Spectroscopic studies and electronic structure description of the high potential type 1 copper site in fungal laccase: Insight into the effect of theaxial ligand

A variety of spectroscopic techniques combined with density functional calc ulations are used to describe the electronic structure of the nonaxially li gated, trigonal planar type I copper site in three fungal laccases with sub stantially different type 1 copper reduction potentials. These methods are also applied to a mutant of the high-potential Polyporus pinsitis laccase i n which the nonligating axial phenylalanine (Phe) is changed to methionine (Met). Optical absorption, circular dichroism, and magnetic circular dichro ism spectroscopies of all three fungal laccases reveal that, relative to th e classic blue copper protein plastocyanin, the ligand field strength at th e type 1 Cu center and the oscillator strength of the charge-transfer trans itions increase. Resonance Raman spectra show that the envelope of Cu-S(Cys ) stretching bands is shifted to higher energy in the fungal laccases, impl ying a stronger Cu-S(Cys) bond. Differences in the EPR spectra of the funga l laccases and plastocyanin are found to result from the increased ligand f ield and decreased 4s mixing into the Cu d(x2-y2) half-tilled, highest occu pied molecular orbital (HOMO). All three fungal laccases display similar sp ectroscopic properties despite their differing reduction potentials. Electr onic absorption, circular dichroism (CD), magnetic circular dichroism (MCD) , resonance Raman, and EPR spectroscopies show significant perturbation of the electronic structure of the fungal laccase type 1 copper site upon muta tion of the axial Phe to Met, consistent with the site becoming more like t hat in plastocyanin, which has an axial Met ligand; the ligand field decrea ses, covalency of the Cu-S(Cys) bond decreases, and the Raman shifts of the Cu-S stretching bands decrease. Density functional calculations on the fun gal laccase site provide insight into the origin of the experimentally obse rved increase in covalency and ligand field strength. These calculations sh ow that it is the elimination of the Met ligand donor interaction that lead s to an increase in the donor strength of the S(Cys). The contribution of t he axial ligand to the reduction potential is discussed.