Low-temperature optical absorption and MCD and CD spectra in conjunction with self-consistent field-Xα-scattered wave (SCF-Xα-SW) calculations are presented for the blue copper active site in plastocyanin. These spectral studies allow quantitative determination of the number, intensities, and MCD parameters of the blue copper absorption bands. Transitions strong in absorption are found to be generally weak in low-temperature MCD (LTMCD), while the opposite is true for weak absorption bands. Xα-SW calculations performed with sphere radii adjusted so that the ground state reproduces the experimental g values clearly separate ligand field and charge-transfer band energies. The results of these calculations are used to further calculate oscillator strengths and MCD parameters. This protocol is tested through application to both D2d and D4h symmetry tetrachlorocuprates where comparison with definitive experimental assignments is possible. The four lowest energy bands in the blue copper spectrum which are weak in absorption but strong in LTMCD are found to be ligand field transitions with the ordering dx2-y2 ← dz2 < ← dxy < ← dxy+yz < ← dxz-yz in the Cs-perturbed, elongated C3υ, blue copper site. The intense blue band at 600 nm is found to be a cysteine S pπ → dx2-y2 transition which gets its intensity from the extremely good overlap between the ground- and excited-state wave functions. The blue band has the correct symmetry to allow for configuration interaction with the dxz+yz orbital and thus gives intensity to this lower energy 780-nm transition. A transition from the pseudo-σ orbital of the thiolate is predicted to have little intensity due to poor overlap with the ground-state dx2-y2 orbital and is responsible for the weak band on the high-energy side of the blue band (at 535 nm). Finally histidine π1 and thioether a1 transitions are assigned to very weak bands in the region of 425-465 nm. This assignment provides further insight into the bonding at the plastocyanin site. The large splitting between the dx2-y2 and dxy orbitals reflects the presence of a quite strong cysteine Spπ interaction with the copper which is responsible for the orientation of the dx2-y2 ground-state orbital. The low energy of the dz2 orbital indicates a reasonable interaction between the copper and the thioether S at a 2.9 Å bond length. Finally, it is determined that the thiolate-copper(II) bond makes a dominant contribution to the electronic structure of the blue copper active site which can be strongly influenced by the orientation of this residue by the protein backbone. Thus, changes in spectral features correlated with changes in the thiolate bond in different blue copper proteins are described.
ASJC Scopus subject areas
- Colloid and Surface Chemistry