Abstract
The absolute amount of plastocyanin (PC), ferredoxin-NADP+-oxidoreductase (FNR), hydrogenase (HYDA1), and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standards in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 1.4:1.2:0.003:0. In anaerobic cells, the ratios changed to 1.1:1.3:0.019:0.027 (PC:FNR:HYDA1:FDX5). Employing sodium dithionite and methyl viologen as electron donors, the specific activity of hydrogenase in whole cells was calculated to be 382 ± 96.5 μmolH2 min−1 mg−1. Importantly, these data reveal an about 70-fold lower abundance of HYDA1 compared to FNR. Despite this great disproportion between both proteins, which might further enhance the competition for electrons, the alga is capable of hydrogen production under anaerobic conditions, thus pointing to an efficient channeling mechanism of electrons from FDX1 to the HYDA1.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andersen B, Scheller HV, Moller BL (1992) The PSI-E subunit of photosystem I binds ferredoxin:NADP + oxidoreductase. FEBS Lett 311(2):169–173
Böhme H (1978) Quantitative determination of ferredoxin, ferredoxin-NADP + reductase and plastocyanin in spinach chloroplasts. Eur J Biochem 83:137–141
Bruska MK, Stiebritz MT, Reiher M (2011) Regioselectivity of H cluster oxidation. J Am Chem Soc 133(50):20588–20603. https://doi.org/10.1021/ja209165r
Burkey KO, Gizlice Z, Carter TE Jr (1996) Genetic variation in soybean photosynthetic electron transport capacity is related to plastocyanin concentration in the chloroplast. Photosynth Res 49(2):141–149. https://doi.org/10.1007/BF00117664
Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25(9):1327–1333. https://doi.org/10.1002/elps.200305844
Chambers MC, Maclean B, Burke R, Amodei D, Ruderman DL, Neumann S, Gatto L, Fischer B, Pratt B, Egertson J, Hoff K, Kessner D, Tasman N, Shulman N, Frewen B, Baker TA, Brusniak MY, Paulse C, Creasy D, Flashner L, Kani K, Moulding C, Seymour SL, Nuwaysir LM, Lefebvre B, Kuhlmann F, Roark J, Rainer P, Detlev S, Hemenway T, Huhmer A, Langridge J, Connolly B, Chadick T, Holly K, Eckels J, Deutsch EW, Moritz RL, Katz JE, Agus DB, MacCoss M, Tabb DL, Mallick P (2012) A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol 30(10):918–920. https://doi.org/10.1038/nbt.2377
Craig R, Beavis RC (2004) TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20(9):1466–1467. https://doi.org/10.1093/bioinformatics/bth092
Deutsch EW, Csordas A, Sun Z, Jarnuczak A, Perez-Riverol Y, Ternent T, Campbell DS, Bernal-Llinares M, Okuda S, Kawano S, Moritz RL, Carver JJ, Wang M, Ishihama Y, Bandeira N, Hermjakob H, Vizcaino JA (2017) The ProteomeXchange consortium in 2017: supporting the cultural change in proteomics public data deposition. Nucleic Acids Res 45(D1):D1100–D1106. https://doi.org/10.1093/nar/gkw936
Eilenberg H, Weiner I, Ben-Zvi O, Pundak C, Marmari A, Liran O, Wecker MS, Milrad Y, Yacoby I (2016) The dual effect of a ferredoxin-hydrogenase fusion protein in vivo: successful divergence of the photosynthetic electron flux towards hydrogen production and elevated oxygen tolerance. Biotechnol Biofuels 9(1):182. https://doi.org/10.1186/s13068-016-0601-3
Eriksson M, Moseley JL, Tottey S, Del Campo JA, Quinn J, Kim Y, Merchant S (2004) Genetic dissection of nutritional copper signaling in chlamydomonas distinguishes regulatory and target genes. Genetics 168(2):795–807. https://doi.org/10.1534/genetics.104.030460
Finkelmann AR, Stiebritz MT, Reiher M (2014) Activation barriers of oxygen transformation at the active site of [FeFe] hydrogenases. Inorg Chem 53(22):11890–11902. https://doi.org/10.1021/ic501049z
Florin L, Tsokoglou A, Happe T (2001) A novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain. J Biol Chem 276(9):6125–6132. https://doi.org/10.1074/jbc.M008470200
Forestier M, King P, Zhang L, Posewitz M, Schwarzer S, Happe T, Ghirardi ML, Seibert M (2003) Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions. Eur J Biochem 270(13):2750–2758
Geer LY, Markey SP, Kowalak JA, Wagner L, Xu M, Maynard DM, Yang X, Shi W, Bryant SH (2004) Open mass spectrometry search algorithm. J Proteome Res 3(5):958–964. https://doi.org/10.1021/pr0499491
Gerber SA, Rush J, Stemman O, Kirschner MW, Gygi SP (2003) Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc Natl Acad Sci USA 100(12):6940–6945. https://doi.org/10.1073/pnas.0832254100
Ghirardi ML(2015) Implementation of photobiological H2 production: the O2 sensitivity of hydrogenases. Photosynth Res 125(3):383–393. https://doi.org/10.1007/s11120-015-0158-1
Ghirardi ML, Togasaki RK, Seibert M (1997) Oxygen sensitivity of algal H2- production. Appl Biochem Biotechnol 63–65:141–151. https://doi.org/10.1007/BF02920420
Girbal L, von Abendroth G, Winkler M, Benton PM, Meynial-Salles I, Croux C, Peters JW, Happe T, Soucaille P (2005) Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl Environ Microbiol 71(5):2777–2781. https://doi.org/10.1128/AEM.71.5.2777-2781.2005
Godaux D, Bailleul B, Berne N, Cardol P (2015) Induction of photosynthetic carbon fixation in anoxia relies on hydrogenase activity and proton-gradient regulation-like1-mediated cyclic electron flow in Chlamydomonas reinhardtii. Plant Physiol 168(2):648–658. https://doi.org/10.1104/pp.15.00105
Graan T, Ort DR (1984) Quantitation of the rapid electron donors to P700, the functional plastoquinone pool, and the ratio of the photosystems in spinach chloroplasts. J Biol Chem 259(22):14003–14010
Happe T, Kaminski A (2002) Differential regulation of the Fe-hydrogenase during anaerobic adaptation in the green alga Chlamydomonas reinhardtii. Eur J Biochem 269(3):1022–1032
Happe T, Naber JD (1993) Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii. Eur J Biochem 214(2):475–481
Harris EH (1989) The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. Academic Press, San Diego
Havlis J, Shevchenko A (2004) Absolute quantification of proteins in solutions and in polyacrylamide gels by mass spectrometry. Anal Chem 76(11):3029–3036. https://doi.org/10.1021/ac035286f
Hemschemeier A, Happe T (2011) Alternative photosynthetic electron transport pathways during anaerobiosis in the green alga Chlamydomonas reinhardtii. Biochim Biophys Acta 1807(8):919–926. https://doi.org/10.1016/j.bbabio.2011.02.010
Hippler M, Drepper F, Farah J, Rochaix JD (1997) Fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii requires PsaF. Biochemistry 36(21):6343–6349. https://doi.org/10.1021/bi970082c
Hippler M, Klein J, Fink A, Allinger T, Hoerth P (2001) Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii. Plant J 28(5):595–606
Hong G, Pachter R (2012) Inhibition of biocatalysis in [Fe-Fe] hydrogenase by oxygen: molecular dynamics and density functional theory calculations. ACS Chem Biol 7(7):1268–1275. https://doi.org/10.1021/cb3001149
Hulsker R, Mery A, Thomassen EA, Ranieri A, Sola M, Verbeet MP, Kohzuma T, Ubbink M (2007) Protonation of a histidine copper ligand in fern plastocyanin. J Am Chem Soc 129(14):4423–4429. https://doi.org/10.1021/ja0690464
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):1210–1213. https://doi.org/10.1038/nature08885
Jacobs J, Pudollek S, Hemschemeier A, Happe T (2009) A novel, anaerobically induced ferredoxin in Chlamydomonas reinhardtii. FEBS Lett 583(2):325–329. https://doi.org/10.1016/j.febslet.2008.12.018
Joliot P, Delosme R (1974) Flash-induced 519 nm absorption change in green algae. Biochim Biophys Acta 357(2):267–284
Kamp C, Silakov A, Winkler M, Reijerse EJ, Lubitz W, Happe T (2008) Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae. Biochim Biophys Acta 1777(5):410–416. https://doi.org/10.1016/j.bbabio.2008.02.002
Katoh S (1977) Encyclopedia of plant physiology,” new series. In: Trebst A, Avron M (eds) Photosynthesis I, Vol. 5, Springer, Berlin, pp 247–252
Kirkpatrick DS, Gerber SA, Gygi SP (2005) The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods 35(3):265–273. https://doi.org/10.1016/j.ymeth.2004.08.018
Kubas A, Orain C, De Sancho D, Saujet L, Sensi M, Gauquelin C, Meynial-Salles I, Soucaille P, Bottin H, Baffert C, Fourmond V, Best RB, Blumberger J, Leger C (2017) Mechanism of O2 diffusion and reduction in FeFe hydrogenases. Nat Chem 9(1):88–95. https://doi.org/10.1038/nchem.2592
Kuhlgert S, Drepper F, Fufezan C, Sommer F, Hippler M (2012) Residues PsaB Asp612 and PsaB Glu613 of photosystem I confer pH-dependent binding of plastocyanin and cytochrome c(6). Biochemistry 51(37):7297–7303. https://doi.org/10.1021/bi300898j
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Lambertz C, Hemschemeier A, Happe T (2010) Anaerobic expression of the ferredoxin-encoding FDX5 gene of Chlamydomonas reinhardtii is regulated by the Crr1 transcription factor. Eukaryot Cell 9(11):1747–1754. https://doi.org/10.1128/EC.00127-10
Lambertz C, Leidel N, Havelius KG, Noth J, Chernev P, Winkler M, Happe T, Haumann M (2011) O2 reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase. J Biol Chem 286(47):40614–40623. https://doi.org/10.1074/jbc.M111.283648
Lien S, Pietro AS (1979) Interaction of plastocyanin and P700 in PSI reaction centre particles from Chlamydomonas reinhardtii and spinach. Arch Biochem Biophys 194(No.1):128–137
Liran O, Semyatich R, Milrad Y, Eilenberg H, Weiner I, Yacoby I (2016) Microoxic Niches within the thylakoid stroma of air-grown Chlamydomonas reinhardtii protect [FeFe]-hydrogenase and support hydrogen production under fully aerobic environment. Plant Physiol 172(1):264–271. https://doi.org/10.1104/pp.16.01063
Melis A, Happe T (2001) Hydrogen production. Green algae as a source of energy. Plant Physiol 127(3):740–748
Melis A, Zhang L, Forestier M, Ghirardi ML, Seibert M (2000) Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii. Plant Physiol 122(1):127–136
Meuser JE, D’Adamo S, Jinkerson RE, Mus F, Yang W, Ghirardi ML, Seibert M, Grossman AR, Posewitz MC (2012) Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: insight into the role of HYDA2 in H(2) production. Biochem Biophys Res Commun 417(2):704–709. https://doi.org/10.1016/j.bbrc.2011.12.002
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. Photosynth Res. https://doi.org/10.1007/s11120-017-0408-5
Mulder DW, Ortillo DO, Gardenghi DJ, Naumov AV, Ruebush SS, Szilagyi RK, Huynh B, Broderick JB, Peters JW (2009) Activation of HydA(DeltaEFG) requires a preformed [4Fe-4S] cluster. Biochemistry 48(26):6240–6248. https://doi.org/10.1021/bi9000563
Mulder DW, Boyd ES, Sarma R, Lange RK, Endrizzi JA, Broderick JB, Peters JW (2010) Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA(DeltaEFG). Nature 465(7295):248–251. https://doi.org/10.1038/nature08993
Mus F, Cournac L, Cardettini V, Caruana A, Peltier G (2005) Inhibitor studies on non-photochemical plastoquinone reduction and H(2) photoproduction in Chlamydomonas reinhardtii. Biochim Biophys Acta 1708(3):322–332. https://doi.org/10.1016/j.bbabio.2005.05.003
Mus F, Dubini A, Seibert M, Posewitz MC, Grossman AR (2007) Anaerobic acclimation in Chlamydomonas reinhardtii: anoxic gene expression, hydrogenase induction, and metabolic pathways. J Biol Chem 282(35):25475–25486. https://doi.org/10.1074/jbc.M701415200
Nawrocki WJ, Santabarbara S, Mosebach L, Wollman FA, Rappaport F (2016) State transitions redistribute rather than dissipate energy between the two photosystems in Chlamydomonas. Nat Plants 2:16031. https://doi.org/10.1038/nplants.2016.31
Onda Y, Matsumura T, Kimata-Ariga Y, Sakakibara H, Sugiyama T, Hase T (2000) Differential interaction of maize root ferredoxin:NADP(+) oxidoreductase with photosynthetic and non-photosynthetic ferredoxin isoproteins. Plant Physiol 123(3):1037–1046
Ong SE, Mann M (2005) Mass spectrometry-based proteomics turns quantitative. Nat Chem Biol 1(5):252–262. https://doi.org/10.1038/nchembio736
Peden EA, Boehm M, Mulder DW, Davis R, Old WM, King PW, Ghirardi ML, Dubini A (2013) Identification of global ferredoxin interaction networks in Chlamydomonas reinhardtii. J Biol Chem 288(49):35192–35209. https://doi.org/10.1074/jbc.M113.483727
Peters JW, Lanzilotta WN, Lemon BJ, Seefeldt LC (1998) X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282(5395):1853–1858
Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975(3):384–394. https://doi.org/10.1016/S0005-2728(89)80347-0
Quinn JM, Barraco P, Eriksson M, Merchant S (2000) Coordinate copper- and oxygen-responsive Cyc6 and Cpx1 expression in Chlamydomonas is mediated by the same element. J Biol Chem 275(9):6080–6089
Quinn JM, Eriksson M, Moseley JL, Merchant S (2002) Oxygen deficiency responsive gene expression in Chlamydomonas reinhardtii through a copper-sensing signal transduction pathway. Plant Physiol 128(2):463–471. https://doi.org/10.1104/pp.010694
Roessler PG, Lien S (1984a) Activation and de novo synthesis of hydrogenase in chlamydomonas. Plant Physiol 76(4):1086–1089
Roessler PG, Lien S (1984b) Purification of hydrogenase from Chlamydomonas reinhardtii. Plant Physiol 75(3):705–709
Rupprecht J, Hankamer B, Mussgnug JH, Ananyev G, Dismukes C, Kruse O (2006) Perspectives and advances of biological H2 production in microorganisms. Appl Microbiol Biotechnol 72(3):442–449. https://doi.org/10.1007/s00253-006-0528-x
Sawyer A, Winkler M (2017) Evolution of Chlamydomonas reinhardtii ferredoxins and their interactions with [FeFe]-hydrogenases. Photosynth Res. https://doi.org/10.1007/s11120-017-0409-4
Sawyer A, Bai Y, Lu Y, Hemschemeier A, Happe T (2017) Compartmentalisation of [FeFe]-hydrogenase maturation in Chlamydomonas reinhardtii. Plant J 90(6):1134–1143. https://doi.org/10.1111/tpj.13535
Schottler MA, Kirchhoff H, Weis E (2004) The role of plastocyanin in the adjustment of the photosynthetic electron transport to the carbon metabolism in tobacco. Plant Physiol 136(4):4265–4274. https://doi.org/10.1104/pp.104.052324
Steinbeck J, Nikolova D, Weingarten R, Johnson X, Richaud P, Peltier G, Hermann M, Magneschi L, Hippler M (2015) Deletion of proton gradient regulation 5 (PGR5) and PGR5-Like 1 (PGRL1) proteins promote sustainable light-driven hydrogen production in Chlamydomonas reinhardtii due to increased PSII activity under sulfur deprivation. Front Plant Sci 6:892. https://doi.org/10.3389/fpls.2015.00892
Stripp ST, Goldet G, Brandmayr C, Sanganas O, Vincent KA, Haumann M, Armstrong FA, Happe T (2009) How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms. Proc Natl Acad Sci USA 106(41):17331–17336. https://doi.org/10.1073/pnas.0905343106
Swanson KD, Ratzloff MW, Mulder DW, Artz JH, Ghose S, Hoffman A, White S, Zadvornyy OA, Broderick JB, Bothner B, King PW, Peters JW (2015) [FeFe]-hydrogenase oxygen inactivation is initiated at the H cluster 2Fe subcluster. J Am Chem Soc 137(5):1809–1816. https://doi.org/10.1021/ja510169s
Terashima M, Specht M, Naumann B, Hippler M (2010) Characterizing the anaerobic response of Chlamydomonas reinhardtii by quantitative proteomics. Mol Cell Proteomics 9(7):1514–1532. https://doi.org/10.1074/mcp.M900421-MCP200
Terauchi AM, Lu SF, Zaffagnini M, Tappa S, Hirasawa M, Tripathy JN, Knaff DB, Farmer PJ, Lemaire SD, Hase T, Merchant SS (2009) Pattern of expression and substrate specificity of chloroplast ferredoxins from Chlamydomonas reinhardtii. J Biol Chem 284(38):25867–25878. https://doi.org/10.1074/jbc.M109.023622
Vizcaino JA, Csordas A, Del-Toro N, Dianes JA, Griss J, Lavidas I, Mayer G, Perez-Riverol Y, Reisinger F, Ternent T, Xu QW, Wang R, Hermjakob H (2016) 2016 update of the PRIDE database and its related tools. Nucleic Acids Res 44(22):11033. https://doi.org/10.1093/nar/gkw880
Volgusheva A, Styring S, Mamedov F (2013) Increased photosystem II stability promotes H2 production in sulfur-deprived Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 110(18):7223–7228. https://doi.org/10.1073/pnas.1220645110
Winkler M, Kuhlgert S, Hippler M, Happe T (2009) Characterization of the key step for light-driven hydrogen evolution in green algae. J Biol Chem 284(52):36620–36627. https://doi.org/10.1074/jbc.M109.053496
Yacoby I, Pochekailov S, Toporik H, Ghirardi ML, King PW, Zhang S (2011) Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro. Proc Natl Acad Sci USA 108(23):9396–9401. https://doi.org/10.1073/pnas.1103659108
Yacoby I, Tegler LT, Pochekailov S, Zhang S, King PW, Riggs PD (2012) Optimized expression and purification for high-activity preparations of Algal [FeFe]-Hydrogenase. PLoS ONE 7(4):e35886
Yang W, Wittkopp TM, Li X, Warakanont J, Dubini A, Catalanotti C, Kim RG, Nowack EC, Mackinder LC, Aksoy M, Page MD, D’Adamo S, Saroussi S, Heinnickel M, Johnson X, Richaud P, Alric J, Boehm M, Jonikas MC, Benning C, Merchant SS, Posewitz MC, Grossman AR (2015) Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism. Proc Natl Acad Sci USA 112(48):14978–14983. https://doi.org/10.1073/pnas.1515240112
Zalutskaya Z, Minaeva E, Filina V, Ostroukhova M, Ermilova E (2017) Regulation of sulfur deprivation-induced expression of the ferredoxin-encoding FDX5 gene Chlamydomonas reinhardtii in aerobic conditions. Plant Physiol Biochem 123:18–23. https://doi.org/10.1016/j.plaphy.2017.11.024
Acknowledgements
We are grateful to Prof. Dr. Thomas Happe from the University of Bochum for providing the plasmid pJJ18. M.H. acknowledges support from the German Science Foundation (DFG, HI 739/13-1).
Funding
This study was funded by the German Science Foundation (DFG, HI 739/13-1).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Nikolova, D., Heilmann, C., Hawat, S. et al. Absolute quantification of selected photosynthetic electron transfer proteins in Chlamydomonas reinhardtii in the presence and absence of oxygen. Photosynth Res 137, 281–293 (2018). https://doi.org/10.1007/s11120-018-0502-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11120-018-0502-3