Electron Spin Echo Envelope Modulation Spectroscopy of the Photoprotective Site in the Peridinin-Chlorophyll Protein

The photoprotective site in the Peridinin-Chlorophyll Protein of the dinoflagellate Amphidinium carterae has been studied with the pulse EPR techniques ESEEM (Electron Spin Echo Envelope Modulation) and HYSCORE (HYperfine Sublevel CORrElation spectroscopy). In previous EPR studies of the PCP antenna complex, a specific peridinin molecule (Per614) has been identified as the only one among the four peridinin molecules surrounding chlorophyll (Chl601) at Van der Waals distance responsible for photoprotection through triplet-triplet energy transfer (TTET) [Di Valentin et al., Biochim. Biophys. Acta-Bioenerg. 2008, 1777, 186-195]. The properties of this peridinin molecule are studied by means of advanced EPR techniques in order to shed light on the requirements for efficient TTET. The TTET mechanism requires an overlap of the wavefunctions of the donor and acceptor molecules and thus establishes strict distance and orientation requirements. Per614 is distinguished from the other peridinins of the pigment cluster by a shorter centre-to-centre distance and by the presence of a unique water molecule (H2O 701) at the interface between Per614 and Chl601, which has been proposed to favour TTET by extending the overlap of the donor and acceptor wavefunctions. The hyperfine interaction between the protons of the water molecule H2O 701 and the peridinin triplet state has been studied in two-pulse and three-pulse ESEEM experiments on untreated and D2O-exchanged PCP samples at cryogenic temperatures. In this work deuterium ESEEM and HYSCORE have been applied to a photo-excited triplet state in a protein complex for the first time. Since the analytical expressions describing the ESEEM signal for a triplet state coupled to I=1 nuclei are not present in literature, they have been derived following a density matrix treatment described in the literature [Mims, Phys. Rev. B 1972, 5, 2409-2419.]. The ESEEM experiments have been combined with state-of-the-art computational methods in order to determine the exact geometry of the photprotective site, in particular the orientation of the water molecule, not defined in the X-ray structure. The modulations observed in the ESEEM experiment have been assigned to the exchangeable protons of H2O 701 and the hyperfine interaction parameters have been extracted from the experimental data through simulations with starting values obtained from DFT calculations at the B3LYP/EPRII level. The constraints imposed on the simulation by the two types of ESEEM experiments and by the orientation selectivity allow an accurate determination of the hyperfine parameters. The spectroscopic parameters indicate an involvement of the water molecule in the TTET from Per614 to Chl601. The geometry and electronic structure of the photoprotective site in the PCP antenna complex will be used in future calculations of the TTET exchange integral by employing wavefunctions validated by experimental data and comprehending the contribution of the bridging water molecule.