As the active-site cysteine pairs of ScoI and CoxB need to be in the dithiol form for copper binding, both proteins were treated with reducing agents before loading with Cu ions. ScoI was incubated overnight at 4°C with 10 mM DTT, 20 mM tris-Cl (pH 8.5), 0.3 M NaCl, and 0.5 mM EDTA. CoxB (20 mM) was incubated with 5 to 10 mM tris(2-carboxyethyl)phosphine (TCEP) overnight at 4°C in 20 mM Mops-NaOH (pH 7.0) and 50 mM NaCl. Reduced proteins were buffer-exchanged against 20 mM Mops-NaOH (pH 6.5), and concentrations were determined via their absorbance at 280 nm. For loading of ScoI with Cu2+, reduced apo-ScoI was mixed with 1.5 meq of CuCl2 and incubated for 15 min at room temperature. For preparation of CoxB·CuA, reduced apo-CoxB (10 to 20 μM) was mixed with 3 meq of CuCl2 and incubated for 5 min at room temperature. The CoxB·CuA–specific pink color formed immediately after mixing. Cu-loaded proteins were desalted and buffer-exchanged against 20 mM Mops-NaOH (pH 7.0) by gel filtration to remove excess, unbound Cu ions and subjected to a final purification step by hydrophobic chromatography. The ScoI·Cu2+ and CoxB·CuA solutions were supplemented with 1 M ammonium sulfate and 1.5 M ammonium sulfate, respectively, and the holo-proteins were separated from the apo-proteins on Butyl Sepharose 4 Fast Flow columns (GE Healthcare). ScoI·Cu2+ and CoxB·CuA were eluted with a linear gradient from 1 or 1.5 M to 0 M ammonium sulfate, respectively. Separation from the apo-protein was monitored using the ScoI·Cu2+–specific absorbance at 360 nm or the CoxB·CuA–specific absorbance at 479 nm. Fractions containing the holo-proteins were pooled, buffer-exchanged against 20 mM Mops-NaOH (pH 7.0) and 50 mM NaCl, concentrated, and stored at 4°C until further use within 48 hours.

Samples with PcuC variants were transferred to an anaerobic chamber (Coy Laboratory); loaded with different amounts of Cu1+ [tetrakis(acetonitrile)copper(I) hexafluorophosphate, Sigma-Aldrich, anaerobically dissolved in dimethyl sulfoxide], Cu2+ (CuCl2 in H2O), or a mixture of both; and incubated overnight at 5°C. Cu-loaded PcuC samples were desalted in the anaerobic chamber by gel filtration over PD MidiTrap G-25 columns (GE Healthcare), their concentration was measured, and protein samples were directly used for the experiments.

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