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Cryo-EM photosystem I structure reveals adaptation mechanisms to extreme high light in Chlorella ohadii

Abstract

Photosynthesis in deserts is challenging since it requires fast adaptation to rapid night-to-day changes, that is, from dawn’s low light (LL) to extreme high light (HL) intensities during the daytime. To understand these adaptation mechanisms, we purified photosystem I (PSI) from Chlorella ohadii, a green alga that was isolated from a desert soil crust, and identified the essential functional and structural changes that enable the photosystem to perform photosynthesis under extreme high light conditions. The cryo-electron microscopy structures of PSI from cells grown under low light (PSILL) and high light (PSIHL), obtained at 2.70 and 2.71 Å, respectively, show that part of light-harvesting antenna complex I (LHCI) and the core complex subunit (PsaO) are eliminated from PSIHL to minimize the photodamage. An additional change is in the pigment composition and their number in LHCIHL; about 50% of chlorophyll b is replaced by chlorophyll a. This leads to higher electron transfer rates in PSIHL and might enable C. ohadii PSI to act as a natural photosynthesiser in photobiocatalytic systems. PSIHL or PSILL were attached to an electrode and their induced photocurrent was determined. To obtain photocurrents comparable with PSIHL, 25 times the amount of PSILL was required, demonstrating the high efficiency of PSIHL. Hence, we suggest that C. ohadii PSIHL is an ideal candidate for the design of desert artificial photobiocatalytic systems.

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Fig. 1: Cryo-EM structures of the C. ohadii PSI complexes that were isolated from the cells grown under LL and HL conditions.
Fig. 2: Pigment changes between the C. ohadii LHCILL and LHCIHL complexes.
Fig. 3: Oxidation/reduction course of \({\mathrm{P}}_{700}/{\mathrm{P}}_{700}^{ \cdot + }\) of the C. ohadii PSILL and PSIHL complexes.
Fig. 4: Photocurrents produced by electrode-bound PSILL and PSIHL.

Data availability

The atomic coordinates of the three supercomplexes have been deposited in the Protein Data Bank, with accession codes 6ZZX (PSILL), 6ZZY (PSIHL), and 7A4P (PSIHL1). The cryo-EM maps have been deposited in the Electron Microscopy Data Bank, with accession codes EMD-11588 (PSILL), EMD-11589 (PSIHL), and EMD-11640 (PSIHL1).

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (AD 458/1-1 and LU 315/17-1) in the framework of a Deutsch–Israelische Projektkooperation, ‘Nanoengineered optobioelectronics with biomaterials and bioinspired assemblies’. W.L. and A.S. thank the Max Planck Society for their financial support. This study was part of the research in the MINERVA Centre for Bio-hybrid Complex Systems. We thank the Electron Microscopy Core Facility at the EMBL for their support. E.N. was supported by the Hebrew University of Jerusalem - Prof. Leonora Reinhold Fellowship. A.F. and M.M.N. were supported by the Research Training Group 2341 ‘MiCon’ also funded by the DFG. We thank F. Weis for his assistance and guidance in data collection. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization and Informatics at the University of California San Francisco, with support from National Institutes of Health P41-GM103311. This work was supported by The Israel Science Foundation (grant no. 569/17) to N.N. and by the German–Israeli Foundation for Scientific Research and Development to N.N., grant no. G-1483-207/2018. Y.S. was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement 723991—CRYOMATH) and by the Zimin Institute for Engineering Solutions Advancing Better Lives. G.S. acknowledges funding by a ‘Nevet’ grant from the Grand Technion Energy Program and a Technion Vice President of Research (VPR) Berman Grant for Energy Research.

Author information

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Authors

Contributions

R.N. designed and performed the functional experiments. G.S. designed the C. ohadii growth and protein purifications. V.L. grew the C. ohadii cells. V.L. and M.F. purified the PSI complexes. Y.L.K. performed the protein-grid attachment for cryo-EM. E.N. collected the cryo-EM data. I.C. and Y.S. processed the cryo-EM data. I.C., N.N. and E.N. built the atomic models. M.F. performed the P700 kinetic measurements. M.R. and M.M.N. designed the PAM and photocurrent experiments. W.S. participated in designing the photocurrent experiments. A.F. and V.H. performed the PAM and photocurrent measurements. A.K. designed the gene sequencing and metabolomics experiments. O.M. performed the gene sequencing experiment. H.T. performed the metabolomics measurements. W.L. and A.S. designed the EPR experiments. A.S. performed the EPR measurements. R.N., M.M.N., W.S, M.R., N.N., A.K., I.W., I.C., E.N. and G.S. wrote the manuscript and all authors contributed to editing.

Corresponding authors

Correspondence to Nathan Nelson, Wolfgang Lubitz or Rachel Nechushtai.

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The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Jean-David Rochaix, Jian-Ren Shen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Analytical assessment and identification of the PSILL and PSIHL purified complexes and their subunits.

(a) The SDS-PAGE of the dissociated PSI complexes: 1µg chlorophyll was loaded into each well. The relevant bands that were used for the MS analysis (Supplementary Fig. 1) are marked 1-12, and these were excised from the gel. The 12 sliced bands were subjected to MS analysis after chymotrypsin digestion. PSI subunit annotations: 1 – PsaI/PsaJ; 2 – PsaK; 3 – PsaG; 4 – PsaH/PsaC; 5 – PsaE/PsaC; 6 – PsaL; 7 – Lhca9/PsaF; 8 – Lhca1/PsaD; 9 – Lhca4; 10 – Lhca2; 11 – Lhca6/Lhca5/Lhca3; 12 – Lhca7. (b) SDS-PAGE of the purified PSILL and PSIHL used for cryo-EM structure determination.

Extended Data Fig. 2 C. ohadii Cryo-EM structure analysis of PSILL.

(a) A zoomed-in view of a PSILL micrograph. (b) Representing 2D classes. (c) Cryo-EM data processing workflow. (d) Local resolution of the complete PSILL (left) and map cut-through (right). (e) Angular distribution of PSILL. (f) Fourier shell correlation (FSC) of the two half-sets postprocessing result.

Extended Data Fig. 3 C. ohadii Cryo-EM structure analysis of PSIHL.

(a) Zoomed-in view of a PSIHL micrograph. (b) Representing 2D classes. (c) Cryo-EM data processing workflow. (d) Local resolution of complete PSIHL (left) and map cut-through (right). (e) Angular distribution of PSIHL. (f) Fourier shell correlation (FSC) of the two half-sets postprocessing result.

Extended Data Fig. 4 Membrane view of the C. ohadii PSILL tructure.

(a) Membrane side view in surface representation of C. ohadii PSILL. (b) Zoomed-in view of the reaction centre, including the primary donor, P700, and all of the electron acceptors (two chlorophyll a molecules, A and A0, Phylloquinone, A1, and three [4Fe-4S] clusters, FX, FA, and FB) of C. ohadii PSILL Core Complex (CC). (c) Stromal view of the irregular stromal loop that was identified in C. ohadii Lhca4, Lhca7, and Lhca8 (circled in red). This shared fold was not observed in the other seven LHCIs complexes. Moreover, this unique loop did not seem to assist in binding or coordinating any prosthetic group. Lhca4 is coloured in blue, Lhca7 in pale-yellow, and Lhca8 in deep teal. (d) Membrane plane view of the three unusual disulfide bonds that were identified in Lhca5 and Lhca6 in the C. ohadii LHCILL complex. The two luminal bonds were previously detected in C. reinhardtii PSI33, and a novel stromal S-S bond was uncovered in C. ohadii Lhca6 (circled in blue). Lhca5 is coloured in cyan, Lhca6 in hot pink, and the disulfide bond is circled in yellow.

Extended Data Fig. 5 The C. ohadii Cryo-EM structure analysis of PSIHL1.

(a) Zoomed-in view of the PSIHL1 micrograph. (b) The Cryo-EM data processing workflow. (c) The local resolution estimation of complete PSIHL1 (left) and map cut-through (right). (d) Angular distribution of PSIHL1. (e) Fourier shell correlation (FSC) of the two half-sets postprocessing result.

Extended Data Fig. 6 Structural features and composition of C. ohadii PSIHL1.

(a) Stromal view of PSIHL1 and (b) luminal view of C. ohadii PSIHL1. The subunits associated with the core-complex (sea green), the first LHCI belt (steel blue), and the second LHCI belt (dark red), were coloured separately. The location of the missing LHCI dimers (Lhca2 and Lhca9, magenta outline), the missing core subunit PsaO (blue outline), and PsaH (firebrick outline) are marked. In PSIHL, the subunit PsaO (blue outline) is missing (Fig. 1).

Extended Data Fig. 7 Major pigment changes between the different LHC´s in C. ohadii PSI.

(a) Identifying chlorophyll a/b. Electron density map surrounding several chlorophylls. (I) Chlorophyll b position 613 from Lhca6HL. The red ellipsoid indicates the carbonyl group of chlorophyll b. (II) Chlorophyll b position 611 from Lhca1LL. The red ellipsoid indicates the carbonyl group of chlorophyll b. (III) Chlorophyll a position 611 from Lhca1HL. The dashed red ellipsoid indicates the methyl group of chlorophyll a. (b) Consistent and variable chlorophyll positions in C. ohadii LHC’s. (I) Membrane plane view from the outer membrane of the LHCI chlorophylls. The recurring positions 601-613 that were found in nearly all LHCI’s (see Extended Data Table 1) are coloured in transparent red, and principally they were found in the LHCI centre. Positions 614-621 appeared intermittently in the C. ohadii LHCI periphery. An example from each chlorophyll position is shown and coloured as follow: Lhca1 is coloured green, Lhca2 in bright-orange, Lhca3 in deep-purple, Lhca4 in blue, Lhca5 in cyan, Lhca6 in hot-pink, Lhca7 in pale-yellow, and Lhca8 in deep-teal. The polypeptide backbones of Lhca1 and Lhca2 are shown as cartoons and are coloured in green and orange, respectively. (II) Membrane plane view rotated by 180˚, from the PSI core to the outer membrane, showing chlorophylls in the interface of the first and second LHCI belts, or the first LHCI belt and the PSI core. (c) Consistent and variable carotenoid positions in the C. ohadii LHC’s. Four carotene positions, namely 504-507, were identified in the C. ohadii. LHCIs; they are located between the PSI core complex and the first LHCI belt, or in the interface between the first and second LHCI belts. Carotenoid positions 501 and 502 are present in all 10 LHCI subunits, whereas position 503 is missing solely in the Lhac2-Lhca9 dimer (I) Membrane plane view from the periphery to PSI of the LHC carotenoids. The recurring positions of 501-503 were found in nearly all of the LHCI’s, apart from 503 in Lhca2 and Lhca9, and they are coloured in green. Positions 504-507 appeared intermittently in the C. ohadii LHCs. The polypeptide backbones of Lhca1 and Lhca2 are shown as cartoons and are coloured in green and orange, respectively. (II) Membrane plane view rotated by 180˚, from the PSI core to the outer membrane, showing carotenoids in the interface of the first and second LHCI belt, or the first LHCI belt and the PSI core. (d) Carotenoids arrangement in LHCILL (left), LHCIHL (middle), and zoomed superposition on Lhca9, from both light conditions (right panel). Carotenoids appearing in both conditions are coloured in orange, while carotenoids unique to LHCILL are coloured in purple, and truncated carotenoids that appear in LHCIHL are coloured in red.

Extended Data Fig. 8 C. ohadii PSILL shows higher functional efficiency than the ‘gold standard’ cyanobacterial PSI complexes.

(a) Short-time decay kinetics of \(P_{700}^{ \cdot + }\) (blue trace) and \(A_1^{ \cdot - }\) (green trace) in the PSI complexes from C. ohadii (solid lines), and the cyanobacteria Synechocystis sp. PCC6803 (dashed lines) in the frozen aqueous solution measured at 120 K by transient W-band EPR after a 532 nm laser pulse that created the radical pair. The \(A_1^{ \cdot - }\) EPR signal of PSI from the C. ohadii decays with 0.25±0.05 ms, about 4 times faster than the Synechocystis sp. PCC6803 PSI (0.95±0.1 ms). (b) Long time decay kinetics of \(P_{700}^{ \cdot + }\) for C. ohadii (blue trace) and cyanobacteria Synechocystis sp. PCC6803 (black trace). The kinetic decay traces were acquired at the maximum EPR absorption of the thermally equilibrated \(P_{700}^{ \cdot + }\) and \(A_1^{ \cdot - }\) cofactors. (c-d) W-band cw EPR spectra of C. ohadii PSILL (c) and Cyanobacteria Synechocystis sp. PCC6803 (d) recorded at 120 K; blue trace: dark-adapted sample; green trace: under continuous illumination at 690 nm; red trace: 10 min. after the 690 nm light was switched off. The full circles mark the EPR lines from a MnMgO standard that was used for the magnetic field calibration, and the squares mark the EPR signals stemming from the Mn2+ contaminations in the sample solution. (e-f) W-band cw EPR spectra of C. ohadii PSILL (e) grown under LL conditions and cyanobacteria Synechocystis sp. PCC6803 (f), both recorded at 120 K. The spectra in the \(P_{{\mathrm{700}}}^{\cdot + }{A_1^{\cdot - }}\) spectral range were corrected by the EPR signal of the dark-adapted sample. (g) Directly-detected time-resolved W-band EPR spectra of the radical cofactors in the PSI complexes from C. ohadii (blue trace), and Synechocystis sp. PCC6803 (black trace) recorded at 120 K and 600 ns after the 532 nm laser flash driving the charge separation event. The spectra are normalised in such a way that the up-field features, which are primarily due to \(P_{{\mathrm{700}}}^{\cdot +}\), have the same signal amplitude. The shape of the spectra reveal that the \(A_1^{ \cdot - }\) quinone acceptor in the PSI particles from C. ohadii is phylloquinone. (h) Fluorescence decay of photo-oxidized \(P_{{\mathrm{700}}}^{\cdot +}\) in PSI from C. ohadii (red) when in comparison to PSI from T. elongatus (black). The bi-exponential curve fitting to the \(P_{{\mathrm{700}}}^ +\) decay profiles was performed. The 830-875 nm absorption signal is plotted in normalised units against time after P700 was oxidized by a 20 µE actinic light pulse at 635 nm and room temperature (RT). DCPIP/NaAsc concentration: 10 µM/ 5 mM. The curves were normalised to a \(P_{{\mathrm{700}}}^ {\cdot +}\) signal amplitude of 1 at t=0. The fast electron recombination phase at \(P_{{\mathrm{700}}}^ {\cdot +}\) appeared to be significantly faster in the case of PSILL from C. ohadii (t2 = 53 ± 1 ms) when compared to the slower recombination rate of PSI from T. elongatus (t1 = 91 ± 1 ms).

Extended Data Fig. 9 Photocathode setup and loading optimisation of C. ohadii PSIHL and PSILL on the Au electrodes.

(a) Electrochemical three-electrode setup for chronoamperometric long-term measurements. The electrodes were immersed in an electrolyte solution (pH 4 buffer with 3 mM methyl viologen). The illumination was achieved by a computer-controlled red LED (λ= 685 nm). CE: counter electrode (1 mm diameter coiled platinum wire), WE: working electrode (gold rod electrodes with 2 mm diameter (CH Instruments, USA and Metrohm, Germany), RE: reference electrode (Ag/AgCl electrode in 3.5 M KCl solution). (b) Scheme of PSIHL and PSILL as immobilised on the Au electrode surface via the P-Os (osmium complex modified polymer49). (c-d) Photocurrents of the loading optimisation for PSIHL (c) and PSILL (d). PSIHL and PSILL showed different photocurrents, when applied at the same concentration. In order to reliably investigate the half-life time of the complexes (Fig. 4), it is necessary to start with a similar signal for the initial photocurrent. Therefore, 5 µl of the PSI/P-Os samples with different PSI amounts were drop casted on the Au working electrode and the resulting photocurrent signals were analysed. The light intensity was set to 1500 µmol photons m −2 s −1 at 685 nm and the samples were polarised at 0 V vs. Ag/AgCl. The illumination and dark phases are marked with yellow and grey backgrounds, respectively.

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Caspy, I., Neumann, E., Fadeeva, M. et al. Cryo-EM photosystem I structure reveals adaptation mechanisms to extreme high light in Chlorella ohadii. Nat. Plants 7, 1314–1322 (2021). https://doi.org/10.1038/s41477-021-00983-1

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