Abstract
An investigation of the photoexcited triplet state of chlorophyll (Chl) a in the water-soluble chlorophyll protein (WSCP) of Brassica oleracea has been carried out by means of electron-nuclear double resonance (ENDOR), achieving a complete assignment of the observed hyperfine couplings corresponding to methine protons and methyl groups of Chl a triplet state. The triplet-state properties, and in particular the hyperfine couplings, were found to be similar to those previously reported for Chl a in the WSCP of Lepidium virginicum. Therefore, the porphyrin ring deformation observed in Brassica oleracea WSCP seems to only slightly affect the spin density of 3Chl a. This may be relevant when considering the robustness of triplet–triplet energy transfer mechanisms, relying on wavefunction overlap, in systems, such as the photosynthetic light-harvesting complexes, in which Chl triplet states with distorted geometries are involved.
Highlights
The water-soluble chlorophyll protein (WSCP) [1] has emerged in recent years as a promising model system for the investigation of tetrapyrroles in a protein environment [2], by virtue of its structural simplicity [3,4,5], high stability [6,7,8], and capability to be reconstituted in vitro with different tetrapyrroles [9,10,11]
We recently showed that WSCP is suitable for obtaining intense and narrow electron-nuclear double resonance (ENDOR) lines of 3Chl a [11, 32], compared to those obtained in frozen solvents or in other Chl-binding proteins [34], probably by virtue of a high rigidity of the Chl-binding sites in WSCP [11]
|D| +|E| transitions (721 and 947 MHz, respectively; note that, as previously reported for 3Chls, the 2|E| transition was too weak to be detected) allowed a precise calculation of the values of the zero-field splitting (ZFS) parameters |D| and |E| (|D|= 0.028 cm−1 and |E|= 0.0038 cm−1), which are close to those previously reported for Lepidium virginicum (Lv) WSCP (|D|= 0.029 cm−1 and |E|= 0.0037 cm−1 [20])
Summary
The water-soluble chlorophyll protein (WSCP) [1] has emerged in recent years as a promising model system for the investigation of tetrapyrroles in a protein environment [2], by virtue of its structural simplicity [3,4,5], high stability [6,7,8], and capability to be reconstituted in vitro with different tetrapyrroles [9,10,11]. To verify if the 3Chl a in Bo WSCP is affected by the tuning properties of the protein-binding sites, a mapping of the spin density over the molecular structure, with a particular focus onto the portions of the molecules more affected by the Chl deformations, has been carried out in this study To this purpose, pulse electron-nuclear double resonance (ENDOR), combined with laser excitation, is the best-suited technique [31]. The first full experimental characterization of Bo WSCP 3Chl a by means of Davies and Mims ENDOR, time-resolved EPR (TR-EPR), and Absorption Detected Magnetic Resonance (ADMR) was carried out This full characterization is aimed to provide a complete picture of the triplet state, in terms of ZFS parameters, spin polarization properties, and proton hfcs, including small (< 5 MHz) contributions from the Mims spectra. A direct comparison between all the magnetic observables of 3Chl a in two species, i.e., Brassica oleracea and Lepidium virginicum, having different ring geometries and local protein environment, has been possible
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