A role for copper in biological time-keeping

A family of cell surface and growth related proteins that oxidize both NADH and hydroquinones and carry out protein disulfide-thiol interchange (ECTO-NOX proteins) exhibits unique characteristics. The two activities they catalyze, hydroquinone or NADH oxidation and protein disulfide-thiol interchange, alternate in CNOX (the constitutive ECTO-NOX), to generate a regular period length of 24 min. For NADH or hydroquinone oxidation each period is defined by maxima that recur at intervals of 24 min. Here, we report that bound Cu II is required to sustain the 24 min oscillation cycle of CNOX. CNOX preparations from plasma membranes of soybean, when unfolded in the presence of the copper chelator bathocuproine and refolded, lose activity. When refolded in the presence of copper, activity is restored. Unexpectedly, however, the released copper is capable of catalyzing NADH (or hydroquinone) oxidation in the absence of protein. Solvated Cu II as the chloride or other salts alone is capable of catalyzing NADH oxidation and the oxidation rates oscillate with an overall period length of 24 min. With Cu IICl 2 the pattern consists of five maxima, two of which are separated by an interval of 6 min and three of which are separated by intervals of 4.5 min [6 min + 4 (4.5 min)]. The period length is independent of temperature and pH. The asymmetry of the oscillatory pattern is retained after solvation of the Cu II salts in D 2O but the overall period length is increased to 30 min. The findings suggest that the bound copper of CNOX and perhaps of ECTO-NOX proteins in general, is essential to maintain the structural changes that underlie the periodic alternations in activity that define the 24 min time-keeping cycle of the protein.