Epr tube

Substance P, [Lys]³-bombesin, and formic acid were purchased from Sigma Aldrich Company Ltd., Dorset, UK. Low concentration Agilent tuning mix was purchased from Agilent Technologies (Santa Clara, CA). Complex 1 was synthesised and characterised as described elsewhere.6 (link) EPR tubes were purchased from Wilmad Labglass. The spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) was obtained from Enzo Life Sciences in high purity. Ultra-pure water was obtained from a Milli-Q UV III system (Milli-Q, Hertfordshire, UK).

Washed cell pellets from 800 ml cultures were resuspended in 2.5 ml of anoxic base salts medium using a pipette. Once pellets were fully resuspended, 2,000 μl, 500 μl, and 10 μl of the cell suspension were subsampled for EPR spectroscopy, AA spectroscopy, and cell density determination, respectively. Samples for EPR and AA spectroscopic analyses were placed into 2.0- or 1.5-ml screw-cap microcentrifuge tubes with O-ring fittings, respectively (Thermo Fisher Scientific). The tubes and their contents were then centrifuged in a fixed-angle rotor at 15,000 × g for 15 min at 4°C. The samples for cell counts were diluted 40-fold and subjected to cell counting immediately. After centrifugation, the supernatant of the samples for use in EPR and AA spectroscopy was removed from the cell pellet using a pipette. Cell pellets for determination of Fe content via AA spectroscopy were stored at −80°C, while those for EPR spectroscopy were resuspended in 300 μl of anoxic base salts medium containing 25% glycerol as a glassing agent. Samples for EPR spectroscopy were transferred into EPR tubes (4-mm OD; Wilmad Lab Glass, NJ, USA), capped with rubber septa, and then immediately removed from the chamber and flash frozen in liquid N₂. Tubes and their contents were stored at liquid N₂ temperatures until spectral acquisition occurred.

Continuous-wave X-band electron paramagnetic resonance (EPR) spectra were recorded using a Bruker ESP-300 spectrometer with an EMX PremiumX microwave bridge and an EMXplus standard resonator in perpendicular mode, equipped with an Oxford Instruments ESR900 continuous helium flow cryostat using a VC40 flow controller for helium gas. Spectra were recorded in 4-mm calibrated quartz EPR tubes (Wilmad LabGlass, Vineland, NJ) under the following conditions: temperature, 12 K; microwave frequency, 9.4 GHz; microwave power, 20 mW; modulation frequency, 100 KH_Z; modulation amplitude, 8.14 G; time constant, 20.48 ms. The cavity background signal was recorded using an EPR tube filled with 100 mM MOPS buffer at pH 7.3 and was subtracted from the experimental spectra. Each spectrum represents the sum of 5 scans. Spectra presented were normalized to the same concentration of FeFe protein (10 mg/ml).