All samples were mixed in an anaerobic chamber and sealed in a silanized microvial (8004-HP-H/iV2μ/SZ, Infochroma) with a polypropylene screw cap (G004-HP-CB-FKSKFK10, Infochroma). Samples were incubated at 10°C overnight before gel filtration at 25°C on an AdvancedBio SEC column (PL1580-5350, Agilent Technologies) equilibrated with 20 mM Mops-NaOH (pH 7.0) and 50 mM NaCl. Gel filtration runs were performed with a high-performance liquid chromatography system (Agilent Technologies, 1100 Series) equipped with a diode array detector, allowing online recording of ultraviolet-visible (UV-Vis) spectra of eluting proteins. The initial concentration of ScoI·Cu2+·CoxB was kept constant (20 μM) in all experiments, and PcuC·Cu1+·Cu2+ was varied between 5 and 60 μM. CuA center formation was followed by recording the CoxB·CuA–specific absorbance at 813 nm. The amount of CoxB·CuA formed was quantified by plotting the peak areas at 813 nm against the molar equivalents of PcuC·Cu1+·Cu2+ and comparing them to the peak area of a 20 μM CoxB·CuA reference sample. Peak areas were determined with PeakFit v4.12 (exponentially modified Gaussian model). Furthermore, the absorbance spectra at the peak maxima recorded at 280 nm were used to quantify the CoxB·CuA center by comparing the increase in the CoxB·CuA–specific absorbance peaks at 367, 479, and 813 nm to the absorbance spectra of the CoxB·CuA reference sample. For the titration of apo-CoxB with PcuC·Cu1+·Cu2+, the initial concentration of apo-CoxB was kept constant (15 μM), and PcuC·Cu1+·Cu2+ was again varied between 0.25 and 3 meq. Titrations were repeated independently at least two times and proved to be reproducible within experimental error.

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