Analysis of resonance Raman data on the blue copper site in pseudoazurin: excited state π and σ charge transfer distortions and their relation to ground state reorganization energy.

The short Cu2+–S(Met) bond in pseudoazurin (PAz) results in the presence of two relatively intense Sp(π) and Sp(σ) charge transfer (CT) transitions. This has enabled resonance Raman (rR) data to be obtained for each excited state. The rR data show very different intensity distribution patterns for the vibrations in the 300–500cm−1 region. Time-dependent density functional theory (TDDFT) calculations have been used to determine that the change in intensity distribution between the Sp(π) and Sp(σ) excited states reflects the differential enhancement of S(Cys) backbone modes with Cu–S(Cys)–Cβ out-of-plane (oop) and in-plane (ip) bend character in their respective potential energy distributions (PEDs). The rR excited state distortions have been related to ground state reorganization energies (λs) and predict that, in addition to M–L stretches, the Cu–S(Cys)–Cβ oop bend needs to be considered. DFT calculations predict a large distortion in the Cu–S(Cys)–Cβ oop bending coordinate upon reduction of a blue copper (BC) site; however, this distortion is not present in the X-ray crystal structures of reduced BC sites. The lack of Cu–S(Cys)–Cβ oop distortion upon reduction corresponds to a previously unconsidered constraint on the thiolate ligand orientation in the reduced state of BC proteins and can be considered as a contribution to the entatic/rack nature of BC sites.