Suprasil epr tubes

All samples were prepared in 50 mM HEPES, 100 mM NaCl, pH 8 under oxygen-free conditions in an anaerobic glovebox (Mbraun, Stratham, NH). For all EPR experiments the final concentration of Cgr2 was either 150 or 200 µM. When required, the samples were reacted with an excess of sodium dithionite (10–20 equivalents) for 20–30 min at 22°C prior to freezing in liquid N₂. Spin quantification was carried out against a Cu²⁺-EDTA standard containing an equimolar concentration of CuSO₄ in 10 mM EDTA (150 or 200 µM), under non-saturating conditions. Samples (250 µL) were loaded into 250 mm length, 4 mm medium wall diameter Suprasil EPR tubes (Wilmad LabGlass, Vineland, NJ) and frozen in liquid N₂ under oxygen-free conditions. EPR spectra were acquired on a Bruker E500 Elexsys continuous wave (CW) X-Band spectrometer (operating at approx. 9.38 GHz) equipped with a rectangular resonator (TE102) and a continuous-flow cryostat (Oxford 910) with a temperature controller (Oxford ITC 503). The spectra were recorded at variable temperatures between 10–40 K at a microwave power of 0.2 mW, using a modulation amplitude of 0.6 mT, a microwave frequency of 9.38 GHz, a conversion time of 82.07 ms, and a time constant of 81.92 ms.

A 1 mM solution of 8-hydroxyquinoline (1 mL, 1.46% v/v DMSO in 1 mM ammonium acetate solution pH 9) was prepared. 0.5 equivalents of an aqueous solution of manganese(ii) chloride tetrahydrate solution was added. EPR measurements of the resulting solution were performed at 100 K using an X/Q-band Bruker Elexsys E580 spectrometer (Bruker, Germany) equipped with a closed-cycle cryostat (Cryogenic Ltd, UK) controlled by a LakeShore temperature controller. Measurements were carried out in an X-band split-ring resonator module with 2 mm sample access (ER 4118X-MS2). Samples were loaded on Suprasil EPR tubes (Wilmad LabGlass) with OD = 1.6 mm, ID = 1.1 mm. Baseline spectra of samples containing only the buffer were also collected and used as a reference. All the spectra presented have been baseline-subtracted.