Ground- and Excited-State Mapping of Plastocyanin from Resonance Raman Spectra of Isotope-Labeled Proteins

Abstract

Resonance Raman (RR) spectra are reported for recombinant poplar plastocyanin, which was isotopically labeled with cysteine-CβD2 and -15N. Abundantly expressed protein and careful low-temperature scanning permitted resolution of an unprecedented number of bands in the 400 cm-1 region and determination of their isotope shifts. These shifts confirm extensive mixing of ligand internal coordinates with the Cu−S(Cys) stretching coordinate and also reveal mixing into the mainly Cu−N(His) stretching mode at 267 cm-1. RR scattering is observed for combinations of this mode with all of the ∼400 cm-1 modes as well as of the ∼400 cm-1 modes with one another. In addition, the isotopic labeling establishes the assignment of higher-frequency RR-enhanced fundamental modes, S−C stretching, methylene twisting and scissoring, and the amide III mode, all of which are localized on cysteine, as well as three imidazole ring modes of histidine. The 1040 cm-1 imidazole peak is definitively assigned to the ligating His87 because it shifts cleanly to 1054 cm-1 upon brief exposure to D2O, which is known to permit selective NH/D exchange of the solvent-exposed imidazole ring of His87. Selective enhancement of His87 modes is ascribed to the orientation of the imidazole ring, which allows mixing of imidazole and sulfur π orbitals in the resonant charge-transfer excited states. Support for this mixing is provided by the RR excitation profiles, which reveal a 20-nm red-shift of the scattering maximum for modes localized on histidine, relative to those localized on cysteine. Both coordinate mixing and RR enhancement of internal ligand modes are ascribed to coplanarity of the atoms in the electron-transfer pathways to the remote (Tyr83) site and their alignment with the imidazole ring of the adjacent (His87) site for electron transfer, a geometry that maximizes the electronic coupling along both pathways.