Two hundred micromolar WT L-PGDS in 20 mM HEPES, 100 mM NaCl, pH 7.5 with 200 μM heme (1 : 1) complex was used to record the X-band (9.3835 GHz) CW EPR spectra with a Bruker Biospin Elexsys E500 EPR spectrometer fitted with a Bruker superhigh Q cavity and a flow-through Oxford cryostat (CF935LT) in conjunction with an Oxford Instruments ITC503 variable-temperature controller. Measurements were made at 7.5 K using a modulation frequency of 100 kHz, modulation amplitude of 0.5 mT and a microwave power of 5 mW. The magnetic field was calibrated with a Bruker ER 036TM Teslameter.
Er 036tm teslameter
Manufactured by Bruker
The ER 036TM Teslameter is a precision instrument designed to measure magnetic field strength. It provides accurate and reliable measurements of magnetic flux density in Tesla (T) or Gauss (G). The core function of the ER 036TM is to quantify the magnitude of a magnetic field, without interpretation or extrapolation of its intended use.
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2 protocols using er 036tm teslameter
1
EPR Spectroscopy of Heme-Bound L-PGDS
2
Continuous-Wave EPR Spectroscopy of Mo(V) Centers
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Continuous-wave X-band (ca. 9 GHz) (CW) electron paramagnetic resonance (EPR) spectra were recorded with a Bruker Elexsys E580 CW/pulsed EPR spectrometer fitted with a super high Q resonator; the microwave frequency and magnetic field were calibrated with a Bruker microwave frequency counter and a Bruker ER 036TM Teslameter, respectively. A microwave power of 20 mW was used and optimal spectral resolution was obtained by keeping the modulation amplitude to a 1/10 of the linewidth. A flow-through cryostat in conjunction with a Eurotherm (B-VT-2000) variable temperature controller provided temperatures of 127–133 K at the sample position in the cavity.
Bruker’s Xepr (version 2.6b.45) software was used to control the data acquisition including, spectrometer tuning, signal averaging, temperature control and visualization of the spectra. Computer simulation of the EPR spectra were performed with the following spin Hamiltonian (Equation 2 )
using the XSophe-Sophe-XeprView (version 1.1.4) computer simulation software suite(Hanson et al., 2004 (link); Hanson et al., 2013 (link)) on a personal computer, running the Mandriva Linux v2010.2 operating system. Further detail on data interpretation and analysis for these experiments is detailed in Appendix 1.
Bruker’s Xepr (version 2.6b.45) software was used to control the data acquisition including, spectrometer tuning, signal averaging, temperature control and visualization of the spectra. Computer simulation of the EPR spectra were performed with the following spin Hamiltonian (
using the XSophe-Sophe-XeprView (version 1.1.4) computer simulation software suite(Hanson et al., 2004 (link); Hanson et al., 2013 (link)) on a personal computer, running the Mandriva Linux v2010.2 operating system. Further detail on data interpretation and analysis for these experiments is detailed in Appendix 1.
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