Reduced apo-CoxB was mixed with equimolar amounts of freshly purified ScoI·Cu2+ in 20 mM Mops-NaOH (pH 7.0) and 50 mM NaCl (final protein concentrations, 20 to 30 μM). The pink ScoI·Cu2+·CoxB complex formed within seconds after mixing and was loaded onto a Superdex 75 (16/60) column equilibrated with 20 mM Mops-NaOH (pH 7.0) and 50 mM NaCl. Fractions containing ScoI·Cu2+·CoxB were pooled, concentrated with ultrafiltration (10 kDa cutoff), and immediately used for further experiments.

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