V500 spectrometer

UV-visible absorbance measurements were performed using a Jasco V500 spectrometer, and CD spectra were measured with a Jasco J810 spectropolarimeter. EPR measurements were made with an X-band Bruker EMX EPR spectrometer equipped with a helium flow cryostat (Oxford Instruments). 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^–1; single scan per spectrum. Relative concentrations of the paramagnetic species were measured using the procedure of spectral subtraction with a variable coefficient56 (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.

UV-visible absorbance measurements were performed using a Jasco V500 spectrometer, and CD spectra were measured with a Jasco J810 spectropolarimeter. EPR measurements were performed at 10 K using a Bruker EMX EPR spectrometer (X-band) equipped with a liquid helium system (Oxford Instruments). Spin concentrations in the protein samples were estimated by double integration of EPR spectra with reference to a 1 mM Cu(II) in 10 mM EDTA standard. For native MS analysis, His-tagged RsrR was exchanged into 250 mM ammonium acetate, pH 8, using PD10 desalting columns (GE Life Sciences), diluted to ~21 μM cluster and infused directly (0.3 mL/h) into the ESI source of a Bruker micrOTOF-QIII mass spectrometer (Bruker Daltonics, Coventry, UK) operating in the positive ion mode. Full mass spectra (m/z 700–3500) were recorded for 5 min. Spectra were combined, processed using the ESI Compass version 1.3 Maximum Entropy deconvolution routine in Bruker Compass Data analysis version 4.1 (Bruker Daltonik, Bremen, Germany). The mass spectrometer was calibrated with ESI-L low concentration tuning mix in the positive ion mode (Agilent Technologies, San Diego, CA).

UV-visible absorbance and CD measurements were made with a Jasco V500 spectrometer and Jasco J810 spectropolarimeter, respectively. Samples were prepared in an anaerobic glovebox (O₂ < 10 ppm) and measured in a 1 cm pathlength anaerobic quartz cuvette. To simulate low iron conditions, the soluble high affinity iron chelator EDTA (Fe²⁺-EDTA, log K = 14.3, Fe³⁺-EDTA, log K = 25.1)²³ was used. For iron chelator and O₂ experiments, protein samples were placed in an anaerobic cuvette, a solution of the iron chelator EDTA was added to a final concentration of 1 mM, and the cluster response was followed via spectroscopy. For the O₂ experiments, protein samples were rapidly diluted with aerobic buffer (25 mM Hepes, 2.5 mM CaCl₂, 50 mM NaCl, 750 mM KCl, pH 7.5) to give the desired O₂ concentration, and the cluster response was immediately followed by spectroscopy.