All EPR samples were prepared in a 10 mm HPEPS buffer with 250 mm NaCl, 0.5 mm TCEP and 10% glycerol (v/v) (pH 7.6) in an aerobic condition. Samples containing ~ 200 μm AbCntA, 75 mm nicotinamide adenine dinucleotide (NADH) were transferred into 4‐mm Suprasil quartz EPR tubes (Wilmad LabGlass, Vineland, NJ, USA) and frozen in liquid N2. Annealing of the samples was performed at room temperature for the specified time‐duration in the figure caption. All EPR samples were measured on a Bruker EMX‐Plus EPR spectrometer (Coventry, UK) equipped with a Bruker ER 4112SHQ X‐band resonator as reported previously [39 (link)]. Sample cooling was achieved using a Bruker Stinger [40 (link)] cryogen‐free system mated to an Oxford Instruments ESR900 cryostat, and temperature was controlled using an Oxford Instruments MercuryITC (Abingdon, UK). The optimum conditions used for recording the spectra are given below; microwave power 30 dB (0.2 mW), modulation amplitude 5 G, time constant 82 ms, conversion time 12 ms, sweep time 120 s, receiver gain 30 dB and an average microwave frequency of 9.383 GHz, temperature 20 K. cw‐EPR spectra for the AbCntA‐WT, single (C206A and C209A) and double (C206AC209A) mutants of CntA were recorded in the presence and absence of CntB + NADH + carnitine, as reported previously [18 (link), 41 (link)].
Esr900 cryostat
Manufactured by Oxford Instruments
Sourced in United Kingdom
The ESR900 cryostat is a laboratory equipment designed for low-temperature research applications. It provides a stable and controlled low-temperature environment for various experiments and measurements. The core function of the ESR900 cryostat is to maintain a desired temperature, typically ranging from 4 Kelvin to 300 Kelvin, for the sample being studied.
Automatically generated - may contain errors
Lab products found in correlation
Elexsys e500 epr spectrometer, by Bruker (5 mentions)
Suprasil quartz epr tubes, by Wilmad (4 mentions)
Er 4112shq x band resonator, by Bruker (3 mentions)
Mercuryitc, by Oxford Instruments (3 mentions)
Er 4122shq super high q cavity, by Bruker (3 mentions)
Elexsys e500 spectrometer, by Oxford Instruments (3 mentions)
Emxplus epr spectrometer, by Bruker (2 mentions)
Itc503, by Oxford Instruments (2 mentions)
Elexsys e500 x band spectrometer, by Oxford Instruments (2 mentions)
Itc503s temperature controller, by Oxford Instruments (2 mentions)
Elexsys e500, by Bruker (2 mentions)
Elexsys e680 spectrometer, by Bruker (1 mentions)
Emx x band spectrometer, by Bruker (1 mentions)
Sm2f32 a, by Thorlabs (1 mentions)
Diode array spectrophotometer, by Bio-Logic (1 mentions)
Elexsys e580 spectrometer, by Bruker (1 mentions)
Itc 503 temperature controller, by Oxford Instruments (1 mentions)
E 101 microwave bridge, by Agilent Technologies (1 mentions)
Esp 300 spectrometer, by Oxford Instruments (1 mentions)
Elexsys 500 x band spectrometer, by Bruker (1 mentions)
28 protocols using esr900 cryostat
1
EPR Spectroscopy of AbCntA Protein
2
Continuous-Wave EPR Spectroscopy at 20K
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Continuous-wave (CW) EPR spectra were obtained at 9.48 GHz using an ELEXSYS E680 spectrometer (Bruker BioSpin GmbH) equipped with a standard TE102 cavity and an ESR900 Cryostat (Oxford Instruments). The measurements were performed at 20 K, modulation amplitude/frequency of 0.5 mT/100 kHz, time constant of 40 ms and microwave power of 0.16 mW.
3
Continuous Wave EPR Spectroscopy of CYP144A1
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Continuous wave X-band electron paramagnetic resonance (EPR) spectra of CYP144A1 proteins were obtained at 10 K using a Bruker ELEXSYS E500 EPR spectrometer equipped with an ER 4122SHQ Super High Q cavity. Temperature control was effected using an Oxford Instruments ESR900 cryostat connected to an ITC 503 temperature controller. Microwave power was 0.5 mW, modulation frequency was 100 KHz and the modulation amplitude was 5 G. EPR spectra were collected for the CYP144A1-FLV and CYP144A1-TRV proteins in the ligand-free state (200 μM) and at the same protein concentration following the addition of the azole inhibitor drug econazole (400 μM).
4
Continuous Wave EPR Analysis of OleT_JE P450s
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Continuous wave X-band EPR spectra for the OleTJE WT and mutant P450s were obtained at 10 K using a Bruker ELEXSYS E500 EPR spectrometer with an ER 4122SHQ Super High Q cavity. Temperature was controlled with an ESR900 cryostat (Oxford Instruments, Abingdon, UK). EPR spectra were collected for WT and OleTJE mutants at concentrations of 100–200 μm for ligand-free, arachidic acid-bound, and imidazole-bound forms. Arachidic acid was added to dilute OleTJE protein until the UV-visible spectrum showed no further optical change toward high spin. The enzyme was then concentrated to an appropriate concentration (200 μm for WT, H85Q, and F79A OleTJE; 150 μm for R245L OleTJE; and 100 μm for the F79W, F79Y, and R245E mutants) (the latter in its substrate-free form), taking into consideration that some of the mutants are prone to precipitation at high concentrations) using a Vivaspin (30,000 molecular weight cutoff).
5
Spectroscopic Analysis of Cellular Components
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MB, EPR, and UV–vis spectra
were collected and analyzed as described.23 (link) MB spectra were collected using a MS4 WRC spectrometer (SEE Co.,
Edina MN), calibrated using α-Fe foil at room temperature. Spectra
were analyzed and simulated using WMOSS software. EPR spectra were
collected at the University of Texas at Arlington on an X-band EMX
spectrometer (Bruker Biospin Corp., Billerica, MA) equipped with a
bimodal resonator (4116DM) and an Oxford Instruments ESR900 cryostat.
Signals were integrated using a custom Matlab (Mathworks.com) program.
Spin concentrations were calculated as described24 (link) using 1.00 mM CuSO4-EDTA as the standard.
UV–vis spectra of whole cells were collected on a Hitachi
U3310 spectrometer with a Head-on photomultiplier tube. Spectra were
simulated using OriginPro software as described.23 (link) Packed-cell samples were diluted 3-fold with ddH2O and analyzed in a 10 mm path length quartz UV–vis cuvette
(Precision cells). After collecting MB spectra, isolated mitochondria
were thawed anaerobically in a glovebox (≤5 ppm of O2, MBraun, Labmaster), diluted 3-fold with SH buffer (0.6 M Sorbitol,
0.02 M HEPES, Fisher Scientific, pH 7.4), and transferred to a 2 mm
path length quartz UV–vis cuvette (Precision cells). The cuvette
was sealed with a rubber septum and brought out of the glovebox and
immediately analyzed by UV–vis.
were collected and analyzed as described.23 (link) MB spectra were collected using a MS4 WRC spectrometer (SEE Co.,
Edina MN), calibrated using α-Fe foil at room temperature. Spectra
were analyzed and simulated using WMOSS software. EPR spectra were
collected at the University of Texas at Arlington on an X-band EMX
spectrometer (Bruker Biospin Corp., Billerica, MA) equipped with a
bimodal resonator (4116DM) and an Oxford Instruments ESR900 cryostat.
Signals were integrated using a custom Matlab (Mathworks.com) program.
Spin concentrations were calculated as described24 (link) using 1.00 mM CuSO4-EDTA as the standard.
UV–vis spectra of whole cells were collected on a Hitachi
U3310 spectrometer with a Head-on photomultiplier tube. Spectra were
simulated using OriginPro software as described.23 (link) Packed-cell samples were diluted 3-fold with ddH2O and analyzed in a 10 mm path length quartz UV–vis cuvette
(Precision cells). After collecting MB spectra, isolated mitochondria
were thawed anaerobically in a glovebox (≤5 ppm of O2, MBraun, Labmaster), diluted 3-fold with SH buffer (0.6 M Sorbitol,
0.02 M HEPES, Fisher Scientific, pH 7.4), and transferred to a 2 mm
path length quartz UV–vis cuvette (Precision cells). The cuvette
was sealed with a rubber septum and brought out of the glovebox and
immediately analyzed by UV–vis.
6
EPR Analysis of Redox-Active Metalloenzyme PhoX
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All PhoXMED193 EPR samples were prepared in activity buffer (pH 8.0). Two hundred fifty–microliter samples of purified PhoXMED193 at 190 μM were prepared with additives at final concentrations of either 5 mM dithionite, 20 mM dithionite, 50 mM EDTA, 1 mM PE, or no additives (no additive control). PhoXMED193 activity buffer was used as a blank. All PhoXMED193 samples were measured on a Bruker EMXplus EPR spectrometer equipped with a Bruker ER 4112SHQ X-band resonator at 10 K. Sample cooling was achieved using a Bruker “Stinger” cryogen free system mated to an Oxford Instruments ESR900 cryostat, and temperature control was maintained using an Oxford Instruments MercuryITC, as reported previously (75 (link)–77 (link)). The EPR spectra were measured with a microwave power of 20 dB (2.2 mW), a modulation amplitude of 5 G, a time constant of 82 ms, a conversion time of 12 ms, a sweep time of 120 s, a receiver gain of 30 dB, and an average microwave frequency of 9.385 GHz, as previously described (29 (link)). Each spectrum was averaged over four to six scans to get a better signal to noise ratio. The analysis of the continuous wave EPR spectra was performed using EasySpin toolbox (5.2.28) for the MATLAB program package (78 (link)).
7
EPR Analysis of Photoreactive Proteins
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All EPR samples were prepared in Tris-HCl buffer pH 7.5. Samples containing ~300 μM of wild-type TtCBD and TtCBD-H132A were transferred into 4 mm outer diameter/3 mm inner diameter Suprasil quartz EPR tubes (Wilmad LabGlass) and frozen in liquid N2. The photoactivation of the TtCBD and TtCBD-H132A was carried out at room temperature under anaerobic conditions. Optical irradiation at 530 nm (500 mW) was accomplished (for the specified duration described in the text/legend) using a Thorlabs Mounted High Power LED (M530L3) with the output beam collimated using a Thorlabs collimation adaptor (SM2F32-A). All EPR samples were measured on a Bruker EMXplus EPR spectrometer equipped with a Bruker ER 4112SHQ X-band resonator. Sample cooling was achieved using a Bruker Stinger34 (link) cryogen-free system mated to an Oxford Instruments ESR900 cryostat, and temperature was controlled using an Oxford Instruments MercuryITC. The optimum conditions used for recording the spectra are given below; microwave power 30 dB (0.22 mW), modulation amplitude 5 G, time constant 82 ms, conversion time 25 ms, sweep time 90 s, receiver gain 30 dB and an average microwave frequency of 9.383 GHz. All EPR spectra were measured as a frozen solution at 20 K, respectively. The analysis of the continuous wave EPR spectra were performed using EasySpin toolbox (5.2.28) for the Matlab program package35 (link).
8
EPR Spectroscopy of Reconstituted Radical Enzymes
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All reconstituted FnY·-β2s
were transferred to the appropriate
EPR tubes and frozen in liquid N2 prior to EPR spectroscopy
at a specific observation temperature. In the case of (2,3,6)F3Y·-β2 only, the sample was aged for 2 min following
addition of O2 during reconstitution of the active cofactor
and then frozen in liquid N2. The reason for this aging
procedure is detailed in theResults . All
9 GHz (X-band) continuous-wave (CW) EPR spectra were collected at
a temperature of 80 K under slow-passage, nonsaturating conditions
using a Bruker (Billerica, MA) ELEXSYS E500 X-band spectrometer equipped
with an Oxford Instruments ESR900 cryostat and an ITC-503 temperature
controller.
were transferred to the appropriate
EPR tubes and frozen in liquid N2 prior to EPR spectroscopy
at a specific observation temperature. In the case of (2,3,6)F3Y·-β2 only, the sample was aged for 2 min following
addition of O2 during reconstitution of the active cofactor
and then frozen in liquid N2. The reason for this aging
procedure is detailed in the
9 GHz (X-band) continuous-wave (CW) EPR spectra were collected at
a temperature of 80 K under slow-passage, nonsaturating conditions
using a Bruker (Billerica, MA) ELEXSYS E500 X-band spectrometer equipped
with an Oxford Instruments ESR900 cryostat and an ITC-503 temperature
controller.
9
EPR Spectroscopy of Altered NpRdhA Proteins
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Samples of 100 μM NpRdhA altered proteins were prepared as isolated or reduced and transferred in volumes of 300 μL into 4 mm Suprasil quartz EPR tubes (Wilmad, Vineland, NJ, USA) under anaerobic conditions. The tubes were anaerobically sealed, frozen and stored in liquid nitrogen. Experimental parameters were as follows: microwave power 0.5 mW, modulation frequency 100 kHz, modulation amplitude 5 G, temperature 30 K. Spectra were obtained using a Bruker ELEXSYS E500 spectrometer (Coventry, UK), Super high Q resonator (ER4122SHQ), Oxford Instruments (Abingdon, UK) ESR900 cryostat and ITC503 temperature controller. Spectra were sums of between 8 and 16 scans.
10
EPR Analysis of Protein Samples
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Samples were prepared for EPR after concentration of protein to ~30 mg/mL in 50 mM Tris pH 7.5, 200 mM NaCl. Samples were transferred into 4 mm Suprasil quartz EPR tubes (Wilmad, USA) and directly frozen and stored in liquid nitrogen. EPR experiments were conducted using the parameters as follows: microwave power 0.5 mW, modulation frequency 100 kHz, modulation amplitude 5 G, temperature 30 K. These parameters are non-saturating for the EPR signals concerned. Spectra were obtained using a Bruker ELEXSYS E500 spectrometer, Super high Q resonator (ER4122SHQ), Oxford Instruments ESR900 cryostat and ITC503 temperature controller.
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