Isocyanide binding to the copper(I) centers of the catalytic core of peptidylglycine monooxygenase (PHMcc)

Binding of the Cu(I)-specific ligands 2,6-dimethylphenyl isocyanide (DIMPI) and isopropyl isocyanide (IPI) to the reduced form of peptidylglycine monooxygenase (PHM) is reported. Both ligands bind to the methionine-containing Cu_M center, eliciting FTIR bands at 2138 and 2174 cm^-1, respectively, but appear unable to coordinate at the histidine-containing Cu_H center in the wild-type enzyme. This chemistry parallels that previously observed for CO binding to the reduced PHM catalytic core (PHMcc). However, in contrast to the CO chemistry, peptide substrate binding did not induce binding of the isocyanide at Cu_H. XAS confirmed the binding of DIMPI at Cu_M via the observation of a short Cu-C interaction at 1.87 Å and by the lengthening of the Cu-S(methionine) bond length by 0.06 Å. Similarly, FTIR studies on DIMPI binding to the M314I and H172A mutant forms of reduced PHMcc confirmed the assignment of the 2138-cm^-1 IR band as a Cu_M-DIMPI complex, but surprisingly also showed DIMPI binding to Cu_H, as indicated by a band at 2148 cm^-1. An inorganic complex, [Cu(1,2-Me₂Im)₂(DIMPI)](PF₆), was synthesized and its crystal structure was determined as a model for the interaction of isocyanides with imidazole-containing Cu(I) complexes. Comparison of EXAFS data for the protein and model suggests that DIMPI probably binds to Cu_M in a tilted fashion, similar to that of ethyl isocyanide binding to myoglobin.