1. Fig. 2 Copper-binding properties of PcuC.
Copper-binding properties of PcuC. (A) ESI mass spectra of apo-PcuC (blue), PcuC·Cu1+·Cu1+ (pink), and PcuC·Cu2+·Cu2+ (violet). (B) EPR spectra (continuous wave, 9.5 GHz, 40 K), normalized to identical protein concentration, of PcuC·Cu2+·Cu2+ (violet), PcuC·Cu1+·Cu1+ in the presence of 50 mM Na-dithionite (gray), PcuC·Cu1+·Cu1+ in the absence of Na-dithionite (pink), and PcuC after incubation with 1.5 (green) or 3 meq (blue) of a Cu1+/Cu2+ mixture (1:1). Copper complexes were prepared under anaerobic conditions, and excess Cu ions were removed by buffer exchange with 20% glycerol, 20 mM Mops-NaOH (pH 7.0), and 50 mM NaCl before EPR measurements. (C) EPR determination of distances between the Cu2+ ions in PcuC·Cu2+·Cu2+ by four-pulse ultra-wideband DEER, with uncertainties within 15% root mean square deviation. The DEER distance distribution shows Cu(II)-Cu(II) interspin distances widely distributed with a full width at half maximum (FWHM) ranging from 1.6 to 2.6 nm. The small peak observed in the 3- to 4-nm distance region is at the edge of resolution for the achievable dipolar evolution time of 1.4 μs (fig. S3), is therefore affected by trace length and noise, and was not interpreted.
Fabia Canonica et al. Sci Adv 2019;5:eaaw8478
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