Continuous flow helium cryostat

The formation of the Fe-S center in ferredoxin was also analyzed by Electron paramagnetic resonance EPR. Apo-ferredoxin was prepared as described above. All the reconstitutions were made in the presence of 30 µM apo-ferredoxin, 4 mM DTT, 2 µM IscS, 2 mM L-cysteine and 300 µM E. coli RIC. The spectra of control reactions that contained all components except apo-ferredoxin or RIC were acquired. Two other control reactions in which RIC was replaced by ferrous iron (200 µM) or apo-RIC (300 µM) were also analyzed. In all the assays, the reaction was initiated by the addition of L-cysteine and let proceed for 2 h in an anaerobic chamber, after which, samples were transferred to EPR tubes and frozen in liquid nitrogen. After recording their spectra, samples were thawed (under anaerobic conditions) and reduced by the addition of a few drops of sodium dithionite solution (100 mM), frozen and analyzed again. EPR spectra were obtained on a Bruker EMX spectrometer equipped with an Oxford Instruments continuous flow helium cryostat and were recorded at 9.34 MHz microwave frequency, 2.0 mW microwave power, 1 mT modulation amplitude and at 17 K.

Pulsed EPR measurements were performed at Q band (34 GHz) and −223 °C on an Elexsys 580 spectrometer (Bruker). Therefore, 15 μl of the freshly prepared samples were loaded into EPR quartz tubes with a 1.6 mm outer diameter and shock frozen in liquid nitrogen. During the measurements, the temperature was controlled by the combination of a continuous-flow helium cryostat (Oxford Instruments) and a temperature controller (Oxford Instruments). The four-pulse DEER sequence was applied^{64 (link)} with observer pulses of 32 ns and a pump pulse of 13–18 ns. The frequency separation was set to 70 MHz and the frequency of the pump pulse to the maximum of the nitroxide EPR spectrum. Validation of the distance distributions was performed by means of the validation tool included in DeerAnalysis^{65 (link)} and varying the parameters “Background start” and “Background density” in the suggested range by applying fine grid. A prune level of 1.15 was used to exclude poor fits. Furthermore, interspin distance predictions were carried out by using the rotamer library approach included in the MMM software package^{19 (link)}. The calculation of the interspin distance distributions is based on the cryo-EM structures of state 1, state 2 and the crystal structure [5MRW]^{4 (link)} for the comparison with the experimentally determined interspin distance distributions.