Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: A biological model for type 1 Cu 2+ and its changes upon ligand binding and conformational transitions

Abstract

Insertion of Cu 2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn 2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD + and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the ear parameters of Type 2 Cu 2+. Ternary complex formation with NAD + converts the Cu 2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%–1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.