§4 Mechanism of Photosynthesis

Photosynthesis comprises two steps: Light reaction and Dark reaction Warburg found photosynthesis can be divided into two steps in 1920 Light reaction Photosynthesis Dark reaction

Flash experiment with Chlorella (小球藻) Radiated continuously Less O2 Light Radiated for 0.0034S , interval 0.0166S, 50cps More O2

Now it’s clear Light reaction: Dark reaction: Primary reaction: absorption, transfer and conversion of light energy to electric energy. Electron transfer and photophosphorylation: turn electric energy into active chemical energy. Dark reaction: Assimilation of CO2, turn active chemical energy to stable chemical energy.

I: Primary reaction The first step of photosynthesis Includes: absorption, transference and conversion of light energy Site: thylakoid

Photosynthetic Unit Photosynthetic unit: the minimum unit for carrying out photosynthetic O2 production Emerson & Arnold (1930) found that it took the cooperative action of around 2400 chlorophyll molecules to produce one molecule of O2. It takes a minimum of 8 photons to produce one molecule of O2. For each photon, about 300 chls are needed.

A Photosynthetic Unit includes: Light harvesting system: includes antenna pigments and proteins (chl bond with proteins), light harvest and transmission Reaction center: converse light energy to electric energy. It includes: Reaction center chl: some special chla Primary electron donor Primary electron acceptor And related proteins

How energy is converted? light DPA → DP*A →DP+A- →D+PA-

Note: resonance energy transfer is quite rapid Note: resonance energy transfer is quite rapid. The energy of 1 red photon can be passed to 200-300 chl (30ps/s) in its life span. The efficiency is up to 90-100%

Antenna complex as energy funnel

II: Electron transfer and Photophosphorylation The electron produced in the primary reaction is transferred through the photosynthetic electron transfer chain, and at last causes the generation of ATP and NADPH.

1. Electron transference needs two photosystems PSI and PSII Discovered by Emerson from his famous Red Drop Phenomenon (红降现象）and Enhancement Effect（双光增益效应）.

685 nm

685nm 650nm 650nm 685nm

Conclusion: Light reaction needs 2 photosystems, one is driven by long wavelength light, another is driven by short wavelength light.

2 PSII, PSI and the chain

A. Four protein complexes in the thylakoid membrane

1) PSII Components P680 Primary acceptor: pheo Primary donor: LHCII Peptides(D1and D2;CP43,CP47,cytb6/f) QA，QB OEC(33,23 and 16kDa) Functions: photolysis of H2O to emit O2 and provide e and proton

Events at the PS II reaction center

Hill Reation The Hill reaction occurs when isolated ‘chloroplasts’ are illuminated in the presence of an electron acceptor ‘A.’ The acceptor is reduced (to AH2) and molecular oxygen (O2) is evolved. A can be NAD(P)+ and so on. 2H2O+2A　　　　　2AH2+O2 light chloroplast

Significance of the discovery Proved that O2 is released from H2O, rather than CO2, as had been previously thought.  Proved that chloroplast play an important role in photosynthesis. Proved that photosynthesis is an oxidation-reduction reaction, and the process of photosynthesis is the transport of activated electron from one substance to another. This established the essence of converting light  energy to chemical energy.

Proof of water oxidation clock (Joliet light flash experiment:5-10s, interval,300ms) Note: maximum O2 evolution comes after the 3rd flash, because during dark relaxation, most OEC returns to S1 state.

How O2 is evolved? ----the “water oxidation clock” OEC contains 4 Mn, thus shows 5 oxidative states: S0，S1，S2，S3，S4. : S0 and S1 are stable states and in the dark, S0 is ¼ and S1 is 3/4， S2，S3 are unstable and converted to S0 and S1 in the dark. For each time P680 absorbs a photon, P680 captures a electron form OEC through Tyr and OEC accumulates a positive charge. Once OEC accumulates 4 charges, it will oxidate H2O and split it to evolve O2.

Structure of Mn-cluster of OEC Note: there are also Cl, O and Ca in this model

2) PSI Components Function：reduce NADP+ to NADPH P700 Primary electron acceptor：A0 Primary electron donor：PC Electron transporter LHCI Peptites(PsaA:82kDa),Psa:83kDa; C,D,E,F…) Function：reduce NADP+ to NADPH

3) cytb6/f complex Components: 4 peptides, of which 3 contain Fe (1 cytb6, 1 cytf, 1 Rieske Fe) Function: Electron transport PQ shuttle

4) ATP Synthase Components: CF0：I II III IV(1:1:12:1) Function: Generate ATP

B: Electron Transfer Chain

Electron transport types: ①Noncyclic electron transport, ATP and NADPH; ②cyclic electron transport, only ATP; ③pseudocyclic electron transport, (ATP and ROS) ③ O2- O2- ① O2 ②

Inhibitors of Electron Transfer Chain PSII cytb6/f PSI DCMU(敌草隆)：QA→QB DBMIB(2,5-二溴-3-甲基-6-异丙基-对-苯醌)：PQ oxidation Paraquat(百草枯)：deviate from FA/FB

C: Proton accumulation in the lumen Two sources: (1) Photolysis of water (2) PQ shuttle

D: Photophosphorylation Equipment: ATP synthase=CF Driving force: ΔμH+ across the thylakoid membrane Mechanism: ① chemiosmotic theory of Michell. ② binding change theory of Boyer

Chemiosmotic theory---- Transmembrane H+ gradient generates ATP

Proved by Jagenhorf

Binding Change Mechanism

Inhibitors of Photophosphorylation Uncoupler: DNP(二硝基苯酚) , FCCP(羰基-氰-对-三氟甲氧基苯腙） ATPase inhibitor: oligomycin（寡霉素）, prevent H+ to across CF0 Inhibitor of the chain: DCMU, DBMIM paraquat

Summary of Light Reaction To evolve 1 molecule O2, 4 electrons are needed, thus theoretically 8 photons are required to evolve 1 O2. 2H2O → 4H+　＋4e + O2 During light reaction, O2 is evolved together with the production of ATP and NADPH, which will be consumed in dark reaction for CO2 assimilation, thus ATP and NADPH are named assimilation power. -2