Identification of the Cyanide Stretching Frequency in the Cyano Derivative of Copper/Zinc-Superoxide Dismutase by Ir and Raman Spectroscopy

Cyanide has been investigated as a potential ligand-directed probe of the coordination chemistry of Cu(II) and Cu(I) active sites via vibrational spectroscopic studies of CN^-coordinated to the metal centers in non-blue copper proteins. Native superoxide dismutase (SOD) was found to bind one CN^-that had IR and Raman frequencies at 2137 cm^-1. The assignment of this band was confirmed by using¹³CN^-, which gave the theoretically expected isotope shift, and by comparison with an inorganic model complex, [Cu(PMAS)]²⁺, which was found to form a monocyano complex with v_CN= 2125 cm^-1. Ultrafiltration experiments definitively identified the 2137-cm^-1band as v_CNof a protein-bound species. These results are consistent with a linear end-on-bonded CN^-on Cu(II)-SOD as proposed from earlier EPR and EXAFS experiments. Replacing H₂O in the medium by D₂O gave no isotope shift of the 2137-cm^-1band, indicating that CN^-is most likely not involved in hydrogen-bonding interactions in the active-site cavity. With increasing concentrations of cyanide, the 2137-cm^-1band became weaker and was accompanied by the appearance of strong vibrational modes characteristic of di-, tri-, and tetracyano Cu(I) complexes arising from Cu removal from the protein. These studies demonstrate the potential importance of cyanides (and isocyanides) as ligand-directed vibrational spectroscopic probes of non-blue copper proteins.