8. Photosynthesis: “Dark Reactions” Read for this lesson: Biology of Plants 6th ed. pp. 126-153 7th ed. pp. 115-139 8th ed. pp. 122-149 (same as for last lesson) Extra reading: [Plant Physiology (Taiz & Zeiger) pp. 195-224]

An Overview of Photo-Synthesis Light Reactions “Dark” Reactions 1 3 CO2 Fixation 2

1) Photoexcitation / De-Excitation of Chlorophyll and”Charge Separation” 1 (~<1%) 2 (~<10%)

Energy transfer and photochemistry Fluorescence Ph Chlorophyll Heat Energy transfer and photochemistry Fluorescence Ph Blue-photon excitation level Red-photon excitation level Ground state red orange yellow green blue Electron energy level

Photoexcitation, Resonance Transfer and Charge Separation 3 1 4 2 Resonance Energy Transfer

Photosystem ‘Antenna’ Resonance Energy Transfer Electron transfer Reaction Centre 4 Electron acceptor

Charge Separation Primary Q (phaeophytin) P 680 / P 680+ In PS II H2O

Charge Separation Primary Fd = Feredoxin P 700 / P700+ In PS I PS II (H2O)

2) Electron Transport in TWO Photosystems

(mid-point) Redox potential (V) +0.8 +0.4 -0.4 -0.8 -1.0 PSII (Pheophytin) QA QB PSI “A” PQ Cyt b6/f PC P700+ Strong oxidant Strong reductant Fd (mid-point) Redox potential (V) (Thylakoid membrane) Ph Stroma Lumen NADP+ NADPH H2O

3) Formation of ATP -1.0V -0.8V Quinone Red-Ox Potential 0.8V

pH ~ 8 pH ~ 4, Proton Motive Force (PMF)

Oxidative phosphorylation Photophosphorylation

PS-I Cyt PS-II Complex 1 Complex 3 Complex 2 4e- 2H2O O2 +4H+ 2H2O

“Dark” reactions

Light Reactions “Dark” Reactions Stroma CO2 Fixation

The Calvin Cycle The C3 Cycle (of CO2 fixation and Reduction) 6CO2 + 6H2O >>> C6H12O6 + 6O2 Melvin Calvin

PGA = phosphoglyceric acid or phosphoglycerate = a 3-carbon compound, therefore the C3 cycle.

Autochromatogram (from paper chromatogrphy Lamp Lollipop flask Chlo-rella Alco-hol Sugars 14CO2 Autochromatogram (from paper chromatogrphy PGA

The Calvin Cycle / C3 Cycle Phosphoglyceric acid (PGA) Ribulose bis-phosphate (RuBP) Triose phosphate (TP)

The Calvin Cycle: Phase 1 Carbon Fixation Phytobenthos C5 2 x C3 PGA P P P RuBP

The Calvin Cycle: Phase 2, Carbon reduction (PGA) P P P P P TP P

TP

The Calvin Cycle – C3 cycle 3 x 1C 3 x 5C 6 x 3C 5 x TP 1 x TP

The Calvin Cycle: Phase 3, Regeneration RuBP P TP

3 CO2 Rubisco 3 RuBP 6 PGA Calvin cycle 6 6 PGAL 5 TP 6 TP TP Sugars 6 ATP Calvin cycle 3 ATP 6 NADPH 6 6 PGAL 5 TP 6 TP TP Sugars

3 CO2 C1 6 PGA 6 Triose-P (TP) C3 C3 3 RuBP C5 C3 C3 C3 C3 C3 C3 C5 Sugars 3 ATP 6 ATP 6 NADPH

3 CO2 (C1) 6 PGA (C3) 6 6 TP (C3) 3 RuBP (C5) TP Sugars 5 TP Rubisco 6 PGAL (C3) 6 ATP 3 ATP 6 NADPH C3 C5 C7 C6 2 C5 C4 Calvin cycle

2H2O / 4e- / O2 / 2NADPH / CO2 / 3ATP needed / X photons Stoichiometry for Photosynthesis Light Reactions: O2 2 H2O (4H+) ~3 ATP 4 e- 2 NADP+ + 4 e- 2 NADPH (+2H+) 2ATP “Dark” Reactions (Calvin cycle): CO2 + RuBP [C6] 2 PGA 2 PGAld (TP) (C5) (C3) (C3) 5/6 ATP 1/6 Sugars 2H2O / 4e- / O2 / 2NADPH / CO2 / 3ATP needed / X photons

Light reactions TP

Why are most terrestrial plants limited by today’s atmospheric CO2 concentration?

The Calvin Cycle: Phase 1 2 x C3 PGA P P P RuBP Rubisco Ribulose bisphosphate carboxylase…

Rubisco, the “Dinosaur” enzyme: BIG: 8L 8S subunits, 560 kDa Prof. J.A. Raven, FRS “Dinosaur” enzyme: BIG: 8L 8S subunits, 560 kDa SLOW: (Rubisco) Km (CO2) = 10-30 mM STUPID: Rubisco acts also as an Oxygenase!

http://web.expasy.org/spotlight/back_issues/038/

Air

(mM)

Rubisco, Ribulose 1,5-bisposphate carboxylase/oxygenase “Dinosaur” enzyme: BIG: 8L 8S subunits, 560 kDa SLOW: (Rubisco) Km (CO2) = 10-30 mM STUPID: Acts also as an Oxygenase! (Rubisco) Ribulose bisphospate Carboxylase/oxygenas

Rubisco Function as an Oxygenase PGA Glycolate

CO2 /or O2 (2 x 3-C) (3-C) (2-C) Ribulose bisphosphate carboxylase = Rubisc…o (Rubisco) = Ribulose bisphosphate carboxylase / oxygenase (3-C) (2-C)

Glycolate Metabolism in Photorespiration Peroxisome Mitochondrion Chloroplast

Chloroplast Peroxisome NH3 Calvin cycle glyoxylate glycine O2 CO2 2 x PGA Rubisco O2 PGA + glycolate glycine NH3 serine + CO2 glycerate Mitochondrion

Rubisco Carboxylase vs. Oxygenase activity AIR: CO2 O2 0.035% 21% 15 mM 9400 mM SOLUBLE: 10 mM 250 mM Rubisco Carboxylase vs. Oxygenase activity (activity = affinity x substrate concentration) CO2 O2 Concentration 1 : 25 Affinity 100 : 1 (Km ~ 10 vs. 1,000 mM) ------------------------------------------------------------------ Activity 100 : 25 48

[CO2] During Evolution [O2] 21% [CO2] 0.038% 49