Spectroscopic investigation of stellacyanin mutants: axial ligand interactions at the blue copper site.
Detailed electronic and geometric structural descriptions of the blue copper sites in wild-type (WT) stellacyanin and its Q99M and Q99L axial mutants have been obtained using a combination of XAS, resonance Raman, MCD, EPR, and DFT calculations. The results show that the origin of the short Cu-S(Cys) bond in blue copper proteins is the weakened axial interaction, which leads to a shorter (based on EXAFS results) and more covalent (based on S K-edge XAS) Cu-S bond. XAS pre-edge energies show that the effective nuclear charge on the copper increases going from O(Gln) to S(Met) to no axial (Leu) ligand, indicating that the weakened axial ligand is not fully compensated for by the increased donation from the thiolate. This is further supported by EPR results. MCD data show that the decreased axial interaction leads to an increase in the equatorial ligand field, indicating that the site acquires a more trigonally distorted tetrahedral structure. These geometric and electronic structural changes, which result from weakening the bonding interaction of the axial ligand, allow the site to maintain efficient electron transfer (high H(DA) and low reorganization energy), while modulating the redox potential of the site to the biologically relevant range. These spectroscopic studies are complemented by DFT calculations to obtain insight into the factors that allow stellacyanin to maintain a trigonally distorted tetrahedral structure with a relatively strong axial Cu(II)-oxygen bond.
DeBeer George
Serena
S
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Basumallick
Lipika
L
Szilagyi
Robert K
RK
Randall
David W
DW
Hill
Michael G
MG
Nersissian
Aram M
AM
Valentine
Joan S
JS
Hedman
Britt
B
Hodgson
Keith O
KO
Solomon
Edward I
EI
D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D018553:Cucumis sativus / Q000737:chemistry / Q000235:genetics; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D010100:Oxygen / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013057:Spectrum Analysis
D008024:Ligands; D008667:Metalloproteins; D010940:Plant Proteins; C003343:stellacyanin protein, plant; D003300:Copper; D010100:Oxygen