ISOCYANIDES AS LIGAND-DIRECTED INDICATORS OF CU(I) COORDINATION IN COPPER PROTEINS - PROBING THE INEQUIVALENCE OF THE CU(I) CENTERS IN REDUCED DOPAMINE-BETA-MONOOXYGENASE

摘要

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-beta-monooxygenase (D beta M) 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)(2)X ... Cu-B(I)(His)(2)Y (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. [References: 49]