A new, unquenched intermediate of LHCII
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100322
ISSN1083-351X
AutoresFei Li, Cheng Liu, Simona Streckaitė, Chunhong Yang, Pengqi Xu, Manuel J. Llansola‐Portoles, Cristian Ilioaia, Andrew A. Pascal, Roberta Croce, Bruno Robert,
Tópico(s)Advanced NMR Techniques and Applications
ResumoWhen plants are exposed to high-light conditions, the potentially harmful excess energy is dissipated as heat, a process called non-photochemical quenching. Efficient energy dissipation can also be induced in the major light-harvesting complex of photosystem II (LHCII) in vitro, by altering the structure and interactions of several bound cofactors. In both cases, the extent of quenching has been correlated with conformational changes (twisting) affecting two bound carotenoids, neoxanthin, and one of the two luteins (in site L1). This lutein is directly involved in the quenching process, whereas neoxanthin senses the overall change in state without playing a direct role in energy dissipation. Here we describe the isolation of an intermediate state of LHCII, using the detergent n-dodecyl-α-D-maltoside, which exhibits the twisting of neoxanthin (along with changes in chlorophyll–protein interactions), in the absence of the L1 change or corresponding quenching. We demonstrate that neoxanthin is actually a reporter of the LHCII environment—probably reflecting a large-scale conformational change in the protein—whereas the appearance of excitation energy quenching is concomitant with the configuration change of the L1 carotenoid only, reflecting changes on a smaller scale. This unquenched LHCII intermediate, described here for the first time, provides for a deeper understanding of the molecular mechanism of quenching. When plants are exposed to high-light conditions, the potentially harmful excess energy is dissipated as heat, a process called non-photochemical quenching. Efficient energy dissipation can also be induced in the major light-harvesting complex of photosystem II (LHCII) in vitro, by altering the structure and interactions of several bound cofactors. In both cases, the extent of quenching has been correlated with conformational changes (twisting) affecting two bound carotenoids, neoxanthin, and one of the two luteins (in site L1). This lutein is directly involved in the quenching process, whereas neoxanthin senses the overall change in state without playing a direct role in energy dissipation. Here we describe the isolation of an intermediate state of LHCII, using the detergent n-dodecyl-α-D-maltoside, which exhibits the twisting of neoxanthin (along with changes in chlorophyll–protein interactions), in the absence of the L1 change or corresponding quenching. We demonstrate that neoxanthin is actually a reporter of the LHCII environment—probably reflecting a large-scale conformational change in the protein—whereas the appearance of excitation energy quenching is concomitant with the configuration change of the L1 carotenoid only, reflecting changes on a smaller scale. This unquenched LHCII intermediate, described here for the first time, provides for a deeper understanding of the molecular mechanism of quenching. During the first steps of the photosynthetic process, solar photons are absorbed by specialized light-harvesting complexes (LHCs), and the resulting excitation energy is transferred to reaction center pigments, where it is converted into a chemical potential. In low-light conditions, most of the photons absorbed lead to a charge separation event at the reaction center (1Scholes G.D. Fleming G.R. Olaya-Castro A. van Grondelle R. Lessons from nature about solar light harvesting.Nat. Chem. 2011; 3: 763-774Crossref PubMed Scopus (1182) Google Scholar, 2Croce R. van Amerongen H. Light harvesting in oxygenic photosynthesis: Structural biology meets spectroscopy.Science. 2020; 369eaay2058Crossref PubMed Scopus (23) Google Scholar). However, when the absorbed energy is in excess of that which can be used for energy transduction, the overaccumulation of excited states can result in damage to the photosynthetic membrane, in particular, via the production of reactive oxygen species. 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Changes in the interaction state of several chlorophylls with the protein host were observed, as well as in the configuration of the neoxanthin molecule (27Ruban A.V. Horton P. Robert B. Resonance Raman spectroscopy of the photosystem II light-harvesting complex of green plants: A comparison of trimeric and aggregated states.Biochemistry. 1995; 34: 2333-2337Crossref PubMed Scopus (53) Google Scholar), and more recently, in that of L1 lutein (14Ilioaia C. Johnson M.P. Liao P.-N. Pascal A.A. van Grondelle R. Walla P.J. Ruban A.V. Robert B. Photoprotection in plants involves a change in lutein 1 binding domain in the major light-harvesting complex of photosystem II.J. Biol. Chem. 2011; 286: 27247-27254Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), consistent with the role of L1 as the quenching species. The amplitude of the neoxanthin change is strictly correlated with the extent of quenching in LHCII as well as in intact chloroplasts and leaves (6Ruban A.V. Berera R. Ilioaia C. van Stokkum I.H.M. Kennis J.T.M. Pascal A.A. van Amerongen H. Robert B. Horton P. van Grondelle R. Identification of a mechanism of photoprotective energy dissipation in higher plants.Nature. 2007; 450: 575-578Crossref PubMed Scopus (662) Google Scholar), and this carotenoid thus appears as a reporter of structural changes leading to quenching, a proposition that has recently been supported by molecular dynamics simulations (28Liguori N. Periole X. Marrink S.J. Croce R. From light-harvesting to photoprotection: Structural basis of the dynamic switch of the major antenna complex of plants (LHCII).Sci. Rep. 2015; 5: 15661Crossref PubMed Scopus (72) Google Scholar). LHC proteins were proposed to be the major site of quenching in plants, which would occur through a subtle equilibrium between two LHC conformations (29Chmeliov J. Gelzinis A. Songaila E. Augulis R. Duffy C.D.P. Ruban A.V. Valkunas L. 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Disentangling protein and lipid interactions that control a molecular switch in photosynthetic light harvesting.Biochim. Biophys. Acta Biomembr. 2017; 1859: 40-47Crossref PubMed Scopus (21) Google Scholar). LHCII purified in the presence of either α- or β-dodecyl-D-maltoside (α-DM or β-DM) displays slightly different electronic properties (31Akhtar P. Dorogi M. Pawlak K. Kovács L. Bóta A. Kiss T. Garab G. Lambrev P.H. Pigment interactions in light-harvesting complex II in different molecular environments.J. Biol. Chem. 2015; 290: 4877-4886Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 33Georgakopoulou S. van der Zwan G. Bassi R. van Grondelle R. van Amerongen H. Croce R. Understanding the changes in the circular dichroism of light harvesting complex II upon varying its pigment composition and organization.Biochemistry. 2007; 46: 4745-4754Crossref PubMed Scopus (78) Google Scholar), and the H-bonding interactions of two bound chlorophyll a molecules are sensitive to the detergent used (34Llansola-Portoles M.J. Li F. Xu P. Streckaite S. Ilioaia C. Yang C. Gall A. Pascal A.A. Croce R. Robert B. Tuning antenna function through hydrogen bonds to chlorophyll a.Biochim. Biophys. Acta Bioenerg. 2019; 1861: 148078Crossref PubMed Scopus (12) Google Scholar). In this work, we have studied the vibrational properties of the different pigments bound to LHCII in the presence of α-DM or β-DM. We show that the Raman signals of Chls a and b and neoxanthin that were previously associated with LHCII quenching are already present in LHCII purified in the presence of α-DM—even though this preparation is unquenched—while aggregation-induced quenching affects the L1 carotenoid-binding site alone. The molecular structure of Chl a, Chl b, lutein and neoxanthin pigments and those of the detergents used for the purification of LHCII are dispayed in Figure 1. As already reported (31Akhtar P. Dorogi M. Pawlak K. Kovács L. Bóta A. Kiss T. Garab G. Lambrev P.H. Pigment interactions in light-harvesting complex II in different molecular environments.J. Biol. Chem. 2015; 290: 4877-4886Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 33Georgakopoulou S. van der Zwan G. Bassi R. van Grondelle R. van Amerongen H. Croce R. Understanding the changes in the circular dichroism of light harvesting complex II upon varying its pigment composition and organization.Biochemistry. 2007; 46: 4745-4754Crossref PubMed Scopus (78) Google Scholar, 34Llansola-Portoles M.J. Li F. Xu P. Streckaite S. Ilioaia C. Yang C. Gall A. Pascal A.A. Croce R. Robert B. Tuning antenna function through hydrogen bonds to chlorophyll a.Biochim. Biophys. Acta Bioenerg. 2019; 1861: 148078Crossref PubMed Scopus (12) Google Scholar), both the carotenoid and chlorophyll absorption regions of LHCII exhibit differences between the α-DM-solubilized versus the β-DM-solubilized protein (Fig. 2A) . In the blue region, the Soret absorption transition of Chl is perturbed—in α-DM, an increase in intensity is seen at 432 nm, concomitant with a loss at 438 nm. The carotenoid contributions appear to lose intensity at 480 and 472 nm and gain intensity at 457 nm. In the Chl Qy region, a new band is present at 660 nm in LHCII isolated in α-DM, while the intensity of the transitions at 676 and 672 nm is reduced. All these changes are more easily observed in the difference spectrum (Fig. 2B). None of these changes in absorption induces any observable differences in fluorescence properties (e.g., Fig. 2C). Resonance Raman spectroscopy has seen extensive application to the assessment of pigment structure and interactions in photosynthetic proteins (35Robert B. Resonance Raman spectroscopy.Photosynth. Res. 2009; 101: 147-155Crossref PubMed Scopus (100) Google Scholar, 36Llansola-Portoles M.J. Pascal A.A. Robert B. Electronic and vibrational properties of carotenoids: From in vitro to in vivo.J. R. Soc. Interface. 2017; 1420170504Crossref PubMed Scopus (39) Google Scholar, 37Gall A. Pascal A.A. Robert B. Vibrational techniques applied to photosynthesis: Resonance Raman and fluorescence line-narrowing.Biochim. Biophys. Acta Bioenerg. 2015; 1847: 12-18Crossref PubMed Scopus (25) Google Scholar) and played a vital role in revealing the modifications to cofactor structure associated with the LHCII "conformational change" model of NPQ (12Pascal A.A. Liu Z. Broess K. van Oort B. van Amerongen H. Wang C. Horton P. Robert B. Chang W. Ruban A. Molecular basis of photoprotection and control of photosynthetic light-harvesting.Nature. 2005; 436: 134-137Crossref PubMed Scopus (476) Google Scholar, 27Ruban A.V. Horton P. Robert B. Resonance Raman spectroscopy of the photosystem II light-harvesting complex of green plants: A comparison of trimeric and aggregated states.Biochemistry. 1995; 34: 2333-2337Crossref PubMed Scopus (53) Google Scholar). The Raman spectra of chlorophyll molecules are particularly rich, containing a number of bands that are sensitive to the chlorophyll conformation and to its interactions with the immediate environment. Upon aggregation-induced quenching in LHCII, two bound Chl a molecules lose a hydrogen bond to their conjugated keto carbonyl group on position C131, while one or two Chls b gain H-bonds to their conjugated formyl group at position C7 (observed for excitations at 413.1 and 441.6 nm, respectively [27Ruban A.V. Horton P. Robert B. Resonance Raman spectroscopy of the photosystem II light-harvesting complex of green plants: A comparison of trimeric and aggregated states.Biochemistry. 1995; 34: 2333-2337Crossref PubMed Scopus (53) Google Scholar]). Chlorophyll resonance Raman spectra of the two LHCII preparations at 77 K are presented in Figure 3. Comparing α-DM-LHCII relative to β-DM-LHCII for the Chl a excitation at 413.1 nm (Fig. 3A), there is a clear increase in contributions on the high-frequency side of the envelope of bands in the 1660 to 1700 cm−1 region, which corresponds to stretching modes of Chl a keto groups conjugated with the macrocycle. This increase around 1690 cm−1 is accompanied by a corresponding decrease at lower frequency at ∼1670 cm−1 (shown by black arrow heads in Fig. 3A). As discussed elsewhere (34Llansola-Portoles M.J. Li F. Xu P. Streckaite S. Ilioaia C. Yang C. Gall A. Pascal A.A. Croce R. Robert B. Tuning antenna function through hydrogen bonds to chlorophyll a.Biochim. Biophys. Acta Bioenerg. 2019; 1861: 148078Crossref PubMed Scopus (12) Google Scholar), this reflects the loss of an H-bond to probably two LHCII-bound Chl a molecules, at the level of their conjugated keto carbonyl group. Excitation at 441.6 nm yields spectra in which Chl b vibrational modes dominate. The high-frequency region thus corresponds to stretching modes of both conjugated carbonyl groups of Chl b – C7-formyl and C131-keto, in the 1620 to 1660 and 1660 to 1700 cm−1 ranges, respectively. When the two preparations are compared at this wavelength (Fig. 3B), an intense contribution is observed for α-DM-LHCII at 1637 cm−1 that is not present in β-DM-LHCII, accompanied by a decrease in the intensity of the contribution at 1666 cm−1 (shown by black arrow heads in Fig. 3B). This indicates that the formyl carbonyl of at least one Chl b, which is free from interactions in β-DM-LHCII, becomes strongly H-bonded in α-DM-LHCII. It is particularly interesting to note that the changes in Chl a and b interactions observed for α-DM-LHCII are strikingly similar to those seen upon aggregation-induced quenching in LHCII (27Ruban A.V. Horton P. Robert B. Resonance Raman spectroscopy of the photosystem II light-harvesting complex of green plants: A comparison of trimeric and aggregated states.Biochemistry. 1995; 34: 2333-2337Crossref PubMed Scopus (53) Google Scholar) (see spectra of aggregates in Fig. 3). 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Chem. 2017; 292: 1396-1403Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and sensitive to the presence of a conjugated allene group (47Llansola-Portoles M.J. Litvin R. Ilioaia C. Pascal A.A. Bina D. Robert B. Pigment structure in the violaxanthin–chlorophyll-a-binding protein VCP.Photosynth. Res. 2017; 134: 51-58Crossref PubMed Scopus (12) Google Scholar, 48Llansola-Portoles M.J. Uragami C. Pascal A.A. Bina D. Litvin R. Robert B. Pigment structure in the FCP-like light-harvesting complex from Chromera velia.Biochim. Biophys. Acta Bioenerg. 2016; 1857: 1759-1765Crossref Scopus (12) Google Scholar). Finally, the ν4 band, around 960 cm−1, arises from C–H out-of-plane wagging motions coupled with C=C torsional modes (38Saito S. Tasumi M. Normal-coordinate analysis of β-carotene isomers and assignments
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