Lectures 12 and 13: QUIZ 5 Flashcards
Light-Dependent Reactions and Photophosphorylation:
Endergonic Reactions:
- H2O oxidized to O2 - NADP+ reduced to NADPH
○ Photophosphorylation: light activation generates gradient to make ATP.
Photosystems and the Z scheme:
○ Photosystem I (PS I, P700)
- makes NADPH (2 e- required)
○ Photosystem II (PSII, P680)
- Replenishes electrons of PSI (takes them from H2O —> 1/2 O2, photolysis)
- maintains proton gradient for ATP synthesis.
Photosystem II:
○ Photons excite P680 of PSII
- e- flow starts
(H2O—> Tyr[r] )—> P680 —> Pheo –> PQa —> PQb
○ Electron hole created when P680+ was generated
- Tyr[r] of water oxidizing complex
- gets e- from H2O via Mn4Ca ion cluster inside the enzyme complex.
○ H2) splitting process releases H+ on lumen side:
- contributes to electrochemical gradient
○ Takes 4 photons (hv) to transfer 4 e-
Water splitting activity of O2 evolving complex:
○ In plants PS II, P680+ needs e- from H2O
○ Problem 1:
- 1 photon doesn’t have enough energy to break H2O bonds
▪ H2O is a bad e- donor
▪ need 4 photons to split 2 H2O —> 4H+ + 4e- + O2
○ e- pass one at a time to P680 via Tyr
- Tyr passes them one at a time (and loses H+ simultaneously) to become radical Tyr
- 4 e- removed from H2O in packets via Mn4Ca cluster of WOC, but pass to Tyr one at a time.
- 4 H+ released (pumped) to lumen.
Cytochrome B6-F and Plastocyanin:
○ PQbH2 (plastoquinone, similar to QH2 in mitochondria)
○ Transfer e- through cyt B6-f complex —> plastocyanin
- Cyt b6-f = complex III
- Plastocyanin (PC) ~ cyt c
○ Uses Q cycle, like complex III
○ 4 H+ pumped from stroma —> lumen per 2 e- transferred.
Photosystem I:
○ Photon excites P700 of PS I e- flow starts
- P700 —> A0 —> A1 —> Fe-S —> Fd
○ Fd (ferredoxin), peripheral protein on stroma side of membrane.
○ Ferredoxin: NADP + oxidoreductase.
- transfers e- from Fd —> NADP +, making NADPH + H+ in stroma
- Enzyme is flavoprotein (picks up 1 e- at a time but passes 2 on)
4PCred + 2 NADP+ + 4H+ –(4 hv)—> 4 PCox + 2 NADPH + 2H+
Noncyclic vs cyclic electron flow:
○ Noncyclic flow: - Fd passes e- to NADP+ ---> NADPH made - O2 made (H2O split) - cF0F1 makes ATP using H+ gradient. ○ Cyclic flow: - Fd passes e- back to cyt b6-f complex. - pump more H+ (larger gradient) - ATP made - NO NADPH NOR O2 made.
Photophosphorylation:
○ Proton gradient across thylakoid membrane drives ADP phosphorylation:
- electrochemical gradient maintained.
- H + flow through cF0 of ATP synthase to make ATP via cF1 (binding change mechanism)
▪ H+ flow from lumen —> stroma
Carbon Fixation Reactions:
○ Formerly “dark” reactions: can occur in dark but mainly in light due to regulation.
- aka Calvin cycle
○ takes places in stroma
○ Use NADPH and ATP from light reactions to fix CO2
- carbon fixation: incorporating CO2 into an organic compound (3PG)
Calvin Cycle three stages
- Carbon fixation: CO2 condenses with RuBP (5C)
- forms two 3PG (6C total)
- Catalyzed by Rubisco in stroma.
◊ Rubisco: ribulose 1,5-bisphosphate carboxylase oxygenase
2. Phosphorylation & Reduction:
- ATP phosphorylates 3PG to make 1,3-BPG
- NADPH reduces 1,3-BPG to DHAP and GAP
◊ GAP/DHP: glycolysis for energy, or gluconeogenesis for sucrose, starch, or cellulose
3. Regeneration of Ribulose 1,5-bisphosphate (RuBP)
- carbon shuffling (similar to non oxidative reactions of PPP)
- also uses ATP
Rubisco Mechanism:
○ Mg2+ is important
1. enediolate formed 2. CO2 polarized by Mg 2+ - gets Nu attacked - 6-C intermediate 3. C-3 hydrolyzed 4. 1st 3BPG cleaved off 5. Protonate carbanion - 2nd 3PG made
Rubisco: Details and Regulation, involving Mg2+ and Carbamoyl-Lys:
○ Mg2+:
- brings reactants together and orients them in active site
- Coordinates 6 O atoms: 3 on enzyme, 2 on RuBP, 1 on CO2
□ Rubisco: 1 on Glu, 1 on Asp, and 1 on carbamoyl-Lys
○ Carbamoyl-Lys: - Enzyme inactive unless Lys is carbamoylated □ needed to bind Mg2+ - Carbamoylation inhibited by RuBP binding tightly to "closed" conformation (no Mg2+) □ Rubisco catalyzes ATP-dependent release of RuBP □ Lys now exposed, can be carbamylated non-enzymatically □ Mg2+ then bonds ---> Rubisco activated
Rubisco: Regulation (cont)
○ Light activation of Rubisco:
- Light activates light-dependent reactions —> electrochemical gradient made
□ stroma more negative (N side, fewer H+, higher pH)
1. Carbamoylation of Lys is faster at higher pH
2. Mg @+ flows from lumen —> stroma due to electrical gradient.
○ Nocturnal inhibitor:
- some plant leaves have a naturally-occurring transition state analog (inhibitor) that keeps it inactive at night:
□ inhibitor: 2-carboxy-D-arabinitol-1-phosphate (2CA1P)
□ Found in potato leaves
- Inhibitor is broken down in light or expelled by rubisco activase.
Fates of GAP
○ 6 GAP made
- 5 return to regenerate RuBP for more C fixation
1. Transported to cytosol as DHAP
- used in glycolysis (for energy)
- converted to sucrose for transport to other tissues (for growth)
○ Sucrose is primary sugar transport in plants.
2. Stays in stroma:
- converted to starch for storage in chloroplast
Calvin Cycle: Energetics
○ Light reactions: - for every 8 photos absorbed… ○ 4 e- transported ○ 2 NADPH and 3 ATP made in stroma ○ 2:3 ratio of NADPH;ATP made ~ 6:9 Calcin cycle needs of NADPH ○ Calvin cycle: - 6 NADPH needed ○ 6 for 6 1,3BPG ---> GAP - 9 ATP needed ○ 6 for 6 3PG ---> 6 1, 3-BPG ○ 3 for 3 Ru5P ---> 3 RuBP (Ru-1,5-BP) - 8 Pi released ○ 6 from 1,3 BPG ---> 6 GAP ○ 2 from 2 phosphatases in C-shuffling
Pi - Triose Phosphate Antiporter:
○ Antiporter in inner chloroplast membrane
○ exchange Pi for DHAP (antiporter)
- DHAP: stroma —> cytosol
- Pi : cytosol —> stroma
Antiporter as a shuffle system:
○ Antiporter may take ATP and electrons from chloroplasts —> cytosol, if needed
- ATP and NADPH cant cross chloroplasts, but… - DHAP can carry e- and potential energy for substrate level phosphorylation.
Light driven reduction of disulfide bonds:
○ 4 enzymes have light sensitive disulfide bonds
- FBPase-1
- Su-1,7-Bpase-1
- Ru5P kinase
- GAPDH
○ Oxidized enzymes ( -S-S) inactive:
- in the dark, all 4 enzymes spontaneously oxidize
○ Light driven e- transport ultimately reduces bond (-SH HS-)
- Ferredoxin (Fd) reduced
- Fd reduces thioredoxin via Fd:thioredoxin reductase
- Thioredoxin reduces the 4 Calvin cycle enzymes, activating them.
Light driven reduction of disulfide bonds:
○ 4 enzymes have light sensitive disulfide bonds
- FBPase-1
- Su-1,7-Bpase-1
- Ru5P kinase
- GAPDH
○ Oxidized enzymes ( -S-S) inactive:
- in the dark, all 4 enzymes spontaneously oxidize
○ Light driven e- transport ultimately reduces bond (-SH HS-)
- Ferredoxin (Fd) reduced
- Fd reduces thioredoxin via Fd:thioredoxin reductase
- Thioredoxin reduces the 4 Calvin cycle enzymes, activating them.
Light driven reduction of disulfide bonds:
○ 4 enzymes have light sensitive disulfide bonds
- FBPase-1
- Su-1,7-Bpase-1
- Ru5P kinase
- GAPDH
○ Oxidized enzymes ( -S-S) inactive:
- in the dark, all 4 enzymes spontaneously oxidize
○ Light driven e- transport ultimately reduces bond (-SH HS-)
- Ferredoxin (Fd) reduced
- Fd reduces thioredoxin via Fd:thioredoxin reductase
- Thioredoxin reduces the 4 Calvin cycle enzymes, activating them.
Stage 1: Carbon Fixation:
○ Ribulose 1,5-BP + CO2 —> intermediate—> 2,3-Phosphoglycerate
○ Rubisco ( ribulose 1,5-bisphosphate carboxylase oxygenase )
- plant enzyme is huge (bacterial version, not so much
□ 8 large subunits with active sites
□ 8 small subunits (function unknown)
- Most abundant enzyme in biosphere:
□ -lots of it because low Kcat (3 s^-1)
□ i.e. it sucks as an enzyme
Stage 2: Phosphorylation & Reduction:
○ Two step process:
1. 3-phosphoglycerate + ATP ---> 1,3-bisphosphoglycerate + ADP - 3-phosphoglycerate kinase 2. 1,3-bisphosphoglycerate + NADPH + H+ ---> GAP + NADP+ + Pi
Stage 3: Regeneration of RuBP
○ Carbon shuffling…reverse of PPP non-ox
○ Start with 5 trioses, end with 3 pentoses (Ru5P)
○ Phosphorylate 3 Ru5P—> 3 RuBP
- Need 3 ATP to do this
Stage 3: Regeneration via C-shuffling:
○ Reversible reaction enzymes:
- Aldolase: steps 1,4
- Transketolase: steps 3,6
- R5P isomerase (R5P —> Ru5P): step 7
- Ru5P epimerase (Xu5P —> Ru5P) : step 8
○ Irreversible reaction enzymes:
- Fructose 1,6 bisphosphate (FBPase-1): Step 2
- Sedoheptulose 1,7-bisphosphatase (SuBPase-1): step 5
- Ribulose 5-phosphate kinase (Ru5P kinase): step 9
The three irreversible reactions make all of stage 3 irreversible.
