Resonance Raman studies of the coupled binuclear copper active site in met azide hemocyanin

Abstract

Spectroscopic studies were performed on the met azide derivative of hemocyanin in order to probe small molecule binding to the coupled binuclear copper active site. The charge transfer spectrum shows a broad band near 380 nm in the absorption and a negative feature near 450 nm in the CD. Resonance Raman results indicate the presence of two enhanced vibrations, the copper—azide stretch at 397 cm −1 and the asymmetric intraazide stretch at 2042 cm −1. The 397 cm −1 peak is maximally enhanced near 500 nm and the 2042 cm −1 peak shows increasing enhancement into the u.v. At least three separate charge transfer transitions are needed to simultaneously fit the absorption, CD and resonance Raman profile, which requires that the azide bridges the binuclear copper active site. Mixed isotope results, using 14N 14N 15N, shows that the 2042 cm −1 peak splits into two peaks at 2023 and 2035 cm −1 and the 397 cm −1 peak shifts to 393 cm −1. A normal coordinate analysis was used to determine that the mixed isotopic splitting is not due to a simple mass effect but requires nonequivalence in bonding within the azide molecule. Combined with previous data, the results indicate that the azide is bound in a μ-1,3 geometry and the nonequivalence in bonding within the azide molecule is due to a strong interaction with residue(s) in the protein pocket.