Te 300 x band spectrometer

The (potential) iron-sulfur cluster of C785_RS13685 and C785_RS21250 was analyzed by electron paramagnetic resonance (EPR) using a JEOL TE-300 X-band spectrometer operating with a 100-kHz field modulation. A temperature-dependent analysis was performed in the range of 10 to 40 K using a LTR-3 liquid helium cryostat (Air Products). The purified enzyme (~40 mg/ml) was dialyzed in 50 mM HEPES-NaOH (pH 7.2) containing 0.1 mM (NH₄)₂Fe(SO₄)₂·6H₂O, 10 mM DTT, and 50% (v/v) glycerol (for C785_RS13685) or 50 mM HEPES-NaOH (pH7.2) containing 1 mM MgCl₂ and 50% (v/v) glycerol (for C785_RS21250). If necessary, C785_RS21250 was anaerobically reduced with a 10-fold excess sodium dithionite (Na₂S₂O₄). EPR spectra were recorded using the following representative conditions; microwave frequency 8.9820 to 8.9995 GHz monitored by internal frequency counter, microwave power; 5.0 mW, 100 kHz field modulation magnitude; 0.40 to 0.63 mT, center field; 280 ± 250 mT or 300 ± 50 mT, sweep time; 4.0 or 8.0 min, time constant; 0.1 sec, and receiver amplitude; 100 to 800. In the present study, g-values were evaluated based on the g-value of the Li-salt of tetracyanoquinodimethane (2.0025) as an external standard. The magnetic field strength of EPR spectra was calibrated using the hyperfine coupling constant (8.69 mT) of the Mn(II) ion doped in MgO powder.

Continuous-wave EPR spectra were obtained on a JEOL TE-300 X-band spectrometer operating with a 100-kHz field modulation. A temperature-dependent analysis was performed at 20 K using an LTR-3 liquid helium cryostat (Air Products). The experimental parameters used to acquire the spectra in Fig. 5 were as follows: microwave frequency; 8.9919–9.0012 GHz monitored by an internal frequency counter, microwave power; 5.0 mW, 100 kHz field modulation magnitude; 0.40–1.25 mT, center field; 280 ± 250 mT, sweep time; 4.0 min, time constant; 0.1 sec, and receiver amplitude; 160–1000. The g values of the paramagnetic species were assessed using Li-tetracyanoquinodimethane powder (g = 2.0025) as an external standard. Magnetic field strength was calibrated using the hyperfine coupling constant (8.69 mT) of the Mn(II) ion-doped in MgO powder. The (aerobically) purified enzyme (~40 mg/ml) was dialyzed in 50 mM potassium phosphate (pH 7.0) containing 1 mM (NH₄)₂Fe(SO₄)₂·6H₂O, 10 mM DTT, and 50% (v/v) glycerol under anaerobic conditions using the AnaeroPack system (Mitsubishi Gas Chemical).

The content of non-heme iron was assessed using a Metallo Assay kit (AKJ Global Technology, Japan). The (potential) iron-sulfur cluster was analyzed by electron paramagnetic resonance (EPR) using a JEOL TE-300 X-band spectrometer operating with a 100-kHz field modulation. A temperature-dependent analysis was performed in the range of 10 to 40 K using a LTR-3 liquid helium cryostat (Air Products). The purified AtLhpI protein (~40 mg/ml) was prepared in 50 mM Tris-HCl (pH8.0) containing 50% (v/v) glycerol for this purpose, and treated with a 10-fold excess amount of Na₂S₂O₄ under a nitrogen atmosphere. EPR spectra were recorded using the following representative conditions; microwave frequency; 8.9995 GHz, microwave power; 5.0 mW, 100 kHz modulation magnitude; 0.63 mT, center field; 280 ± 250 mT, sweep time; 4.0 min, time constant; 0.1 sec, and receiver amplitude; 500. In the present study, g-values were evaluated based on the g-value of the Li-salt of tetracyanoquinodimethane (2.0025) as an external standard. The magnetic field strength of EPR spectra was calibrated using the hyperfine coupling constant (8.69 mT) of the Mn(II) ion doped in MgO powder.