The experiment parameters are provided in figure legend.
707 sq 250 m
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The 707-SQ-250 M is a laboratory equipment product manufactured by Wilmad. It is a precision instrument designed for use in scientific research and analytical applications. The core function of this product is to provide accurate measurements and data collection.
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10 protocols using 707 sq 250 m
1
Low-Temperature EPR Analysis of DnfA
2
EPR Spectroscopy of clMagR Protein
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The EPR spectra of the clMagR samples were recorded at the X-band on a Bruker EMX plus 10/12 spectrometer using an Oxford ESR-910 liquid helium cryostat. Briefly, 200 μL of 1 mmol/L purified protein (oxidized) was mixed with 50 μL of glycerol in TBS buffer (20 mmol/L Tris, 150 mmol/L NaCl, pH 8.0). The reduced proteins were prepared by adding 10 mmol/L Na
2S
2O
4 to the samples. The protein samples were transferred into a quartz EPR tube (Wilmad 707-SQ-250 M) and frozen in liquid nitrogen. The EPR signals were monitored at different temperatures (10 K, 25 K, 45 K, and 60 K) with a microwave frequency of 9.40 GHz, microwave power of 2 mW, modulation amplitude of 2.0 G, and receive gain of 1.0×10
4.
2S
2O
4 to the samples. The protein samples were transferred into a quartz EPR tube (Wilmad 707-SQ-250 M) and frozen in liquid nitrogen. The EPR signals were monitored at different temperatures (10 K, 25 K, 45 K, and 60 K) with a microwave frequency of 9.40 GHz, microwave power of 2 mW, modulation amplitude of 2.0 G, and receive gain of 1.0×10
4.
3
Photochemical Catechol Degradation Kinetics
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Solid particles (80 mg) with different concentrations of catechol were poured into thin-wall quartz tubes (707-SQ-250M, Wilmad, USA). These particles were left in the dark for 3 days or subjected to light irradiation for 180 min in pure N2 or O2. The irradiation was provided by an arc light source (LOT-Oriel GmbH & CO. KG, Germany) equipped with a 100 W mercury lamp (USH-102D, Ushio, Japan). Two glass filters were used to restrict the emissions below 280 ± 10 nm or below 340 ± 20 nm (JB280 and JB340, Ceaulight, Beijing, China). The tubes were filled with a continuous flowing gas consisting of different N2:O2 ratios (100:0, 70:30, 50:50, 30:70, and 0:100). The gases (N2 and O2) were highly pure (≥99.999%), and the water content of the gases was less than 3 mg/m3. Because of the surface adsorbed O2 and O2 presence before N2 inflow, it is not practical to completely remove O2. Therefore, for the atomosphere condition of 100% N2, it accutually refers to O2-limited condition. As reference samples, solid particles were placed in the same atmosphere environment without UV/Vis light irradiation. The concentration of catechol was analyzed after 0, 10, 30, 60, 90, 120, 150, and 180 min of irradiation. All of the samples were run in duplicate.
4
Characterization of Cu(II)-(AB)2 Complex
5
EPR Spectroscopy of Oxidized NCOA4
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X-band (∼9.6 GHz) EPR spectra were recorded using EMX plus 10/12 spectrometer (Bruker), equipped with Oxford ESR-910 liquid helium cryostat. Briefly, 1 mM oxidized NCOA4 (as-isolated NCOA4) in Tris buffer (50 mM Tris, pH 8) and 10% (v/v) glycerol were transferred into a 4 mm diameter quartz EPR tube (Wilmad 707-SQ-250 M) and frozen in liquid nitrogen. EPR signals of oxidized NCOA4 were recorded at various temperatures (10 K, 25 K, and 45 K). Parameters for recording the EPR spectra were typically 2 G modulation amplitude, 9.40 GHz microwave frequency, and 2 mW incident microwave power; sweep time was 64 s.
6
X-band EPR Analysis of MagR Proteins
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X-band (9.6 GHz) EPR spectra of the MagR proteins were recorded using an EMX plus 10/12 spectrometer (Bruker, USA), equipped with an Oxford ESR-910 liquid helium cryostat. Briefly, 1 mmol/L oxidized proteins (as-purified) in buffer E (20 mmol/L Tris, 150 mmol/L NaCl, 5 mmol/L d-desthiobiotin, pH8.0) were mixed in a total volume of 200 μL with 50 μL of glycerol, respectively. The 1 mmol/L reduced proteins were obtained by adding 2 μL of Na2S2O4 (1 mol/L) to the protein samples. The protein samples were then transferred into a 4 mm diameter quartz EPR tube (Wilmad 707-SQ-250 M, USA) and frozen in liquid nitrogen. The EPR signals of the oxidized and reduced proteins were recorded at different temperatures (10, 25, 45, and 60 K). The EPR conditions were: microwave frequency, 9.4 GHz; microwave power, 2 mW; modulation frequency, 100 kHz; modulation amplitude, 2 G; receive gain, 1.0×104.
7
Measuring Ascorbate Radical in CSF
8
EPR Spectroscopy Characterization of Samples
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EPR measurements
were performed using a Bruker X-band ELEXSYS E-500 CW EPR spectrometer
equipped with an in-cavity cryostat (Bruker/ColdEdge Technologies).
The magnetic field was calibrated with a 2,2-diphenyl-1-picrylhydrazyl
crystalline sample. All measurements were recorded at room temperature
using a Bruker super-high-Q resonator (ER4122SHQE/0113). Approximately
30 μL of the sample was loaded into a quartz capillary tube
(Wilmad-LabGlass, WG-221T-RB), which was sealed with epoxy and placed
inside an X-band EPR tube (Wilmad-LabGlass, 707-SQ-250M). Typical
EPR parameters were as follows: microwave power, 1 mW; modulation
frequency, 100 kHz; modulation amplitude, 10 G; time constant, 81.92
ms; and conversion time, 81.92 ms. All spectra were manually baseline
corrected.
were performed using a Bruker X-band ELEXSYS E-500 CW EPR spectrometer
equipped with an in-cavity cryostat (Bruker/ColdEdge Technologies).
The magnetic field was calibrated with a 2,2-diphenyl-1-picrylhydrazyl
crystalline sample. All measurements were recorded at room temperature
using a Bruker super-high-Q resonator (ER4122SHQE/0113). Approximately
30 μL of the sample was loaded into a quartz capillary tube
(Wilmad-LabGlass, WG-221T-RB), which was sealed with epoxy and placed
inside an X-band EPR tube (Wilmad-LabGlass, 707-SQ-250M). Typical
EPR parameters were as follows: microwave power, 1 mW; modulation
frequency, 100 kHz; modulation amplitude, 10 G; time constant, 81.92
ms; and conversion time, 81.92 ms. All spectra were manually baseline
corrected.
9
EPR Spectroscopy of Iron-Sulfur Proteins
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The cells expressing clMagRWT, clMagR3M, ecIscA, EntB, FepA or none, were harvested and resuspended in LB medium containing 20 mM deferoxamine and incubated at 37°C for anther 15 min as described by the Imlay's group in 2002 [35 (link)]. Then, the cells were washed with 10 mM diethylenetriaminepentaacetic acid once, and 20 mM cold Tris-HCl (pH = 7.4) twice and resuspended in 20 mM cold Tris-HCl (pH = 7.4). 200 μL of the above samples were mixed with 50 μL of glycerol and transferred to an EPR tube (Wilmad 707-SQ-250 M, USA) and frozen in liquid nitrogen until EPR measurements. The EPR signals were monitored at different temperatures (10 K) with a microwave frequency of 9.40 GHz, a microwave power of 2 mW, a modulation amplitude of 2.0 G and a receive gain of 1.0 × 104.
10
EPR Spectroscopy of Oxidized and Reduced Proteins
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X-Band (∼9.6 GHz) EPR spectra were recorded on an EMX plus 10/12 spectrometer (Bruker, Billerica, MA), equipped with Oxford ESR910 Liquid Helium cryostat. For the oxidized protein samples, 200 μL of 1 mM as-isolated purified protein were mixed with 50 μl of glycerol in TBS buffer (20 mM Tris, 150 mM NaCl, pH 8.0). For reduced protein samples, 10 mM sodium dithionite (Na2S2O4) was added to the above protein solutions. Then, the protein samples were transferred into 4 mm diameter quartz EPR tubes (Wilmad 707-SQ-250 M) and frozen in liquid nitrogen. The EPR signals of oxidized and reduced proteins were recorded at different temperatures (10 K, 25 K, 45 K, and 60 K) with a modulation amplitude of 2 G, a microwave frequency of 9.40 GHz, an incident microwave power of 2 mW, and a sweep time of 25.60 s.
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