Electron Nuclear Double Resonance of the Chlorophyll Triplet State in the Water-Soluble Chlorophyll Protein from Brassica oleracea: Investigation of the Effect of the Binding Site on the Hyperfine Couplings

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.

Primary donor triplet states of Photosystem I and II studied by Q-band pulse ENDOR spectroscopy

“…From a comparison of the number of contacts between the different Chl binding sites, it appears that those of the two WSCPs are characterized by a low number of contacts in the vicinity of all three methyl groups, in good agreement with the fact that the ENDOR lines of the three freely rotating methyl groups can easily be observed in these two systems (Agostini et al 2017(Agostini et al , 2019a(Agostini et al , 2020.…”

“…We attribute the higher resolution achieved here to a combination of longer RF and MW pulses (200 ns inversion π-pulse) and higher magnetic field (1.2 T vs 0.35 T), where off-diagonal elements of the hyperfine tensors A can be neglected. In the case of 3 Chl a in WSCP, Mims ENDOR spectra were also recorded (Agostini et al 2017(Agostini et al , 2020, which are characterized by an intrinsically higher resolution of small hfcs but suffer from blind spots (Gemperle and Schweiger 1991). The corresponding hyperfine structure in the proximity of the free proton line resembles the one for 3 P680 at Q-band.…”

“…The determination of the hfcs allows the most precise quantification of the extent of triplet delocalization and provides a means to assign the triplet to a specific chlorophyll. The technique has been extensively used to study the electron spin density distribution of the triplet state for porphyrin derivatives and (bacterio) chlorophylls and also for photosynthetic primary donors (Kay et al 1995;Di Valentin et al 1996;Lendzian et al 1998Lendzian et al , 2003Marchanka et al 2009;Tait et al 2015;Richert et al 2017;Agostini et al 2017Agostini et al , 2019aAgostini et al , 2020Barbon et al 2020). The monomeric or dimeric nature of the special pair, and the influence of the protein surroundings on the excitation sharing, could be assessed by mapping the distribution of the unpaired electrons in the molecular system 3 P865 and 3 P960 in the bacterial RCs of Rhodobacter sphaeroides and Blastochloris viridis, respectively.…”

“…From the comparison of the well resolved 1 H-ENDOR of 3 Chl a in various Chl-binding proteins (PSI, PSII, and WSCP, see Table 2), it clearly appears that while the signals for the three methine α protons (5, 10, 20) are usually intense and observed in triplet ENDOR (Di Valentin et al 1996;Lendzian et al 2003;Agostini et al 2017Agostini et al , 2019aAgostini et al , 2020, the signals of the β protons of the three methyl groups (2, 7, 12) have an intensity that seems to significantly vary in the investigated protein complexes, leading to the difficulty to detect methyl group 2 in 3 P680 (D1D2Cytb 559 ) (Fig. 3) and the massive weakening and broadening of methyl group 12 in 3 P700 (Fig.…”

“…Photosynthetic pigments, i.e., photosynthetic primary donors, antenna Chls and carotenoids have been extensively investigated in their triplet states by transient and pulse ENDOR spectroscopy in order to derive the hfcs of magnetic nuclei like 1 H and thus the spin density distribution in the triplet state (Di Valentin et al 1996;Lendzian et al 1998Lendzian et al , 2003Niklas et al 2007;Salvadori et al 2012;Carbonera et al 2014b;Marchanka et al 2014). ENDOR spectra have also been reported for many porphyrins, e.g., Kay et al (1995), Tait et al (2015), Richert et al (2017), Barbon et al (2020) and (bacterio)chlorophyll model systems dissolved in organic solvents or inserted in the protein environment of the Water-Soluble Chlorophyll Protein (WSCP) (Marchanka et al 2009;Agostini et al 2017Agostini et al , 2019aAgostini et al , 2020. In the specific case of 3 P680, the "primary donor" of Photosystem II (PSII), X-band 1 H-ENDOR spectra have been reported (Di Valentin et al 1996;Lendzian et al 2003) while no ENDOR data are available so far for 3 P700, the primary donor of Photosystem I (PSI).…”