Structure of a dimeric photosystem II complex from a cyanobacterium acclimated to far-red light
2022; Elsevier BV; Volume: 299; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2022.102815
ISSN1083-351X
AutoresChristopher J. Gisriel, Gaozhong Shen, David A. Flesher, Vasily Kurashov, John H. Golbeck, Gary W. Brudvig, Muhamed Amin, Donald A. Bryant,
Tópico(s)Light effects on plants
ResumoPhotosystem II (PSII) is the water-splitting enzyme central to oxygenic photosynthesis. To drive water oxidation, light is harvested by accessory pigments, mostly chlorophyll (Chl) a molecules, which absorb visible light (400–700 nm). Some cyanobacteria facultatively acclimate to shaded environments by altering their photosynthetic machinery to additionally absorb far-red light (FRL, 700–800 nm), a process termed far-red light photoacclimation or FaRLiP. During far-red light photoacclimation, FRL-PSII is assembled with FRL-specific isoforms of the subunits PsbA, PsbB, PsbC, PsbD, and PsbH, and some Chl-binding sites contain Chls d or f instead of the usual Chl a. The structure of an apo-FRL-PSII monomer lacking the FRL-specific PsbH subunit has previously been determined, but visualization of the dimeric complex has remained elusive. Here, we report the cryo-EM structure of a dimeric FRL–PSII complex. The site assignments for Chls d and f are consistent with those assigned in the previous apo-FRL-PSII monomeric structure. All sites that bind Chl d or Chl f at high occupancy exhibit a FRL-specific interaction of the formyl moiety of the Chl d or Chl f with the protein environment, which in some cases involves a phenylalanine sidechain. The structure retains the FRL-specific PsbH2 subunit, which appears to alter the energetic landscape of FRL-PSII, redirecting energy transfer from the phycobiliprotein complex to a Chl f molecule bound by PsbB2 that acts as a bridge for energy transfer to the electron transfer chain. Collectively, these observations extend our previous understanding of the structure-function relationship that allows PSII to function using lower energy FRL. Photosystem II (PSII) is the water-splitting enzyme central to oxygenic photosynthesis. To drive water oxidation, light is harvested by accessory pigments, mostly chlorophyll (Chl) a molecules, which absorb visible light (400–700 nm). Some cyanobacteria facultatively acclimate to shaded environments by altering their photosynthetic machinery to additionally absorb far-red light (FRL, 700–800 nm), a process termed far-red light photoacclimation or FaRLiP. During far-red light photoacclimation, FRL-PSII is assembled with FRL-specific isoforms of the subunits PsbA, PsbB, PsbC, PsbD, and PsbH, and some Chl-binding sites contain Chls d or f instead of the usual Chl a. The structure of an apo-FRL-PSII monomer lacking the FRL-specific PsbH subunit has previously been determined, but visualization of the dimeric complex has remained elusive. Here, we report the cryo-EM structure of a dimeric FRL–PSII complex. The site assignments for Chls d and f are consistent with those assigned in the previous apo-FRL-PSII monomeric structure. All sites that bind Chl d or Chl f at high occupancy exhibit a FRL-specific interaction of the formyl moiety of the Chl d or Chl f with the protein environment, which in some cases involves a phenylalanine sidechain. The structure retains the FRL-specific PsbH2 subunit, which appears to alter the energetic landscape of FRL-PSII, redirecting energy transfer from the phycobiliprotein complex to a Chl f molecule bound by PsbB2 that acts as a bridge for energy transfer to the electron transfer chain. Collectively, these observations extend our previous understanding of the structure-function relationship that allows PSII to function using lower energy FRL. Photosystem II (PSII) in oxygenic phototrophs is the multisubunit membrane protein complex that couples light absorption with water oxidation to generate reducing equivalents for carbon fixation (1Blankenship R.E. Molecular Mechanisms of Photosynthesis.3rd Ed. John Wiley & Sons, Ltd, Southern Gate2021Google Scholar, 2Vinyard D.J. Ananyev G.M. Charles Dismukes G. Photosystem II: the reaction center of oxygenic photosynthesis.Annu. Rev. Biochem. 2013; 82: 577-606Crossref PubMed Scopus (281) Google Scholar). A complete PSII holocomplex is typically found in a dimeric configuration, each monomer comprising about 20 subunits that bind many cofactors (3Shen J.-R. The structure of photosystem II and the mechanism of water oxidation in photosynthesis.Annu. Rev. Plant Biol. 2015; 66: 23-48Crossref PubMed Scopus (456) Google Scholar). Chlorophyll (Chl) molecules account for 35 of these cofactors, most of which serve primarily to harvest light. Upon light absorption, energy is transferred through Chl molecules until its arrival at a series of cofactors that comprise the electron transfer chain. Charge separation occurs when an electron is transferred to a plastoquinone on the acceptor side of the complex. The hole left on the donor side triggers the catalytic advancement of a Mn4CaO5-6 metallocofactor called the oxygen-evolving complex (OEC) that extracts electrons from water (4Lubitz W. Chrysina M. Cox N. Water oxidation in photosystem II.Photosynth. Res. 2019; 142: 105-125Crossref PubMed Scopus (113) Google Scholar, 5Vinyard D.J. Brudvig G.W. Progress toward a molecular mechanism of water oxidation in Photosystem II.Annu. Rev. Phys. Chem. 2017; 68: 101-116Crossref PubMed Scopus (132) Google Scholar). Many characteristics and properties of PSII are highly conserved and nearly invariant among all oxygen-evolving phototrophs, but like all bioenergetic systems, some differences have evolved among species, typically due to environmental pressures within specific ecological niches (6Sheridan K.J. Duncan E.J. Eaton-Rye J.J. Summerfield T.C. The diversity and distribution of D1 proteins in cyanobacteria.Photosynth. Res. 2020; 145: 111-128Crossref PubMed Scopus (14) Google Scholar). Whereas most oxygenic phototrophs use primarily visible light (VL, 400–700 nm) to drive PSII function, some cyanobacteria have evolved to additionally use lower energy, far-red light (FRL, 700–800 nm) for this purpose (7Viola S. Roseby W. Santabarabara S. Nürnberg D. Assunção R. Dau H. et al.Impact of energy limitations on function and resilience in long-wavelength Photosystem II.eLife. 2022; https://doi.org/10.1101/2022.04.05.486971Crossref Scopus (0) Google Scholar). The best characterized of these strategies is a facultative response mechanism to shaded environments called far-red light-photoacclimation or FaRLiP (8Gan F. Zhang S. Rockwell N.C. Martin S.S. Lagarias J.C. Bryant D.A. Extensive remodeling of a cyanobacterial photosynthetic apparatus in far-red light.Science. 2014; 345: 1312-1317Crossref PubMed Scopus (272) Google Scholar, 9Gan F. Bryant D.A. Adaptive and acclimative responses of cyanobacteria to far-red light.Environ. Microbiol. 2015; 17: 3450-3465Crossref PubMed Scopus (117) Google Scholar, 10Gan F. Shen G. Bryant D.A. Occurrence of far-red light photoacclimation (FaRLiP) in diverse cyanobacteria.Life. 2014; 5: 4-24Crossref PubMed Scopus (125) Google Scholar). Cyanobacteria capable of FaRLiP contain a unique gene cluster, most of which encodes proteins involved in Chl f biosynthesis and FRL-specific paralogs of photosystem subunits. Upon expression of the FaRLiP gene cluster, five subunits of PSII become FRL-specific: the subunits PsbA (i.e., D1), PsbB (i.e., CP47), PsbC (i.e., CP43), PsbD (i.e., D2), and also PsbH, a PSII subunit with a single transmembrane α-helix (FRL-specific isoforms of these subunits are called PsbA3, PsbB2, PsbC2, PsbD3, and PsbH2, respectively) (8Gan F. Zhang S. Rockwell N.C. Martin S.S. Lagarias J.C. Bryant D.A. Extensive remodeling of a cyanobacterial photosynthetic apparatus in far-red light.Science. 2014; 345: 1312-1317Crossref PubMed Scopus (272) Google Scholar, 9Gan F. Bryant D.A. Adaptive and acclimative responses of cyanobacteria to far-red light.Environ. Microbiol. 2015; 17: 3450-3465Crossref PubMed Scopus (117) Google Scholar, 10Gan F. Shen G. Bryant D.A. Occurrence of far-red light photoacclimation (FaRLiP) in diverse cyanobacteria.Life. 2014; 5: 4-24Crossref PubMed Scopus (125) Google Scholar, 11Ho M.-Y. Niedzwiedzki D.M. MacGregor-Chatwin C. Gerstenecker G. Hunter C.N. Blankenship R.E. et al.Extensive remodeling of the photosynthetic apparatus alters energy transfer among photosynthetic complexes when cyanobacteria acclimate to far-red light.Biochim. Biophys. Acta - Bioenerg. 2020; 1861148064Crossref Scopus (37) Google Scholar). Additionally, whereas all 35 Chls bound by PSII (per monomer) are Chl a when cells are grown in VL, the FRL-specific PSII complex (FRL-PSII) binds a mixture of different Chl types in those same positions: about 30 Chl a, 4 Chl f, and 1 Chl d (8Gan F. Zhang S. Rockwell N.C. Martin S.S. Lagarias J.C. Bryant D.A. Extensive remodeling of a cyanobacterial photosynthetic apparatus in far-red light.Science. 2014; 345: 1312-1317Crossref PubMed Scopus (272) Google Scholar, 9Gan F. Bryant D.A. Adaptive and acclimative responses of cyanobacteria to far-red light.Environ. Microbiol. 2015; 17: 3450-3465Crossref PubMed Scopus (117) Google Scholar, 10Gan F. Shen G. Bryant D.A. Occurrence of far-red light photoacclimation (FaRLiP) in diverse cyanobacteria.Life. 2014; 5: 4-24Crossref PubMed Scopus (125) Google Scholar, 11Ho M.-Y. Niedzwiedzki D.M. MacGregor-Chatwin C. Gerstenecker G. Hunter C.N. Blankenship R.E. et al.Extensive remodeling of the photosynthetic apparatus alters energy transfer among photosynthetic complexes when cyanobacteria acclimate to far-red light.Biochim. Biophys. Acta - Bioenerg. 2020; 1861148064Crossref Scopus (37) Google Scholar, 12Ohkubo S. Miyashita H. A niche for cyanobacteria producing chlorophyll f within a microbial mat.ISME J. 2017; 11: 2368-2378Crossref PubMed Scopus (49) Google Scholar). These FRL-specific alterations allow PSII to achieve water oxidation using lower energy FRL, decreasing the low energy limit that was canonically thought to be required to drive oxygenic photosynthesis (13Nürnberg D.J. Morton J. Santabarbara S. Telfer A. Joliot P. Antonaru L.A. et al.Photochemistry beyond the red limit in chlorophyll f–containing photosystems.Science. 2018; 360: 1210-1213Crossref PubMed Scopus (164) Google Scholar). An understanding of the molecular basis of FRL-PSII function is of great interest because it may allow for the engineering of shade tolerance into crops (14Mascoli V. Bersanini L. Croce R. Far-red absorption and light-use efficiency trade-offs in chlorophyll f photosynthesis.Nat. Plants. 2020; 6: 1044-1053Crossref PubMed Scopus (25) Google Scholar, 15Wolf B.M. Blankenship R.E. Far-red light acclimation in diverse oxygenic photosynthetic organisms.Photosynth. Res. 2019; 142: 349-359Crossref PubMed Scopus (31) Google Scholar). To gain the requisite understanding, various spectroscopic, phylogenetic, and structural investigations have been performed (7Viola S. Roseby W. Santabarabara S. Nürnberg D. Assunção R. Dau H. et al.Impact of energy limitations on function and resilience in long-wavelength Photosystem II.eLife. 2022; https://doi.org/10.1101/2022.04.05.486971Crossref Scopus (0) Google Scholar, 8Gan F. Zhang S. Rockwell N.C. Martin S.S. Lagarias J.C. Bryant D.A. Extensive remodeling of a cyanobacterial photosynthetic apparatus in far-red light.Science. 2014; 345: 1312-1317Crossref PubMed Scopus (272) Google Scholar, 13Nürnberg D.J. Morton J. Santabarbara S. Telfer A. Joliot P. Antonaru L.A. et al.Photochemistry beyond the red limit in chlorophyll f–containing photosystems.Science. 2018; 360: 1210-1213Crossref PubMed Scopus (164) Google Scholar, 16Zamzam N. Rakowski R. Kaucikas M. Dorlhiac G. Viola S. Nürnberg D.J. et al.Femtosecond visible transient absorption spectroscopy of chlorophyll-f-containing photosystem II.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 23158-23164Crossref PubMed Scopus (9) Google Scholar, 17Gisriel C.J. Cardona T. Bryant D.A. Brudvig G.W. Molecular evolution of far-red light-acclimated photosystem II.Microorganisms. 2022; 10: 1270Crossref PubMed Scopus (5) Google Scholar, 18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f.J. Biol. Chem. 2022; 298101424Google Scholar, 19Mascoli V. Bhatti A.F. Bersanini L. van Amerongen H. Croce R. The antenna of far-red absorbing cyanobacteria increases both absorption and quantum efficiency of Photosystem II.Nat. Commun. 2022; 13: 3562Crossref PubMed Scopus (5) Google Scholar). To identify directly the sites to which Chls d and f bind, the structure of a monomeric apo-FRL-PSII complex isolated from a marine, mesophilic cyanobacterium, Synechococcus sp. PCC 7335 (hereafter Synechococcus 7335), was solved at a global resolution of 2.25 Å using single-particle cryo-EM (18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f.J. Biol. Chem. 2022; 298101424Google Scholar). Compared to a monomer of a typical PSII holocomplex found in a dimeric configuration, the monomeric apo-FRL-PSII complex lacked various transmembrane subunits, the extrinsic subunits (i.e., PsbO, PsbQ, PsbU, and PsbV), and the OEC; however, it contained all four Chl-binding subunits which allowed for the identification of 33 of the 35 Chl sites. By comparing sequences to determine FRL-specific residues, identifying common Chl d/f chemical environments and employing quantitative map assessments, four Chl f molecules and one Chl d molecule were assigned, which was consistent with the content of those pigments determined by cofactor analysis (18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f.J. Biol. Chem. 2022; 298101424Google Scholar). The assignments were subsequently corroborated by a larger phylogenetic comparison of FRL-PSII sequences, which also provided insight into their evolution (17Gisriel C.J. Cardona T. Bryant D.A. Brudvig G.W. Molecular evolution of far-red light-acclimated photosystem II.Microorganisms. 2022; 10: 1270Crossref PubMed Scopus (5) Google Scholar). A disadvantage of the apo-FRL-PSII structure was that it lacked PsbH2, the FRL-specific isoform of PsbH. Because all other PSII and photosystem I (PSI) subunits encoded by the FaRLiP gene cluster have associated molecular structures (18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f.J. Biol. Chem. 2022; 298101424Google Scholar, 20Gisriel C.J. Shen G. Kurashov V. Ho M.-Y. Zhang S. Williams D. et al.The structure of Photosystem I acclimated to far-red light illuminates an ecologically important acclimation process in photosynthesis.Sci. Adv. 2020; 6eaay6415Crossref PubMed Scopus (41) Google Scholar, 21Kato K. Shinoda T. Nagao R. Akimoto S. Suzuki T. Dohmae N. et al.Structural basis for the adaptation and function of chlorophyll f in photosystem I.Nat. Commun. 2020; 11: 238Crossref PubMed Scopus (57) Google Scholar, 22Gisriel C.J. Huang H.-L. Reiss K.M. Flesher D.A. Batista V.S. Bryant D.A. et al.Quantitative assessment of chlorophyll types in cryo-EM maps of photosystem I acclimated to far-red light.BBA Adv. 2021; 1100019Google Scholar, 23Gisriel C.J. Flesher D.A. Shen G. Wang J. Ho M.-Y. Brudvig G.W. et al.Structure of a photosystem I-ferredoxin complex from a marine cyanobacterium provides insights into far-red light photoacclimation.J. Biol. Chem. 2022; 298101408Google Scholar), PsbH2 is therefore the last FaRLiP-specific protein to have its structure determined. To gain insight into the molecular structure of the FRL-PSII holocomplex, we have altered the purification procedure of FRL-PSII from Synechococcus 7335 allowing us to isolate a dimeric complex that maintains the PsbH2 subunit, and we solved its cryo-EM structure to a global resolution of 2.6 Å. Whereas PsbH found in VL-absorbing PSII is involved in tuning the energy of the lowest energy Chl a molecule found in PSII, PsbH2 lacks this interaction. This suggests that during FaRLiP, the energetic landscape of the antenna Chls is altered to favor energy transfer to Chl f. Because identifying Chls d and f in cryo-EM maps is challenging (24Gisriel C.J. Wang J. Brudvig G.W. Bryant D.A. Opportunities and challenges for assigning cofactors in cryo-EM density maps of chlorophyll-containing proteins.Commun. Biol. 2020; 3: 408Crossref PubMed Scopus (18) Google Scholar), the new cryo-EM map allows for a reassessment of the Chl type assignments. We performed the strategies previously utilized for this purpose, and observe that two sites in FRL-PSII exhibit CH-O interactions with the formyl moiety of Chl f. Because all Chl d and Chl f molecules exhibit FRL-specific interactions between their formyl moiety and the protein environment, we additionally developed a Python-based tool that can be used to search any set of Chl-containing atomic coordinates for potential H-bond donors to similar formyl moieties. The H-bond search tool is useful for making an initial assessment of structures that bind formylated Chl molecules among the "bulk" Chl a. The structural observations are interpreted in the context of other FaRLiP investigations that enhance our understanding of this widespread photobiological acclimation mechanism. FRL-PSII was isolated by immobilized metal affinity chromatography from Synechococcus 7335-psbC2-[His]10 cells as described in the Experimental procedures. The room temperature absorbance spectrum shows a FRL absorbance peak maximal at 724 nm, and the 77 K fluorescence emission spectrum (excitation wavelength, 440 nm) shows maximal emission at 739 nm, both characteristic of FRL-PSII complexes characterized previously (Fig. S1) (8Gan F. Zhang S. Rockwell N.C. Martin S.S. Lagarias J.C. Bryant D.A. Extensive remodeling of a cyanobacterial photosynthetic apparatus in far-red light.Science. 2014; 345: 1312-1317Crossref PubMed Scopus (272) Google Scholar, 11Ho M.-Y. Niedzwiedzki D.M. MacGregor-Chatwin C. Gerstenecker G. Hunter C.N. Blankenship R.E. et al.Extensive remodeling of the photosynthetic apparatus alters energy transfer among photosynthetic complexes when cyanobacteria acclimate to far-red light.Biochim. Biophys. Acta - Bioenerg. 2020; 1861148064Crossref Scopus (37) Google Scholar, 13Nürnberg D.J. Morton J. Santabarbara S. Telfer A. Joliot P. Antonaru L.A. et al.Photochemistry beyond the red limit in chlorophyll f–containing photosystems.Science. 2018; 360: 1210-1213Crossref PubMed Scopus (164) Google Scholar, 18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f.J. Biol. Chem. 2022; 298101424Google Scholar). SDS-PAGE showed bands typical for PSII complexes including core subunits, extrinsic subunits, small peripheral transmembrane subunits, and assembly factors (Fig. S2). Mass spectrometry of tryptic peptides was performed which identified various PSII subunits (Table S1). It showed that the FRL-specific subunits PsbA3, PsbB2, PsbC2, PsbD3, and PsbH2 were far more abundant than were their VL-subunit isoforms. It also showed that the PsbO1, PsbV1, and PsbF2 isoforms were more abundant than PsbO2, PsbV2, and PsbF1, respectively, none of which are encoded in a FaRLiP gene cluster. To make an initial assessment of the oligomeric state, the FRL-PSII complexes were negatively stained with uranyl acetate and imaged by transmission electron microscopy (Fig. S3). The images showed relatively monodisperse single particles whose size was similar to those in a sample of dimeric PSII from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803), suggesting that the FRL-PSII sample had maintained a dimeric configuration. Finally, pigment analysis was performed by solvent extraction and subsequent HPLC (Fig. S4). The results were consistent with previously published pigment analyses (8Gan F. Zhang S. Rockwell N.C. Martin S.S. Lagarias J.C. Bryant D.A. Extensive remodeling of a cyanobacterial photosynthetic apparatus in far-red light.Science. 2014; 345: 1312-1317Crossref PubMed Scopus (272) Google Scholar, 11Ho M.-Y. Niedzwiedzki D.M. MacGregor-Chatwin C. Gerstenecker G. Hunter C.N. Blankenship R.E. et al.Extensive remodeling of the photosynthetic apparatus alters energy transfer among photosynthetic complexes when cyanobacteria acclimate to far-red light.Biochim. Biophys. Acta - Bioenerg. 2020; 1861148064Crossref Scopus (37) Google Scholar, 13Nürnberg D.J. Morton J. Santabarbara S. Telfer A. Joliot P. Antonaru L.A. et al.Photochemistry beyond the red limit in chlorophyll f–containing photosystems.Science. 2018; 360: 1210-1213Crossref PubMed Scopus (164) Google Scholar, 18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f.J. Biol. Chem. 2022; 298101424Google Scholar) that suggest the FRL-PSII complexes contain ∼1 Chl d, 4 Chl f, 30 Chl a, and two pheophytin a per FRL-PSII complex. The FRL-PSII sample was plunge-frozen in dim green light and imaged for single-particle cryo-EM as described in Experimental procedures (Fig. S5). Micrographs showed monodisperse single particles that after processing yielded a 3D-reconstruction with a global resolution of 2.6 Å with local resolutions spanning the range 2.4 to 4.0 Å (Fig. S6). The map exhibited an obvious dimeric configuration and was therefore processed using C2 symmetry. In each monomer of the FRL-PSII dimer, the following subunits were identified and modeled: PsbA3, PsbB2, PsbC2, PsbD3, PsbE, PsbF2, PsbH2, PsbI, PsbK, PsbL, PsbM, PsbO1, PsbT, PsbU, PsbV1, and PsbX (Fig. S7). These subunits were modeled to coordinate 35 Chl molecules, eight carotenoids, 11 diacyl lipids, two pheophytin a molecules, two plastoquinones, two Cl anions, one heme b, one heme c, one non-heme Fe cation, one bicarbonate anion, one OEC, one n-dodecyl β-D-maltoside (β-DM) molecule, and 296 water molecules. The overall structure and three representative map regions are shown in Figure 1. The cryo-EM data and modeling statistics are reported in Table S2. Relative to previously determined structures of dimeric cyanobacterial PSII holocomplexes (25Umena Y. Kawakami K. Shen J.-R. Kamiya N. Crystal structure of oxygen-evolving Photosystem II at a resolution of 1.9 Å.Nature. 2011; 473: 55-60Crossref PubMed Scopus (3074) Google Scholar, 26Gisriel C.J. Wang J. Liu J. Flesher D.A. Reiss K.M. Huang H.-L. et al.High-resolution cryo-EM structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803.Proc. Natl. Acad. Sci. U. S. A. 2022; 119e2116765118Crossref PubMed Scopus (36) Google Scholar), the structure lacks PsbJ, PsbQ, PsbY, Ycf12, and PsbZ. The extrinsic subunits PsbO1, PsbU, and PsbV1, and the OEC exhibit heterogeneity in their occupancy and positions (Text S1, Figs. S8, S9 and Table S3); therefore, their atomic coordinates should be considered unreliable. In the 2.25-Å global resolution structure of apo-FRL-PSII, one site that binds a Chl d molecule at high occupancy was assigned in the ChlD1 position of the electron transfer chain (18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f.J. Biol. Chem. 2022; 298101424Google Scholar). This is also observed in the dimeric FRL-PSII structure, in which the formyl moiety of Chl d accepts an H-bond from the hydroxyl moiety of the sidechain from PsbA3-Thr155 (Fig. 1C), which is in turn H-bonded to PsbA3-Tyr120. Importantly, both PsbA3-Thr155 and Tyr120 are FRL-specific and are conserved in the PsbA3 sequences from all cyanobacterial species for which data are available (17Gisriel C.J. Cardona T. Bryant D.A. Brudvig G.W. Molecular evolution of far-red light-acclimated photosystem II.Microorganisms. 2022; 10: 1270Crossref PubMed Scopus (5) Google Scholar). The presence of Chl d in the ChlD1 site, and additionally the FRL-specific H-bond to PD2 (18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f.J. Biol. Chem. 2022; 298101424Google Scholar) which is also conserved in the dimeric FRL-PSII structure (Fig. 1B), seem likely to be essential for tuning the redox potentials of the electron transfer chain cofactors so that FRL can drive water oxidation. Four sites that bind Chl f molecules at high occupancy were assigned in the apo-FRL-PSII structure: three in the PsbB2 subunit in sites 605, 608, and 614 and one in the PsbC2 subunit in site 507. To identify possible Chl f molecules in the dimeric FRL-PSII map, we performed cone scans as described previously (22Gisriel C.J. Huang H.-L. Reiss K.M. Flesher D.A. Batista V.S. Bryant D.A. et al.Quantitative assessment of chlorophyll types in cryo-EM maps of photosystem I acclimated to far-red light.BBA Adv. 2021; 1100019Google Scholar) (Fig. S10). In brief, the cryo-EM map around each Chl is scanned at the C7 substituent (always a methyl moiety) and the C2 substituent (a formyl moiety in Chl f or a methyl moiety in Chl a) at a defined bond geometry and distance (Fig. S11). The C7 cone scan data are used to generate a 3σ null distribution representative of a methyl moiety. When the C2 cone scan data exceed this null distribution, it suggests the presence of a formyl moiety (rather than methyl) and that the site may thus be occupied by Chl f. In PsbB2, all three previously assigned sites, 605, 608, and 614, had C2 cone scan signals greater than the null distribution, and all exhibit FRL-specific interactions that confer Chl f–binding specificity (Fig. 2). Near the C2 position of Chl 605, the sidechain of PsbB2-Phe33 is found which in PsbB1 (VL-PSII) is instead a Trp residue. It was previously proposed that the smaller Phe33 allows additional space for the presence of a formyl moiety of the Chl f at site 605 (18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and f.J. Biol. Chem. 2022; 298101424Google Scholar). The dimeric FRL-PSII structure is consistent with this hypothesis, but we additionally note that the oxygen atom of the formyl moiety is nearly in-plane and within H-bonding distance of the PsbB2-Phe33 aromatic ring. We propose that Chl f–binding affinity is conferred by the protein for site 605 by a CH-O interaction via the Phe sidechain. Interestingly, another CH-O interaction may also confer some binding affinity for Chl f. In the cone scan analysis of site 614, the profile shows two peaks, only one of which is greater than the methyl distribution. This direction corresponds to a formyl moiety positioned to accept an H-bond from the indole nitrogen atom of the FRL-specific residue PsbB2-Trp462. However, the second peak in the cone scan corresponds to a position toward another Phe sidechain, PsbB2-Phe245, which is also FRL-specific. Modeling the formyl moiety in this direction places its oxygen atom in plane and within H-bonding distance of the aromatic ring, consistent with a CH-O interaction. We hypothesize that the formyl moiety of Chl f 614 occupies two configurations: a primary one directed toward PsbB2-Trp462 and a secondary one that is lower occupancy directed toward PsbB2-Phe245 (Fig. 2A). Thus, it may be that two CH-O interactions participate in Chl f-binding in FRL-PSII, in sites 605 and 614. Another interesting observation is that in structures of FRL-PSI, there was only one Chl f molecule that did not exhibit an obvious H-bond, that in site B38 (22Gisriel C.J. Huang H.-L. Reiss K.M. Flesher D.A. Batista V.S. Bryant D.A. et al.Quantitative assessment of chlorophyll types in cryo-EM maps of photosystem I acclimated to far-red light.BBA Adv. 2021; 1100019Google Scholar, 23Gisriel C.J. Flesher D.A. Shen G. Wang J. Ho M.-Y. Brudvig G.W. et al.Structure of a photosystem I-ferredoxin complex from a marine cyanobacterium provides insights into far-red light photoacclimation.J. Biol. Chem. 2022; 298101408Google Scholar). This site was suggested to form a Chl f dimer that had been observed spectroscopically with the Chl in site B37 (27Tros M. Mascoli V. Shen G. Ho M.-Y. Bersanini L. Gisriel C.J. et al.Breaking the red limit: efficient trapping of long-wavelength excitations in chlorophyll-f-containing photosystem I.Chem. 2020; 7: 155-173Abstract Full Text Full Text PDF Scopus (12) Google Scholar). Near the C2 position of B38, a Trp found in VL sequences is instead a Phe found in FRL sequences (Fig. S12). Rather than a CH-O interaction, the orientation of the Phe sidechain is more consistent with a CHO–π interaction between the formyl oxygen atom of the Chl f and the conjugated system of the Phe sidechain. Thus, it appears that Phe sidechains may commonly participate in conferring Chl f–binding specificity. Consistent with previous observations, both Chls PsbB2 608 and PsbC2 507 also exhibit FRL-specific H-bonding interactions near their C2 positions (17Gisriel C.J. Cardona T. Bryant D.A. Brudvig G.W. Molecular evolution of far-red light-acclimated photosystem II.Microorganisms. 2022; 10: 1270Crossref PubMed Scopus (5) Google Scholar, 18Gisriel C.J. Shen G. Ho M.-Y. Kurashov V. Flesher D.A. Wang J. et al.Structure of a monomeric photosystem II core complex from a cyanobacterium acclimated to far-red light reveals the functions of chlorophylls d and
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