The initial concentration of CoxB (reduced with TCEP and desalted) was kept constant (40 μM) and mixed with 0.5 to 5.0 meq of ScoI·Cu2+ in 20 mM Mops-NaOH (pH 7.0) and 50 mM NaCl. After incubation at 10°C (overnight), samples were analyzed by gel filtration on a Superdex 75 10/300 column (GE Healthcare) equilibrated with the same buffer, and eluted proteins were detected via their absorbance at 280 nm. Titrations were repeated independently at least two times and proved to be reproducible within experimental error.

For absorbance titration of CoxB with ScoI·Cu2+, the initial apo-CoxB concentration was kept constant (15 μM) and the ScoI·Cu2+ concentration was varied between 0 and 75 μM. The absorbance signal at 520 nm was plotted against added equivalents of ScoI·Cu2+. Data were fitted to a noncovalent binding equilibrium according to the following equation, where A is the monitored absorbance signal, A0 and A are the absorbance values of 0 and 100% complex formation, KD is the dissociation constant, [C]0 is the total concentration of apo-CoxB, and [S]0 is the total concentration of ScoI·Cu2+A=A0(A0A)·[C0]+[S0]+KD([C0]+[S0]+KD)24·[C0]·[S0]2·[C0]

Fitting the data yielded a KD value of 1.54 ± 0.8 × 10−7 M, showing that the concentration of apo-CoxB was at least two orders of magnitude above the KD value, thus not allowing accurate KD determination. As we could not lower protein concentrations due to the low sensitivity of the absorbance measurements, we confined the analysis to the estimation that the KD value of the ScoI·Cu2+·CoxB complex is below 10−7 M. Absorbance titrations were repeated independently at least two times, each confirming the 1:1 stoichiometry with the sharp kink in the titration profile (Fig. 1F).

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