Module 0220502 Membrane Biogenesis and Transport Lecture 11 Light-Driven Proton Translocation Dale Sanders 23 February 2009
Aims: By the end of the lecture you should understand How electrons and protons are translocated by Photosystems I and II and by the cytochrome b 6 f complex The basic mechanism of proton transport through bacteriorhodopsin
Reading As for the last lecture: Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3 A detailed review on structural aspects of the components of the photosynthetic electron transport chain: Cogdell, R.J. & Lindsay, J.G. (2000) New Phytol. 145: 167-196 The crystal structure of Photosystem I is reported by Amunts A. et al. (2007) Nature 447: 58-63 For bacteriorhodopsin, an excellent review is by Lanyi, J.K. (2004) Annu. Rev. Physiol. 66: 665-688
Components of the Photosynthetic Electron Transport Chain 3 macromolecular complexes: Photosystem II Cytochrome b 6 f Photosystem I All these are transport complexes that help set up the PMF A number of other components: Plastoquinone, Plastocyanin, Fe/S centres Serve to ferry electrons between the complexes
An Overview of the Arrangement of the Redox Chain -1.0 E' o (V) PSII *P680 Pheophytin *P700 (FeS) PSI F d F p NADP + 0 +1.0 H 2 O OEC P680 PQ cytb FeS cytb cytf PC Cyt b 6 f P700 OEC = Oxygen evolving complex PQ = Plastoquinone PC = Plastocyanin F d = Ferredoxin F p = Flavoprotein (FAD) Note: Free energy is liberated as e - pass downhill on the oxidizing side of each Photosystem.
Charge Separation and H + Translocation by the Photosynthetic Redox Chain As with Complex III of mitochondria, H + per se is not translocated across the thylakoid membrane by redox reactions. Instead: e - is functionally equivalent to: N H P N P (Stroma) (Lumen) H + Each of the major protein complexes of the photosynthetic redox chain translocates e - towards stroma
This vectorial e - transport gives rise to a biphasic change in Δψ in response to light: a) Fast phase: Primary charge separation at PS II and PS I Photosystem Stroma A e RC Lumen b) Slow phase: Associated with cyt b 6 reduction if the PQ pool is pre-reduced (pre-flash in absence of oxidant). Stroma b 6 f complex cytb haem e cytb haem Lumen
How do we know that Δψ changes in response to light? There is an in-built voltmeter(!!) in the form of Carotenoids: accessory pigments, reside in the membrane lipid. e.g. α-carotene H 3 C CH 3 CH 3 CH 3 H 3 C CH CH 3 3 CH 3 isoprenoid chain H 3 C CH 3 Absorbance at 515 nm increases in response to Δψ Electrochromic shift : Results from very high electrical field strength across biological membranes e.g. 45 mv across 4.5 nm hydrophobic phase = 100,000 V/cm
Dye methods give excellent time resolution: Time course of change in Δψ when chloroplasts flashed with light: 0.1% A 515 a b c 50 ms a. Fast rise, complete in < 20 ns PS II, PS I b. Slow rise, complete in 20 ms b 6 f Complex. Present only when chloroplasts pre-flashed in absence of oxidant. c. Slow decay, due to compensatory charge movement of Mg 2+, K +, Cl Light H + Cl K + Mg 2+
Vectorial Location of Redox Chain Components is Responsible for H + Translocation The pathway of an e - pair from H 2 0 to NADP + : Stroma N 2H + Q cycle 2H + H + + NADP + Fd NADPH Double light flash Q B PQ PQH 2 PQH 2 cyt b cyt b PQ PQ P680 2PQH 2 cyt f P700 H 2 O ½O 2 + 2H + 4H + PC Lumen P i.e. Net translocation, per 2 e is 6 H + Since stoichiometry of ATP synthase is 4 H + /ATP, each 2 e - passing through redox chain can energize synthesis of (6/4) = 1.5 ATP or each mol of NADP + reduced is accompanied by formation of 1.5 mol ATP
Structure-Function Relationships of the H + -Translocating Protein Complexes of Photosynthesis PS I Structure determined from pea 17 subunits: PsaA and PsaB bind reaction centre components 6 chlorophylls + 2 quinones + 3 [4Fe-4S] centres Form 2 electron wires across membrane Stroma 3 [4Fe-4S] centres 1 pair quinones 3 pairs of chlorophylls Lumen P700 Special pair Amunts A. et al. (2007) Nature 447: 58-63
Cyt b 6 f complex: Analogous to Complex III, including presence of Rieske [Fe-S] centre PS II: Not yet crystallized from higher plants, but homologue has been crystallized, and structure determined, from photosynthetic purple non-s bacterium Rhodopseudomonas viridis
2e e Photosynthesis in Purple Non-Sulphur Bacteria: A Cyclic System Periplasm P 2e - haems cyt c 2 2e - 4H + RC P870 QH 2 Cytosol N Q 2H + Q pool 2H + cyt bc 1 complex Q - No net reductant e recycled by cyt c 2 - Q cycle makes effective H + pump in cyt bc 1 complex - Reaction centre: 3 polypeptides (+ cyt c 2 ) H (heavy) M (medium) L (light)
Rhodopseudomonas photosynthetic reaction centre Cyt c 2 Periplasm Membrane M Subunit L Subunit Cytoplasm H Subunit Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3
Transmembrane spans: H: 1 M: 5 L: 5 The pathway of electrons through the reaction centre M & L are homologous to each other and to the D1 and D2 proteins of higher plant PS II bacteriochl dimer (P870) bacteriochl a (bchla) L subunit bacteriopheophytin (BP) L subunit Q A = menaquinone (MQ) M subunit Q B = ubiquinone (empty) L subunit
Bacteriorhodopsin: a Light-Driven H + Pump from Halobacterium halobium H. halobium: An archaebacterium, extreme halophile living in saturated brines At low O 2 tension synthesizes purple membrane : Patches of cell membrane with just 1 protein in paracrystalline array Bacteriorhodopsin (br) M r = 26,000, 7 transmembrane helices Chromophore: retinal
A Pink Salt Lake in California
Retinal is liganded via protonated Schiff Base linkage to Lys 216 H 3 C CH 3 CH 3 CH 3 H + N lys 216 CH 3 3D crystals for X-ray crystallography AND combination of: Site directed mutagenesis. Time resolved spectroscopic techniques Have yielded a good working model for br
Bacteriorhodopsin - Structure Cytoplasm Asp 96 Lys 216 Asp 85 Retinal Medium Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3
The Proton-Pumping Mechanism of br THE C-T MODEL Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3
SUMMARY 1. The e- transport chain of thylakoids is a H+ pump. 2. H+ pumping occurs through movement of e- away from lumen, and H towards it via a Q cycle. Further H+ release in lumen from H2O. 3. e- translocation by PS I and PS II is accomplished by strategically positioned chl molecules and Fe/S centres (PS I). 4. Bacteriorhodopsin is a light-driven H+ pump which undergoes conformational changes as a result of absorption of photons by retinal.