Nitrogen cycle (I): biological processes

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1 Nitrogen Fixation and Regulation of Photosynthesis for Nitrogen Fixation Hendrik Küpper, Advanced Course on Bioinorganic Chemistry & Biophysics of Plants, summer semester 2012

2 Part I: Nitrogen fixation

3 Nitrogen cycle (I): biological processes Im Wasser An Land von: von:

4 Nitrogen cycle (II): intermediate steps NO 3-3 Nitrite oxidase Nitrate reductase NO 2 - NO H 2 NOH Anammox N 2 O N 2 NH 3

5 Organisms involved in nitrogen fixation Anabaena Trichodesmium Gloeothece Azotobacter Azolla Rhizobien

6 Total equation of biological nitrogen fixation N 2 + 8H MgATP + 8e - 2NH 3 +H 2 +16MgADP+16P + P i

7 Mechanism of biological nitrogen fixation: nitrogenase of cyanobacteria

8 The iron protein

9 Nitrogenase: P-Cluster

10 Nitrogenase: M-Cluster Structure of Mayer and Smith, 1999, 1.6 Å resolution: discovery of the "central nitrogen", meanwhile re-identified as central carbon (Spatzal Thomas; Aksoyoglu Muege; Zhang Limei; et al. 2011, Science 334; Lancaster Kyle M.; Roemelt Michael; Ettenhuber Patrick; et al., 2011, Science 334)

11 Nitrogenase: Mechanism of nitrogen reduction

12 Nitrogenase: putative intermediates of nitrogen reduction Barney B_et al (2006) Breaking the N 2 triple bond- insights into the nitrogenase mechanism. DaltonTransactions

13 Nitrogenase: central C Spatzal T et al et Einsle O (2011) Science334, 940- Lancaster KM_et al et Neese F et DeBeer S (2011) Science334_974-

14 Nitrogenase: putative intermediates of nitrogen reduction

15 Part II: Regulation of photosynthesis for nitrogen fixation

16 Evolution of biological nitrogen fixation in comparison to photosynthesis Berman-Frank I_Lundgren P_Falkowski P_2003_Research in Microbiology154_

17 Strategies of photosynthesis regulation for nitrogen fixation in cyanobacteria Berman-Frank I_Lundgren P_Falkowski P_2003_Research in Microbiology154_

18 Unicellular cyanobacteria

19 Regulation of photosynthesis for nitrogen fixation in unicellular cyanobacteria (II) glycogen storage begins max. photosynthetic capacity light period: carbon + energy storage down regulation of photosynthesis up regulation of photosynthesis maximum glycogen storage nitrogen fixation begins dark period: nitrogen fixation i it fi ti maximum nitrogen fixation maximum respiration

20 Heterocyst forming cyanobacteria vegetative Zellen Pro-Heterocyste Heterocyste From: Culture service of Instituto de Bioquímica Vegetal y Fotosíntesis, Sevilla, Spain From:El-Shehawy et al 2003 Physiol Plant 119 (1), 49-55

21 Heterocyst differentiation: distribution ib ti maps of chlorophyll h ll fluorescence kinetic parameters Maximal fluorescence quantum yield (F m ) 25 µm 13 h 36 h 55 h 112 h Photosystem II activity (F v /F m ) Ferimazova N, Setlík I, Küpper H, unpublished

22 Heterocyst differentiation: changes in parameters of chlorophyll fluorescence kinetics Ferimazova N, Setlík I, Küpper H, unpublished

23 Trichodesmium: anoxygenic photosynthesis th energizing i nitrogen fixation in the same cells during the photoperiod

24 Trichodesmium Marine filamentous, non-heterocystous cyanobacteria contribution of Trichodesmium to marine N 2 fixation: 30-50% Nitrogen fixation is confined to the photoperiod p and occurs simultaneously with oxygenic photosynthesis. How nitrogenase is protected from damage by photosynthetically produced O 2 has remained an enigma. Surface blooms in the Arafura Sea Colonies tuft and puff formation

25 Trichodesmiumbloom Trichodesmium Relative F v/ F m Aktivitätszyklus von Trichodesmium Nitrogen fix xation (nmol C2H2 µg c hl a -1 h -1 ) colonies: Tuft and Puff GP (µm O2 µg chl a -1 h -1 ) Local time (h) Da ark respirat tion O2 µg chl a -1 h -1 ) (µm Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294,

26 Co-Localisation of nitrogenase and PSII in Trichodesmium D1 protein (green) and Nitrogenase (rot). Big picture: Overlay, small picture: only nitrogenase (Immunostain) Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294,

27 Inhibitor-Tests: Need of PSII-activity for nitrogen fixation in Trichodesmium Influence of DCMU (10 µm), ascorbic acid (100 µm), and DTT (100 µm) were tested for cultures incubated under aerobic (white columns) and anaerobic (blue columns) conditions. Changes in nitrogenase activity as measured by acetylene reduction. Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294,

28 Proof of Mehler-reaction during nitrogen fixation Staining with DAB (Diaminobenzochinon) shows intracellular distribution of H 2 O 2 as brown stain in all cells Mehler-reaction: reaction: H 2 O + 2O 2 --> H 2 O 2 +O 2 Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294,

29 Diurnal cycle of activity: Distribution of F 0 values cal cells) 10 low F 0 normal bright type I "very bright" = bright type II neighbour ring identi 5 Non-diazotrophic period groups of (cells or g 0 10 Diazotrophic period Number of objects F 0 Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135,

30 Diurnal cycle of activity: correlation between bright cells, pigment content and nitrogenase activity ctivity duction) ogenase a hylene red Nitro ((ml et s % Bright cell centration n (µm) Chl con min -1 ) culture) -1..(ml Nitrogenase activity % bright cells Chl :00 09:00 12:00 15:00 18:00 21:00 00:00 control 5% oxygen 50% oxygen Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135,

31 PSII-activity Trichodesmium: reversibility of changes in fluorescence yield Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135,

32 Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135,

33 Hypothesis about the regulation of photosynthesis for nitrogen fixation in Trichodesmium Licht initiates photosynthesis Reduction of the PQ pool Induction of a state 2 --> state 1-transition cells have a high h F 0 ( bright cells type I ) due to surplus phycobiliproteins Energy and reduction equivalent for CO 2 - assimilation Stimulation of pseudocyclic electron transport High respiration rates in early light period Oxygen consumption exceeds oxygen production: Opportunity for nitrogen fixation Carbohydrates are consumed Respiration decreases, transition to the normal F 0 inactive or quenching state Intracellular oxygen rises Nitrogenase becomes inhibited, no further nitrogen fixation until the following day, cells return to "normal F 0 active" state

34 Purification of Trichodesmium phycobiliproteins for deconvoluting spectrally resolved in vivo fluorescence kinetics and absorption spectra Phycourobilin isoforms Diazotrophic cell Phycobiliprotein purification + characterisation: Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, Method of deconvolution: Küpper H, Seibert S, Aravind P (2007) Analytical Chemistry 79, Fluoresce ence (normali ised to red m aximum) Phycourobilin isoforms (II) Phycoerythrin isoforms Phycocyanin isoforms Allophycocyanin red maximum m) Fluo orescence (no ormalised to antum yield Relative fluo orescence qu ence quantum yield ative fluoresc Rela basic fluorescence yield F 0 PSII activity F v

35 Deconvolution of spectrally resolved in vivo fluorescence kinetics shows reversible coupling of individual phycobiliproteins Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787,

36 Deconvolution of spectrally resolved in vivo fluorescence kinetics shows reversible coupling of individual phycobiliproteins Basic dark-adapted fluorescence yield F 0 Non-photochemical changes in fluorescence yield quenching normal F 0 active bright I =diazotrophic Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787,

37 Sequence of uncoupling events in a bright II cell Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787,

38 Reversible coupling of individual phycobiliproteins......as a basis for diazotrophic photosynthesis Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787,

39 Iron limitation: it ti photosynthetic components remain active... Küpper H, Šetlík I, Seibert S, Prášil O, Šetlikova E, Strittmatter M, Levitan O, Lohscheider J, Adamska I, Berman-Frank I (2008) New Phytologist 179,

40 Iron limitation: rescue of photosynthetic components......by sacrificing nitrogenase PsaC new PE isoform Küpper H, Šetlík I, Seibert S, Prášil O, Šetlikova E, Strittmatter M, Levitan O, Lohscheider J, Adamska I, Berman-Frank I (2008) New Phytologist 179,

41 Light limitation: acclimation by reversible phycobiliprotein coupling: basic fluorescence yield F 0 relative frequency Andresen E, Adamska I, Šetlikova E, Lohscheider J, Šimek M, Küpper H, (2010) New Phytologist 185,

42 Light limitation: acclimation by reversible phycobiliprotein coupling (II): maximal PSII activity F v Andresen E, Adamska I, Šetlikova E, Lohscheider J, Šimek M, Küpper H, (2010) New Phytologist 185,

43 Conclusions For Trichodesmium, photosystem II activity is essential for providing energy for nitrogen fixation. Regulation of PSII activity for nitrogen fixation is achieved mainly by quickly reversible (un)coupling of individual phycobiliproteins. The nitrogen fixing activity state is characterised by a particularly large PSIIassociated antenna, which is achieved mainly by coupling of additional units of phycourobilin (PUB) isoforms. Therefore, acclimation to light limitation ( low light stress ) involves enhanced synthesis mainly of phycourobilin, which is then mainly coupled to PSII. Synthesis and levels of other photosynthetic components decrease. Because of their vital importance, when adverse conditions require a choice, Trichodesmium in contrast to other cyanobacteria does not sacrifice its phycobilisomes, but rather its nitrogenase. Stress leads to expression of alternative phycobiliprotein isoforms

44 All slides of my lectures can be downloaded from my workgroup homepage Department of Biology Workgroups Küpper lab, or directly and on the ILIAS website