Electron Transport and Carbon Fixation in Chloroplasts

Transcription

1 Module : Molecular Biology and Biochemistry of the Cell Lecture 17 Electron Transport and Carbon Fixation in Chloroplasts Dale Sanders 10 March 2009

2 Objectives By the end of the lecture you should understand: 1. What are the identities of the components of the photosynthetic electron transport chain. 2. How electron transport occurs between these components in terms of (a) their redox potentials (E o ); (b) their distributions in the membrane; (c) associated H + flux. 3. The nature of cyclic electron transport. 4. How CO 2 is initially fixed by Rubisco. 5. How 3-PGA is assimilated to hexose.

3 Reading As in my previous lectures, all the topics today are well covered by the standard big biochemistry textbooks. One such text is Voet & Voet (2004) Biochemistry (3 rd Ed.) Chapter 24 and especially pp Also useful for a more in-depth treatment is Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3 Chapter 6. Buchanan BB et al. (2000) Biochemistry and Molecular Biology of Plants. Chapter 12, pp

4 PS II and PS I act in Series to Catalyse Electron Flow Between H 2 O and NADP + Pathway of electrons from H 2 O NADP + : H 2 O NADP + + H + PS II Redox components PS I Redox components ½O 2 +2H + NADPH Light and Resonance Energy Transfer Light and Resonance Energy Transfer

5 The Identities of the Redox Components of the Thylakoid Electron Transport Chain: Overview of redox chain components -1.0 E' o (V) *P680 Pheophytin *P700 (FeS) Fd H 2 O OEC P680 PQ cytb FeS cytb cytf PC P700 Fp NADP + OEC = Oxygen evolving complex; PQ = Plastoquinone PC = Plastocyanin: Fd = Ferredoxin Fp = Flavoprotein (FAD)

6 A more detailed look at the redox chain components 1. PS II Reaction Centre Comprises a supramolecular complex: several distinct proteins binding redox chain components: (i) P680: Chl a dimer (ii) Pheophytin (Chl a without Mg 2+ ) E' V (iii) 2 molecules of plastoquinone, bound to specific proteins. Q A, Q B tight loose

7 Plastoquinone (PQ) ( ( oxidized (quinone) reduced (quinol)

8 Oxidation of Q B is prevented by a number of herbicides (e.g. dichlorophenyldimethylurea (DCMU)) Once reduced to plastoquinol (PQH 2 ) the Q B molecule diffuses into 2. The PQ Pool: A large number of molecules of PQ, freely dissolved in the hydrophobic portion of the thylakoid membrane.

9 3. The Cytochrome b 6 f complex A supramolecular complex, accepting e - from PQ comprises: - 2 spectroscopically distinct b-type cytochromes (cytochromes b 6 ) - Cytochrome f - an Fe 2 S 2 centre (Rieske protein) - bound PQ

10 Structurally and functionally, the cytochrome b 6 f complex is very similar to Complex III (Cytochrome bc 1 ) of mitochondria: (i) both are inhibited by Antimycin A (ii) both accept e - from a quinol (iii) Cyt f is similar structurally to cyt c 1 (iv) the b-type cytochrome (actually 2 haem groups bound to a single apoprotein) shows extensive sequence homology to cyt b of mitochondria (v) both contain a high potential (E 0 = mv) Fe 2 S 2 centre All these factors point to a common evolutionary origin of Complex III and the Cytochrome b 6 f complex

11 4. Plastocyanin (PC) A small, water-soluble copper-containing protein located in lumen of thylakoid. 5. PS I Reaction Centre Oxidizes PC The 3 rd supramolecular complex, comprising (bound to proteins): (i) (ii) (iii) (iv) P700: Chl a dimer 6 additional Chls 2 quinones, 3 Fe 4 S 4 centres All help move electrons across membrane to next component

12 6. Ferredoxin (Fd) A small protein with an Fe 2 S 2 centre. Loosely associated with the STROMAL side of the thylakoid membrane. 7. Ferredoxin NADP oxidoreductase A flavoprotein, containing FAD Also located on the STROMAL side of the thylakoid membrane.

13 8. Oxygen-evolving complex OEC 3 proteins; associated with PS II on LUMINAL side of membrane Active centre: 4 tightly-bound Mn 2+ ions Catalyses reaction: 2H 2 O O 2 + 4H e - The e - are passed, one at a time via tyrosine residues, to oxidized P680 + reaction centres.

14 The Useful Products of Photosynthetic e - Transport 1. NADPH: Subsequently used in reduction of CO 2 2. PMF: e - transport chain pumps H + into lumen, hence ATP is synthesised

15 Magnitude of PMF Δψ = +20 mv ΔpH = 3.5 units (lumen acid) Since PMF = Δψ + 59 (ph o ph i ) Thus PMF = = mv (lumen + ve) Note: PMF is inverted compared with mitochondria and so is orientation of ATP synthase: ATP made on outside of thylakoid membrane, in stroma STOICHIOMETRIES: For redox chain, 6H + / 2e - For ATP synthase, 4H + /ATP i.e. for each pair of e - passing through chain: 1 NADPH and 1.5 ATP are produced

16 Cyclic e - Transport and Variable ATP/NADPH Production Observation: Light of wavelength >680 nm results in a PMF, but not net production of reducing equivalents. Interpretation: Stroma Lumen

17 Interpretation: PSI is excited by long wavelength light. Electrons are recycled through ferredoxin, b 6 f complex and plastocyanin b 6 f complex is a H + pump Note: Net production of NADPH is not possible because no reductant (i.e. H 2 O ) is available. But ATP can be produced. Cyclic electron transport might provide plants with a way of producing >1.5 ATP/NADPH if demand for ATP is high.

18 Lateral Heterogeneity and Plastoquinone Diffusion Cyt b 6 f complex and PS I are in stromal lamellae; PS II is in granal lamellae Question: How are reducing equivalents transferred from PS II to Cyt b 6 f complex?? Answer: Plastoquinone: A very mobile molecule, which diffuses in the plane of the membrane. PQH 2 PQH 2 PSI PSII

19 Carbon Fixation The 1 st reaction: catalysed by Ribulose 1,5- bisphosphate carboxylase/oxygenase (Rubisco) C O P C O P C O P CO 2 + C = O C OH C OH Rubisco C OH C + O O C OH C O O C O P Ribulose 1,5 - (3 phosphoglycerate) x 2 bisphosphate

20 Rubisco Reaction Energetics: ΔG O = - 52 kj/mol, hence spontaneous The Protein: Very low turnover rate (about 3 s 1 ) hence very abundant THE most abundant in the world A large, allosteric enzyme: 8 large (L) subunits.. M r = 55,000 8 small (S) subunits..m r = 13,000 L subunits S subunits Control: Mg 2+ released from thylakoid lumen in exchange for H + during electron transport activates Rubisco in stroma

21 3 Phosphoglycerate ATP ADP 1,3 Bisphosphoglycerate NADPH NAD + Assimilation of Hexoses Glyceraldehyde 3 Phosphate Dihydroxyacetone phosphate Fructose 1,6-Bisphosphate P i Fructose 6-Phosphate Phosphoglycerate kinase Glyceraldehyde 3 Phosphate DH NADP + - specific Aldolase Fructose 1,6-Bisphosphatase

22 Note: 1. These stromal reactions are a reversal of glycolysis, except that FBPase provides a unique step. 2. These reactions mirror those of gluconeogenesis (in liver) except that Glyceraldehyde 3-phosphate dehydrogenase is NADP + -specific.

23 SUMMARY 1. The photosynthetic e - transport chain comprises 3 macromolecular complexes (PS II, Cyt b 6 f complex, PS I) and associated redox components. 2. PHS e - transport chain components can be arranged according to E O and to position in membrane. 3. PHS e - transport chain is a H + pump.

24 5. Cyclic e - transport involves PS I and cyt b 6 f: ATP production but no reductant. 6. Rubisco catalyses CO 2 fixation leading to formation of PGA. 7. PGA is assimilated to hexose in stromal reactions analogous to gluconeogenesis.