Isocyanides as Ligand-Directed Indicators of Cu(I) Coordination in Copper Proteins. Probing the Inequivalence of the Cu(I) Centers in Reduced Dopamine-β-monooxygenase

The use of isocyanides as ligand-directed probes of Cu(I) coordination in proteins has been investigated. Reaction of 2,6-dimethylphenyl isocyanide (DIMPI) with reduced dopamine-β-monooxygenase (DβM) indicates the initial formation of monoisocyanide complexes at each of the two coppers (Cu_A and Cu_B) with different frequencies (2148 and 2129 cm^-1) indicative of inequivalent Cu(I) coordination at each copper. However, further addition of DIMPI leads to formation of a species containing multiple isocyanide ligands, believed to be a trisisocyanide adduct with a single IR band at 2160 cm^-1. This titration behavior can be interpreted by the active site model Cu_A^I (His)₂X–CuB^I (His)₂Y (X = His; Y = Met) where the first stage of the reaction with isocyanide is the formation of a mono-DIMPI four-coordinate complex at each Cu, giving rise to the two observed IR bands (2148 and 2129 cm^-1) provided the protein ligands X and Y are different. The second stage is the displacement of protein-bound ligands by the isocyanide to form a protein-bound bis or tris complex (2160 cm^-1). Closely analogous chemistry involving the reaction of DIMPI with deoxyHc is described, which illustrates the generality of isocyanides as probes of Cu(I) coordination in copper proteins. A model system [Cu^I(MePY2)(DIMPI)]ClO4, II, is also described in which identical isocyanide-binding chemistry can be demonstrated, thus validating the conclusions on the protein systems. The crystal structure of II is described, and the clean conversion of II to a trisisocyanide complex is demonstrated by FTIR and FT Raman spectroscopy.