Spectroscopic Studies and Electronic Structure Description of the High Potential Type 1 Copper Site in Fungal Laccase: Insight into the Effect of the Axial Ligand

A variety of spectroscopic techniques combined with density functional calculations are used to describe the electronic structure of the nonaxially ligated, trigonal planar type 1 copper site in three fungal laccases with substantially different type 1 copper reduction potentials. These methods are also applied to a mutant of the high-potential Polyporus pinsitis laccase in which the nonligating axial phenylalanine (Phe) is changed to methionine (Met). Optical absorption, circular dichroism, and magnetic circular dichroism spectroscopies of all three fungal laccases reveal that, relative to the classic blue copper protein plastocyanin, the ligand field strength at the type 1 Cu center and the oscillator strength of the charge-transfer transitions increase. Resonance Raman spectra show that the envelope of Cu−S(Cys) stretching bands is shifted to higher energy in the fungal laccases, implying a stronger Cu−S(Cys) bond. Differences in the EPR spectra of the fungal laccases and plastocyanin are found to resu...