Abstract
Photosynthetic light-dependent reactions occur in thylakoid membranes where embedded proteins capture light energy and convert it to chemical energy in the form of ATP and NADPH for use in carbon fixation. One of these integral membrane proteins is Photosystem I (PSI). PSI catalyzes light-driven transmembrane electron transfer from plastocyanin (Pc) to oxidized ferredoxin (Fd). Electrons from reduced Fd are used by the enzyme ferredoxin-NADP+ reductase (FNR) for the reduction of NADP+ to NADPH. Fd and Pc are both small soluble proteins whereas the larger FNR enzyme is associated with the membrane. To investigate electron shuttling between these diffusible and embedded proteins, thylakoid photoreduction of NADP+ was studied. As isolated, both spinach and cyanobacterial thylakoids generate NADPH upon illumination without extraneous addition of Fd. These findings indicate that isolated thylakoids either (i) retain a “pool” of Fd which diffuses between PSI and membrane bound FNR or (ii) that a fraction of PSI is associated with Fd, with the membrane environment facilitating PSI-Fd-FNR interactions which enable multiple turnovers of the complex with a single Fd. To explore the functional association of Fd with PSI in thylakoids, electron paramagnetic resonance (EPR) spectroscopic methodologies were developed to distinguish the signals for the reduced Fe-S clusters of PSI and Fd. Temperature-dependent EPR studies show that the EPR signals of the terminal [4Fe-4S] cluster of PSI can be distinguished from the [2Fe-2S] cluster of Fd at > 30 K. At 50 K, the cw X-band EPR spectra of cyanobacterial and spinach thylakoids reduced with dithionite exhibit EPR signals of a [2Fe-2S] cluster with g-values gx = 2.05, gy = 1.96, and gz = 1.89, confirming that Fd is present in thylakoid preparations capable of NADP+ photoreduction. Quantitation of the EPR signals of P700+ and dithionite reduced Fd reveal that Fd is present at a ratio of ~ 1 Fd per PSI monomer in both spinach and cyanobacterial thylakoids. Light-driven electron transfer from PSI to Fd in thylakoids confirms Fd is functionally associated (< 0.4 Fd/PSI) with the acceptor end of PSI in isolated cyanobacterial thylakoids. These EPR experiments provide a benchmark for future spectroscopic characterization of Fd interactions involved in multistep relay of electrons following PSI charge separation in the context of photosynthetic thylakoid microenvironments.
Similar content being viewed by others
Data availability
Data are available upon request from the authors.
References
Andersen B, Scheller HV, Moller BL (1992) The PSI-E subunit of photosystem I binds ferredoxin: NADP+ oxidoreductase. FEBS 311:169–173
Benz JP, Stengel A, Lintala M, Lee YH, Weber A, Philippar K, Gugel IL, Kaieda S, Ikegami T, Mulo P, Soll J, Bolter B (2009) Arabidopsis Tic62 and ferredoxin-NADP(H) oxidoreductase form light-regulated complexes that are integrated into the chloroplast redox poise. Plant Cell 21:3965–3993
Bergner SV, Scholz M, Trompelt K, Barth J, Gabelein P, Steinbeck J, Xue H, Clowez S, Fucile G, Goldschmidt-Clermont M, Fufezan C, Hippler M (2015) State transition-dependent phosphorylation is modulated by changing environmental conditions, and its absence trigger remodeling of photosynthetic protein complexes. Plant Physiol 168:615–634
Bohme H (1978) Quntitative determination of ferredoxin, ferredoxin-NADP+ reductase and plastocyanin in spinach chloroplasts. Eur J Biochem 83:137–141
Brahmachari U, Pokkuluri PR, Tiede DM, Niklas J, Poluektov OG, Mulfort KL, Utschig LM (2020) Interprotein electron transfer biohybrid system for photocatalytic H2 production. Photosyn Res 143:183–192
Breyton C, Nandha B, Johnson GN, Joliot P, Finazzi G (2006) Redox modulation of cyclic electron flow around photosystem I in C3 plants. Biochem 45:13465–13475
Bruns CM, Karplus PA (1995) Refined crystal structure of spinach ferredoxin reductase at 1.7 A resolution: oxidized, reduced, and 2′-phospho-5′-AMP bound states. J Mol Biol 247:125–145
Carrillo N, Vallejos RH (1982) Interaction of ferredoxin-NADP oxidoreductase with the thylakoid membrane. Plant Physiol 69:210–213
Caspy I, Borovikova-Sheinker A, Klaiman D, Shkolnisky Y, Nelson N (2020) The structure of a triple complex of plant photosystem I with ferredoxin and plastocyanin. Nat Plants 6:1300–1305
Clark RD, Hawkesford MJ, Coughlan SJ, Bennett J, Hind G (1984) Association of ferredoxin-NADP+ oxidoreductase with the chloroplast cythochrome B-F complex. FEBS Lett 174:137–142
Colvert KK, Davis DJ (1983) Effect of pH, salt, and coupling state on the interaction of ferredoxin with the chloroplast membrane. Arch Biochem Biophys 225:936–943
Forti G, Grubas PMG (1985) Two sites of interaction of ferredoxin with thylakoids. FEBS Lett 186(2):149–152
Forti G, Cappelletti A, Nobili RL, Garlaschi FM, Gerola PD, Jennings RC (1983) Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids. Arch Biochem Biophys 221:507–513
Gisriel CJ, Flesher DA, Shen G, Wang J, Ho MH, Brudvig GW, Bryant DA (2021) Structure of a photosystem I-ferredoxin complex from a marine cyanobacterium provides insights into far-red light photoacclimation. J Biol Chem 298:101408
Gomez-Lojero C, Perez-Gomez B, Shen G, Schluhter WM, Bryant DA (2003) Interaction of ferredoxin: NADP+ oxidoreductase with phycobilisoms and phycobilisome substructres of the cyanobacterium Synechococcus sp. strain PCC 7002. Biochemistry 42:13800–13811
Guedeney G, Corneille S, Cuine S, Peltier G (1996) Evidence for an association of ndh B, ndh J gene products and ferredoxin-NADP-reductase as components of a chloroplastic NAD(P)H dehydrogenase complex. FEBS Lett 378:277–280
Hanke G, Mulo P (2013) Plant type ferredoxins and ferredoxin-dependent metabolism. Plant, Cell Environ 36:1071–1084
Hanke GT, Okutani S, Satomi Y, Takae T, Suzuki A, Hase T (2005) Multiple iso-proteins of FNR in Arabidopsis: evidence for different contributions to chloroplast function and nitrogen assimilation. Plant, Cell Environ 28:1146–1157
Iwai M, Takizawa K, Tokutsu R, Okamuro A, Takahashi Y, Minagawa J (2010) Isolation of the elusive supercomplex that drives cyclic electron flow in photosynthesis. Nature 464(7292):1210-U1134
Joliot P, Johnson GN (2011) Regulation of cyclic and linear electron flow in higher plants. Proc Natl Acad Sci USA 108:13317–13322
Juric S, Hazler-Pilepic K, Tomasic A, Lepedus H, Jelicic B, Puthiyaveetil S, Bionda T, Vojta L, Allen JF, Schleiff E, Fulgosi H (2009) Tethering of ferredoxin: NADP+ oxidoreductase to thylakoid membranes is mediated by novel chloroplast protein TROL. Plant J 60:783–794
Korn A, Ajlai G, Lagoutte B, Gall A, Setif P (2009) Ferredoxin: NADP+ oxidoreductase association with phycocyanin modulates its properties. J Biol Chem 284:31789–31797
Kramer M, Rodriguez-Heredia M, Saccon F, Mosebach L, Twachtmann M, Krieger-Liszkay A, Duffy C, Knell RJ, Finazzi G, Hanke GT (2021) Regulation of photosynthetic electron flow on dark to light transition by ferredoxin: NADP(H) oxidoreductase interactions. eLife 10:e56088
Lelong C, Setif P, Lagoutte B, Bottin H (1994) Identification of the amino-acids involved in the functional interaction between Photosystem-I and ferredoxin from Synechocystis Sp-Pcc-6803 by chemical cross-linking. J Biol Chem 269(13):10034–10039
Li F, Wei X, Zhang L, Liu C, You C, Zhu Z (2022) Installing a green engine to drive an enzyme cascade: a light-powered in vitro biosystem for poly(3-hydroxybutyrate) synthesis. Angew Chem Int Ed 61:e202111054
Liu LN (2016) Distribution and dynamics of electron transport complexes in cyanobacterial thylakoid membranes. Biochim Biophys Acta Bioenerg 1857(3):256–265
Liu H, Weisz DA, Zhang MM, Cheng M, Zhang B, Zhang H, Gerstenecker GS, Pakrasi HB, Gross ML, Blankenship RE (2019) Phycobilisomes Harbor FNRL in Cyanobacteria. Mbio 10:e00669-e619
Marco P, Elman T, Yacoby I (2019) Binding of ferredoxin NADP+ oxidoreductase (FNR) to plant photosystem I. Biochim Biophys Acta Bioenerg 1860:689–698
Matthijs HCP, Coughlan SJ, Hind G (1986) Removal of ferredoxin: NADP+ oxidoreductase from thylakoid membranes, rebinding to depleted membranes, and identification of the binding site. J Biol Chem 261:12154–12158
Medina M (2009) Structural and mechanistic aspects of flavoproteins: photosynthetic electron transfer from photosystem I to NADP+. FEBS J 276(15):3942–3958
Miller TE, Beneyton T, Schwander T, Diehl C, Girault M, McLean R, Chotel T, Claus P, Cortina NS, Baret J-C, Erb TJ (2020) Light-powered CO2 fixation in a chloroplast mimic with natural and synthetic parts. Science 368:649–654
Moal G, Lagoutte B (2012) Photo-induced electron transfer from photosystem I to NADP+: characterization and tentative simulation of the in vivo environment. Biochim Biophys Acta 1817:1635–1645
Mosebach L, Heilmann C, Mutoh R, Gabelein P, Steinbeck J, Happe T, Ikegami T, Hanke G, Kurisu G, Hippler M (2017) Association of ferredoxin: NADP+ oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii. Photosyn Res 134:291–306
Mulo P (2011) Chloroplast-targeted ferredoxin-NADP+ oxidoreductase (FNR): structure, function and location. Biochim Biophys Acta 1807:927–934
Mulo P, Medina M (2017) Interaction and electron transfer between ferredoxin-NADP+ oxidoreductase and its partners: structural, functional, and physiological implications. Photosyn Res 134:265–280
Mustardy L, Buttle K, Steinbach G, Garab G (2008) The three-dimensional network of the thylakoid membranes in plants: quasihlical model of the granum-stroma assembly. Plant Cell 20:2552–2557
Nawrocki WJ, Bailleul B, Pico D, Cardol P, Rappaport F, Wollman FA, Joliot P (2019) The mechanism of cyclic electron flow. Biochim Biophys Acta Bioenerg 1860:433–438
Okutani S, Hanke GT, Satomi Y, Takae T, Kurisu G, Suzuki A, Hase T (2005) Three maize leaf ferredoxin: NADPH oxidoreductases vary in subchloroplast location, expression, and interaction with ferredoxin. Plant Physiol 139:1451–1459
Omairi-Nasser A, de Gracia AG, Ajlani G (2011) A larger transcript is required for the synthesis of the smaller isoform of ferredoxin: NADP oxidoreductase. Mol Microbiol 81:1178–1189
Quiles MJ, Cuello J (1998) Association of ferredoxin-NADP oxidoreductase with the chloroplastic pyridine nucleotide dehydrogenase complex in barley leaves. Plant Physiol 117:235–244
Rupp H, Rao KK, Hall DO, Cammack R (1978) Electron spin relaxation of iron-sulfur proteins studied by microwave power saturation. Biochim Biophys Acta 537:255–269
Schluchter WM, Bryant DA (1992) Molecular characterization of Ferredoxin-NADP+ oxidoreductase in cyanobacteria: cloning and sequence of the petH gene of Synechococcus sp. PCC 7002 and studies on the gene product. Biochem 31:3092–3102
Serre L, Vellieux FMD, Medina M, Gomez-Morena C, Fontecilla-Camps JC, Frey M (1996) X-ray structure of the ferredoxin: NADP+ reductase from the cyanobacterium Anabaena PCC 7119 at 1.8 A resolution, and the crystallographic studies of NADP+ binding at 2.25 A resolution. J Mol Biol 263:20–39
Takahashi H, Okamuro A, Minagawa J, Takahashi Y (2014) Biochemical characterization of photosystem I-associated light-harvesting complexes I and II isolated from state 2 cells of Chlamydomonas reinhardtii. Plant Cell Physiol 55:1437–1449
Thomas JC, Ughy B, Lagoutte B, Ajlani G (2006) A second isoform of the ferredoxin: NADP oxidoreductase generated by an in-frame initiation of translation. Proc Natl Acad Sci USA 103:18368–18373
Tong X, Kim E-J, Lee JK (2022) Sustainability of in vitro light-dependent NADPH generation by the thylakoid membrane of Synechocystis sp. PCC6803. Microbial Cell Factories 21:94
Utschig LM, Silver SC, Mulfort KL, Tiede DM (2011) Nature-driven photochemistry for catalytic solar hydrogen production: A Photosystem I-transition metal catalyst hybrid. J Am Chem Soc 133(41):16334–16337
Utschig LM, Soltau SR, Mulfort KL, Niklas J, Poluektov O (2018) Z-scheme solar water splitting via self-assembly of photosystem I-catalyst hybrids in thylakoid membranes. Chem Sci 9:8504–8512
Utschig LM, Brahmachari U, Mulfort KL, Niklas J, Poluektov OG (2022) Biohybrid photosynthetic charge accumulation detected by flavin semiquinone formation in ferredoxin-NADP+ reductase. Chem Sci 13:18368–18373
Utschig LM, Zaluzec NJ, Malavath T, Ponomarenko NS, Tiede DM (2023) Solar water splitting Pt-nanoparticle photosystem I thylakoid systems: catalyst identification, location and oligomeric structure. Biochim Biophys Acta Bioenerg 1864:148974
van Thor JJ, Geerlings TH, Matthijs HCP, Hellingwerf KJ (1999a) Kinetic evidence for the PsaE-dependent transient ternary complex photosystem I/ferredoxin/ferredoxin: NADP+ reductase in a cyanobacterium. Biochem 38:12735–12746
van Thor JJ, Gruters OW, Matthijs HC, Hellingwerf KJ (1999b) Localization and function of ferredoxin: NADP+ reductase bound to the phycobilisomes of Synechocystis. EMBO J 18:4128–4136
Vassiliev IR, Antonkine ML, Golbeck JH (2001) Iron-sulfur clusters in Type I reaction centers. Biochim Biophys Acta 1507:139–160
Zhang H, Whitelegge JP, Cramer WA (2001) Ferredoxin: NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex. J Biol Chem 276:38159–38165
Acknowledgements
The authors thank A. Wagner for growth of the cyanobacteria and J. Bindra for assistance with EPR spectra collection. This work is supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, Biosciences, as well as the Photon Sciences Division, under Contract No. DE-AC02-06CH11357.
Author information
Authors and Affiliations
Contributions
LMU conceived the project. LMU and CLD prepared the thylakoids and conducted all the biochemistry experiments. LMU prepared the EPR samples. JN and OGP performed the EPR experiments and analysis. LMU wrote the manuscript. All authors discussed the results and contributed to manuscript editing.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Statement of informed consent, human/animal rights
No conflicts, informed consent, human or animal rights are applicable.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Utschig, L.M., Duckworth, C.L., Niklas, J. et al. EPR studies of ferredoxin in spinach and cyanobacterial thylakoids related to photosystem I-driven NADP+ reduction. Photosynth Res (2024). https://doi.org/10.1007/s11120-023-01072-4
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11120-023-01072-4