Esr 9 helium flow cryostat

EPR measurements were conducted with an EMX 6/1 spectrometer (Bruker) operating at X-band. The sample temperature was controlled with an ESR-9 helium flow cryostat (Oxford Instruments). Spectra were recorded at 40 K and 2 mW microwave power and at 13 K and 5 mW microwave power from 300 to 380 mT. Other EPR conditions were: microwave frequency, 9.360 GHz; modulation amplitude, 0.6 mT; time constant, 0.164 s; scan rate, 17.9 mT min⁻¹. 300 µL complex I (2.5–3.5 mg mL⁻¹) in buffer A were reduced with a 2000 fold molar excess NADH (10–14 mM) and shock frozen at 150 K in 2-methylbutane/methylcyclohexane (1:5; v:v).
To determine whether H₂O₂ oxidizes or damages cluster N1b, complex I was incubated with 1 mM H₂O₂ for 5 min. The excess H₂O₂ was removed by concentrating the sample by ultrafiltration (Amicon Ultra-15, MWCO: 100 kDa, Millipore; 3800 g, 4 °C, rotor A-4-44, centrifuge 5804R, Eppendorf) and subsequent tenfold dilution in buffer A with 5 mM MgCl₂, 10% (v/v) glycerol and 0.005% (w/v) LMNG. This procedure was repeated two times. An EPR spectrum of the concentrated sample reduced by a 2000 fold molar excess NADH was recorded.