Itc 503 temperature controller

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.

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.

100 µl aliquots of an apo-HydA1 protein solution (50 µM in 50 mM Tris–HCl, pH 8.0, 150 mM KCl) were mixed with 100 µl of a [2Fe]^adt solution (40 µM in 50 mM Tris–HCl, pH 8.0, 150 mM KCl), resulting in final concentrations of 25 µM protein and 20 µM [2Fe]^adt complex. The mixing was carried out inside of EPR tubes in the glovebox and the reaction mixture was either frozen immediately or incubated for a defined time period before freezing in an isopropanol cold well cooled by liquid nitrogen from the outside. After freezing, the samples were stored in liquid nitrogen until the EPR measurements. CW EPR measurements were carried out with an X-band EMX Micro EPR spectrometer (Bruker) using an ER049X SuperX microwave bridge in a Bruker SHQ0601 resonator equipped with a continuous-flow cryostat and an ITC 503 temperature controller (Oxford Instruments). The spectrometer was controlled by the Xenon software package (Bruker). Spectra were recorded with a 15 G modulation amplitude and a 100 kHz modulation frequency with 1 mW microwave power at 10 K at a microwave frequency of 9.38 GHz. Shown spectra represent the average of two magnetic field scans.

The composition of the iron–sulfur clusters of Huc was analysed by EPR spectroscopy. 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). Samples of Huc isolated in air were prepared under a neat argon atmosphere or flash-frozen following incubation under 1 atm H₂, and X-band EPR spectra were collected in the temperature range 40–7 K, at varying microwave powers. Preliminary simulations of the Ni signals in the EPR spectra were carried out using EasySpin v.6.0.0-dev47 (ref. ^{78 (link)}).

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).

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.