Exploring Local Vibrational Structure in Protein-Bound Chlorophyll <i>a</i> : Isotope-Enrichment Experiments and Electrostatic Analysis

Local chlorophyll (Chl) vibrations play an essential role in biological photosynthesis by facilitating rapid energy transfer between pigments. In research studies, they also provide a useful spectroscopic probe of the local protein environment that surrounds each pigment. However, measuring the complete vibrational absorption spectrum of a protein-bound Chl molecule is much more difficult than, for example, Chl in neat solvent due to overlap with protein vibrations that typically drown out Chl vibrational features. Resonance Raman and fluorescence spectroscopies provide a way around this problem for Franck-Condon active vibrations, but these often rely on cryogenic measurement conditions and fail to capture vibrational signatures from, e.g., ester-group vibrations that lack coupling to a convenient electronic transition. In the present contribution, we use ¹³C-enrichment of the protein backbone to shift protein background signals to lower frequency, providing a largely clean spectral window in which to study local Chl <i>a</i> C═O stretch modes. A room-temperature absorption spectrum for Chl <i>a</i> in the water-soluble chlorophyll protein (WSCP) of <i>Lepidium virginicum</i> is thus extracted as a difference between protein-plus-pigment and protein-only vibrational spectra. Excellent agreement in the molecular fingerprint region with the vibrational spectrum of Chl <i>a</i> in organic solvents confirms that the resulting spectrum represents the response of the protein-bound Chl <i>a</i> molecule. Furthermore, the ester group resonance is observed to shift in response to the S53P mutation that eliminates a 17³ ester group hydrogen bond from the protein environment. Finally, we analyze these experimental results using MD-based electrostatic analysis, finding that electric-field mapping at the C atom of the ester group provides a satisfactory explanation for the observed frequency shifts between organic solvent and protein environment. MD analysis further suggests that a red-shifted ester peak observed experimentally for the S53P mutant results from solvation of the 17³ Chl ester group due increase water penetration into the Chl-binding pocket relative to wild-type WSCP. We anticipate that these results will prove useful both for benchmarking future simulation work and as a reference for interpreting Chl vibrational spectra as a probe of pigment-protein interactions.