X-ray Absorption Spectroscopic Studies of the Blue Copper Site: Metal and Ligand K-Edge Studies To Probe the Origin of the EPR Hyperfine Splitting in Plastocyanin

X-ray absorption spectra for the oxidized blue copper protein plastocyanin and several Cu(II) model complexes have been measured at both the Cu K-edge and the ligand K-edges (Cl and S) in order to elucidate the source of the small parallel hyperfine splitting in the EPR spectra of blue copper centers. Assignment and analysis of a feature in the Cu K-edge X-ray absorption spectrum at ∼8987 eV as the Cu 1s → 4p + ligand-to-metal charge-transfer shakedown transition has allowed for quantitation of 4p mixing into the ground-state wave function as reflected in the 1s → 3d (+4p) intensity at ∼8979 eV. The results show that distorted tetrahedral (D_2d) CuCl₄ ²⁻ is characterized by <4% Cu 4p_z mixing, while plastocyanin has only Cu 4p_xy mixing. Thus, the small parallel hyperfine splitting in the EPR spectra of D_2d CuCl₄ ²⁻ and of oxidized plastocyanin cannot be explained by 12% 4p_z mixing into the (Formula Omitted) orbital as had been previously postulated. Data collected at the Cl K-edge for CuCl₄ ²⁻ show that the intensity of the ligand pre-edge feature at ∼2820 eV reflects the degree of covalency between the metal half-occupied orbital and the ligands. The data show that D_2d CuCl₄ ²⁻ is not unusually covalent. The source of the small parallel splitting in the EPR of D_2d CuCl₄ ²⁻ is discussed. Experiments at the S K-edge (∼2470 eV) show that plastocyanin is characterized by a highly covalent Cu-S(cysteine) bond relative to the cupric-thiolate model complex [Cu(tet b)(o-SC₆H₄CO₂)]·H₂O. Self-consistent-field-Xa-scattered-wave calculations have been used to understand copper-thiolate bonding in this model complex and to quantify the covalency reflected in the S K-edge intensity data. The XAS results demonstrate that the small parallel hyperfine splitting in the EPR spectra of blue copper sites reflects the high degree of covalency of the copper-thiolate bond.