Spectroscopic characterization of the Leu513His variant of fungal laccase: effect of increased axial ligand interaction on the geometric and electronic structure of the type 1 Cu site.
A variety of spectroscopic techniques, combined with density functional calculations, are used to describe the electronic structure of the Leu513His variant of the type 1 Cu site in Myceliophthora thermophila laccase. This mutation changes the type 1 Cu from a blue to a green site. Electron paramagnetic resonance (EPR), optical absorption, circular dichroism, and magnetic circular dichroism (MCD) spectroscopies reveal that, relative to the trigonal planar blue type 1 Cu site in wild-type fungal laccase, the covalency and the ligand field strength at the Leu513His green type 1 Cu center decrease. Additionally, there is a significant reorientation of the d(x)()()2(-)(y)()()2( )singly occupied MO, such that the overlap with the Cys sulfur valence orbital changes from pi to sigma. A density functional study in which internal coordinates are systematically altered reveals that these changes are due to the increased strength of the axial ligand (none to His), leading to a tetragonal distortion and elongation of the equatorial Cu-ligand bonds. These calculations provide insight into the experimental differences in the EPR parameters, charge-transfer absorption spectrum, and ligand-field MCD spectrum between the axial-His variant and blue Cu centers (plastocyanin and the type 1 site in fungal laccase). There are also significant differences between the green site in the Leu513His variant and other naturally occurring, green type 1 Cu sites such as in nitrite reductase, which have short axial Cu-S(Met) bonds. The large difference in EPR parameters between these green type 1 sites derives from a change in ligand field excitation energies observed by MCD, which reflects a decrease in ligand field strength. This is associated with different steric interactions of a His vs an axial Met ligand in a tetragonally distorted type 1 site. Changes in the electronic structure of the Cu site correlate with the difference in reactivity of the green His variant relative to blue wild-type fungal laccase.
Palmer
Amy E
AE
Department of Chemistry, Stanford University, Stanford, California 94305 and Novozymes Biotech, Davis, California 95616, USA.
Szilagyi
Robert K
RK
Cherry
Joel R
JR
Jones
Aubrey
A
Xu
Feng
F
Solomon
Edward I
EI
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003080:Cold Temperature; D003116:Color; D003300:Copper / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D005658:Fungi / Q000201:enzymology; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry* / Q000235:genetics; D007930:Leucine / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation / Q000502:physiology; D013379:Substrate Specificity
D008024:Ligands; D006639:Histidine; D003300:Copper; D004247:DNA; D042845:Laccase; D007930:Leucine