Helium flow cryostat

EPR measurements were performed using an X/Q-band Bruker Elexsys E580 Spectrometer (Bruker BioSpin GmbH, Germany) equipped with a closed-cycle cryostat (Cryogenic Ltd., UK) and X-band split-ring resonator module (ER 4118X-MD5), or a Bruker EMXMicro spectrometer with a helium flow cryostat (Oxford Instruments, UK). All measurements were performed at X-band (9.7 GHz). The magnetic field was calibrated at room temperature with a Bruker strong pitch standard (g = 2.0028). Baseline spectra of the empty resonator, of samples containing only buffer, as well as of oxidised complex I were found to be identical; all the CW spectra presented have been baseline subtracted. Continuous-wave EPR measurement conditions were 100 kHz modulation frequency, 7 G modulation amplitude; other measurement conditions are given in figure legends.

Absorbance kinetic data at A_{386 nm} were recorded via a fibre optic link, as previously described (Crack et al., 2007 (link)). EPR measurements were made with an X-band Bruker EMX EPR spectrometer equipped with a helium flow cryostat (Oxford Instruments). Unless stated otherwise, EPR spectra were measured at 10 K at the following instrumental settings: microwave frequency, 9.471 GHz; microwave power, 3.18 mW; modulation frequency, 100 kHz; modulation amplitude, 5 G; time constant, 82 ms; scan rate, 22.6 G/s; single scan per spectrum. Relative concentrations of the paramagnetic species were measured using the procedure of spectral subtraction with a variable coefficient (Svistunenko et al., 2006 (link)) and converted to absolute concentrations by comparing an EPR spectrum second integral to that of a 1 mM Cu(II) in 10 mM EDTA standard, at non-saturating values of the microwave power. The RirA EPR signal saturation was studied by taking EPR measurements at 13 values of microwave power, ranging from 0.2 µW to 200 mW, at eight temperature values, ranging from 4 K to 50 K. The EPR sample was equilibrated at every new temperature for at least 8 min before the power set of spectra measurements commenced.

X-band EPR spectra were recorded using a Bruker-Biospin EleXsys E500 spectrometer equipped with a standard rectangular Bruker EPR cavity fitted to an Oxford Instruments helium flow cryostat. Redox poised samples were prepared as previously described9 (link)52 (link). Redox potentials are given in the text with respect to the standard hydrogen electrode.

Samples and standards were measured at the Stanford Synchrotron Radiation Lightsource (SSRL), California at the beamline 7–3, with the SPEAR electron storage ring containing 500 mA at 3.0 GeV, using a Si (220) double crystal monochromator. Samples were maintained at 10 K using a helium flow cryostat (Oxford instruments, Abingdon, UK) with sample cuvettes inclined at 45° to the incident X-ray beam. For each sample, 7 to 11 scans each of about 35 min duration were accumulated. The incident and transmitted X-ray intensities were monitored using nitrogen-filled gas ionization chambers operating at 1.2 atmospheres with a sweeping voltage of 1600 V, while the Cd K-edge X-ray absorption was measured as the X-ray K_α fluorescence excitation spectrum using an array of 30 germanium detectors [24 (link)] with Soller slits and six absorption-length silver metal filters to preferentially eliminate scattered X-rays so as to maintain the detector count rates in the pseudo-linear regime. The energy was calibrated with reference to the lowest energy K-edge inflection of cadmium metal foil which was assumed to be 26714.0 eV. Data were collected using the Web version of the XAS Collect data acquisition software (SSRL, Stanford, CA, USA) [25 (link)].