Structure of a PSI–LHCI–cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions Janina Steinbecka,b, Ian L. Rossb, Rosalba Rothnagelb, Philipp Gäbeleina, Stefan Schulzea,1, Nichole Gilesc, Rubbiya Alib,2, Rohan Drysdaleb, Emma Siereckic, Yann Gambinc, Henning Stahlbergd, Yuichiro Takahashie, Michael Hipplera,3, and Ben Hankamerb,3 aInstitute of Plant Biology and Biotechnology, University of Münster, 48143 Münster, Germany; bInstitute for Molecular Bioscience, University of Queensland, St. Lucia, QLD 4072, Australia; cEuropean Molecular Biology Laboratory Single Molecule Science, Lowy Cancer Research Centre, University of New South Wales, Sydney, NSW 2052, Australia; dCenter for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, CH-4058 Basel, Switzerland; and eResearch Institute for Interdisciplinary Science, Okayama University, 700-8530 Okayama, Japan Edited by Krishna K. Niyogi, Howard Hughes Medical Institute and University of California, Berkeley, CA, and approved August 23, 2018 (received for review June 13, 2018) Photosynthetic linear electron flow (LEF) produces ATP and how these membrane protein complexes can contribute to both NADPH, while cyclic electron flow (CEF) exclusively drives photophos- functional modes. Extensive biochemical and biophysical analy- phorylation to supply extra ATP. The fine-tuning of linear and cyclic ses using the green alga Chlamydomonas reinhardtii suggest that electron transport levels allows photosynthetic organisms to balance efficient CEF depends on the formation of a CEF supercomplex – – light energy absorption with cellular energy requirements under consisting of PSI, cyt b6f, and subunits ferredoxin NADP oxidore- constantly changing light conditions. As LEF and CEF share many ductase (FNR), Proton Gradient Regulation-Like 1 (PGRL1), An- electron transfer components, a key question is how the same indi- aerobic Response 1 (ANR1), and Calcium Sensor (CAS). The CEF vidual structural units contribute to these two different functional supercomplex is proposed to enhance CEF over LEF when stromal electron carriers are reduced (excess NADPH) and ATP is limiting modes. Here, we report the structural identification of a photosystem I (7–10). However, structural evidence for this supercomplex in C. – – PLANT BIOLOGY (PSI) light harvesting complex I (LHCI) cytochrome (cyt) b6fsupercom- reinhardtii Chlamydomonas reinhardtii is lacking, probably due to its putative dynamic nature. plex isolated from the unicellular alga un- Here, under CEF-inducing anaerobic conditions, a sucrose der anaerobic conditions, which induces CEF. This provides strong density gradient (SDG) fraction with CEF activity (7, 8) was evidence for the model that enhanced CEF is induced by the formation isolated from C. reinhardtii, and a PSI–light harvesting complex I of CEF supercomplexes, when stromal electron carriers are reduced, to (LHCI)–cyt b6f-containing CEF supercomplex within it, was generate additional ATP. The additional identification of PSI–LHCI– structurally characterized. The physical association between LHCII complexes is consistent with recent findings that both CEF en- PSI–LHCI and cyt b6f was supported using single molecule hancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluores- Significance cence correlation spectroscopy indicates a physical association be- tween cyt b f and fluorescent chlorophyll containing PSI–LHCI super- 6 To optimize photosynthetic performance and minimize pho- complexes. Single particle analysis identified top-view projections of tooxidative damage, photosynthetic organisms evolved to ef- the corresponding PSI–LHCI–cyt b f supercomplex. Based on molecular 6 ficiently balance light energy absorption and electron transport modeling and mass spectrometry analyses, we propose a model in with cellular energy requirements under constantly changing which dissociation of LHCA2 and LHCA9 from PSI supports the forma- light conditions. The regulation of linear electron flow (LEF) tion of this CEF supercomplex. This is supported by the finding that and cyclic electron flow (CEF) contributes to this fine-tuning. a Δlhca2 knockout mutant has constitutively enhanced CEF. Here we present a model of the formation and structural molec- ular organization of a CEF-performing photosystem I (PSI)–light cyclic electron flow | supercomplex | photosystem I | cytochrome b f | 6 harvesting complex I (LHCI)–cytochrome (cyt) b f supercomplex Chlamydomonas reinhardtii 6 from the green alga Chlamydomonas reinhardtii.Suchastruc- tural arrangement could modulate the distinct operation of LEF hotosynthesis captures solar energy and stores it in the form and CEF to optimize light energy utilization, despite the same Pof chemical energy, which is essential to support life on individual structural units contributing to these two different Earth. Photosynthetic electron transport operates in two modes: functional modes. linear (LEF) and cyclic electron flow (CEF). LEF yields ATP and NADPH, while CEF exclusively drives ATP production (1). Author contributions: J.S., I.L.R., M.H., and B.H. designed research; J.S., I.L.R., P.G., and Fine-tuning LEF and CEF maintains the ATP/NADPH equi- N.G. performed research; H.S. and Y.T. contributed new reagents/analytic tools; J.S., I.L.R., R.R., P.G., S.S., R.A., R.D., E.S., Y.G., M.H., and B.H. analyzed data; and J.S., I.L.R., M.H., and librium and efficient carbon assimilation (2, 3). CEF also plays B.H. wrote the paper. an important role in photoprotection (4, 5) as it maintains the The authors declare no conflict of interest. Δ necessary pH across the thylakoid membrane to allow energy- This article is a PNAS Direct Submission. dependent nonphotochemical quenching and to control the rate Published under the PNAS license. limiting step of LEF (6). The dynamic tuning between LEF and 1Present address: Department of Biology, University of Pennsylvania, Philadelphia, CEF is therefore essential for efficient photosynthesis. PA 19104. LEF involves in-series activity of photosystem II (PSII), cyto- 2Present address: Centre for Microscopy and Microanalysis, University of Queensland, chrome (cyt) b6f, and photosystem I (PSI), while CEF involves St. Lucia, QLD 4072, Australia. only PSI and cyt b6f. During CEF, electrons released by PSI are 3To whom correspondence may be addressed. Email: [email protected] or reinjected into the photosynthetic electron transport chain at the [email protected]. plastoquinone (PQ) pool or at the stromal side of the cyt b6f This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. complex. The fact that LEF and CEF share many electron 1073/pnas.1809973115/-/DCSupplemental. transfer components (e.g., PSI and cyt b6f) raises the question of www.pnas.org/cgi/doi/10.1073/pnas.1809973115 PNAS Latest Articles | 1of6 Downloaded by guest on September 23, 2021 fluorescence (SMF) correlation spectroscopy. Immunoblot and A mass spectrometry (MS) analyses also clearly identified PSI, LHCI, cyt b6f as well as FNR, PGRL1, ANR1, and CAS. Their structural organization was characterized using crosslinking, MS, and single particle analysis (SPA). In Chlamydomonas PSI–LHCI, the LHCA2 and LHCA9 subunits are located at its PSAG/H side (11) similar to the recently resolved PSI structure of a red alga (12). Our CEF data suggest a dynamic dissociation/association model, in which LHCA2 and LHCA9 dissociate from PSI–LHCI enabling CEFsupercomplexformation. B Results Identification of a PSI–LHCI–cyt b6f Supercomplex. CEF super- complexes of C. reinhardtii were isolated from anaerobically cul- C tured cells (7, 8). Isolated thylakoid membranes were solubilized with n-dodecyl α-D-maltoside (α-DDM) and fractionated using SDG centrifugation (7, 8). Immunoblot analysis identified a high molecular weight SDG fraction containing the major CEF super- complex components PSI and cyt b6f(Fig.1B and C). Previous Fig. 2. Cytochrome b6f is physically associated with chlorophyll fluorescent work had demonstrated CEF activities in the same SDG fractions, proteins in the CEF supercomplex sucrose density region revealed by SMF but it remained possible that this was due to (i) colocalization of cyt coincidence analysis. (A) Coincident events, i.e., the simultaneous bursts of green and red fluorescence, indicative of the physical association of DyLight b6f and PSI in small residual membrane patches (since α-DDM is a mild detergent) or (ii) comigration of separate cyt b fandPSI 488-labeled cyt f and chlorophyll fluorescent proteins, are most abundant in 6 the CEF supercomplex region of the SDG. The frequency of coincident events supercomplexes on the SDG (e.g., due to the presence of other relative to total fluorescent events recorded over a period of 60 s is plotted molecular partners such as LHCII trimers, ATPase dimers, or for selected fractions over the corresponding SDG. A false positive rate of NDH). We therefore used SMF correlation spectroscopy (SI Ap- 5% was applied to exclude the possible random excitation of two single pendix,Figs.S1–S3) to demonstrate a single molecule, physical in- fluorescent proteins as experimentally examined previously (13). (B) Pooled – SI teraction between cyt b6fandPSILHCI complexes (Fig. 2 and CEF supercomplex fractions from five SDGs of a cyt f His6-tag strain were Appendix,Fig.S3;seeSI Appendix for more details). Total fluo- concentrated, labeled with DyLight 488–trisNTA,