Potential contaminations with Cu2+ ions in the buffer used for BCS titration experiments [20 mM Mops-NaOH (pH 7.0) and 50 mM NaCl] were removed by filtering the buffer through a 5-ml HisTrap agarose column (GE Healthcare) that had been extensively washed with 250 mM EDTA and deionized water. Apo-PcuC was then transferred into this buffer (PD-10 desalting column), and the absence of Cu ions in the apo-PcuC preparation was verified by ESI mass spectrometry. A stock solution of Cu1+(BCS)2 was prepared by mixing tetrakis(acetonitrile)copper(I) hexafluorophosphate with 2 meq BCS acid (B1125, Sigma-Aldrich). The mixture was centrifuged, and the concentration of Cu1+(BCS)2 in the supernatant was determined via its specific absorbance at 483 nm (13,000 M−1 cm−1). Titration experiments were performed under anaerobic conditions at a constant initial Cu1+(BCS)2 concentration of 15 μM, mixed with 0 to 4 meq of apo-PcuC, apo-PcuC ΔC, apo-PcuC ΔHHM, apo-PcuC ΔC ΔHHM, or apo-ScoI. The samples were sealed and incubated for 1 hour or overnight at 25°C. Then, the absorbance at 483 nm of each sample was recorded and plotted against the molar equivalents of the respective protein added to Cu1+(BCS)2.

For the reverse experiments, PcuC variants were loaded with 1 or 2 meq of Cu1+ as described above. Then, full-length PcuC or the PcuC ΔC variants (constant concentration of 15 μM each) were mixed with 0 to 5 meq of BCS stock under anaerobic conditions and incubated overnight at 25°C. The recorded absorbance values at 483 nm were plotted against the molar BCS/protein ratio. Two independent sets of experiments confirmed reproducibility.

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