dark means light-indendent! The Dark Reaction: Carbon Fixation Requires Rubisco: Ribulose-1,5-bisphosphate carboxylase RuBPi + C 6 x 3PG MW = 550, 000 g/mol Subunits eight small (14,000 g/mol) eight large (53,000 g/mol) 4 mm in stroma (50 mg/ml) Mg + required in active and allosteric sites Allosteric site lysine is bound to C Mg lysine-nh + C + lysine-nh-c - + H + (carbamate nonsubstrate C ) Mechanism of C Fixation: Rubisco Step 1 Ribulose 1,5-Bisphosphate Carboxylase rubisco Fig. 0-3 First Step: C adds to enediol form of RuBPi (not HC - or lysine-nh-c -!) 1
Mechanism of C Fixation: Rubisco Step H adds to b-keto intermediate Fig. 0-3 Mechanism of C Fixation: Rubisco Step 3 Hydrated intermediate breaks down to x 3PG Fig. 0-3 **C3 plants** (nontropical) All steps catalyzed by rubisco! Enzyme makes up ~50% by wt total chloroplast protein Most abundant protein in biosphere 10 11 tons C fixed per year!
Why is rubisco reaction energetically favorable? 1 3 Fig. 0-3 RuBPi + C 6 x 3PG DG o = -35 kj/mol Step 1: highly unfavorable Step 3: highly favorable Regulation of Enzymatic Activity of Rubisco (stroma) Rubisco ph max ~ 8 (H + pumped into lumen from stroma) With hv, as H + pumped into lumen, Mg + pumped out into stroma As hv8, NADPH 8, ATP 8, [Mg + 8 H + 9], Rubisco activity 8 C fixation does not take place in the dark! At night, glycolysis and oxidative phosphorylation meet energy needs of the plant. Rubisco activity is controlled by a redox system that is dependent upon [NADPH] 3
Fig. 0-36 Redox regulation of Rubisco ox active Rubisco (red) NADPH Where is this protein found? 4 Cu + plastocyanin 4 Cu + SUMMA RY F ELECTRN TRA NSFER IN PS I T NADP + Chl +. -. a Chl a 4 hn RXN Chla Chla CTR 4 Fe + 4 Fe 3+ 4 Chla Ao 4 PQ A 1 Fe 4 S 4 Fd 4 Fe 3+ 4 Fe + red NADPH NADP + + H + NADP + inactive Rubisco (ox) What controls reduction of Fd? hv + PSI What glycolytic enzymes must be activated? deactivated? FBPi phosphatase 8, PFK9 Allosteric regulation of Rubisco allosteric subunit RuBPi At high C, no ATP or activase is required. At low C, ATP required to help displace a RuBPi which blocks the lys and prevents carbamylation allosteric subunit + RuBPi RuBPi binds in both the active site and the allosteric site? Fig. 0-33 4
From where comes the ribulose 1,5-bisphosphate (RuBPi)? Understanding of C 5 sugar synthesis requires a knowledge of how the pentose phosphate pathway works for carbon fixation! Pentose-Pi pathway: Pentose Pi Shunt Functions: oxidative portion Produces NADPH for biosynthesis (fatty acids, steroids). Produces Ribose (C 5 ) for RNA and cofactors (ATP, NAD, FAD, etc.). Provides a breakdown pathway for C 5 sugars. Provides a pathway to regenerate Ru-5-Pi (C 5 ) for carbon fixation. regenerative (nonoxidative) portions Ribulose 5-phosphate is produced in 3 steps! Ribulose 1,5-bisphosphate is synthesized how? Ru-5-Pi + ATP Ru-1,5-BPi + ADP 5
Pentose-Pi pathway (oxidative): Step 1 Pi H glucos e-6-pi-de hydrogenase NADP + NADPH + H + H 1 Pi H glucos e-6-pi 6-Pi- glucono- d-lactone Typical hydride transfer mechanism to NADP + What H- is transferred? What is the source of G-6-Pi? Comes from phosphorylation of glucose and from G-1-Pi from glycogen phosphorylase breakdown of glycogen using Pi Pentose-Pi pathway (oxidative): Step lactonase Pi H H H + H Pi - 6-Pi-glucono-d-lactone 6-Pi-gluconate Typical hydrolysis of an ester bond. Where is the ester? 6
Pentose-Pi pathway (oxidative): Step 3 H Pi H 6-Pi-gluconate NADP + Typical hydride transfer mechanism to NADP + What H- is transferred? - In what other pathway was a b-keto acid formed? Name of enzyme? CAC: isocitrate dehydrogenase! isocit + NAD + a-kg + C + NADH 3 6-phosphogluconate dehydrogenase Pi H 3 - unstable b -keto acid H + Pi H ribulose-5-pi C 5 NADPH + H + C ketose Pi H Phosphorylation of Ribulose-5-Pi to Bisphosphate ribulose-5-pi C 5 Pi + ATP Pi + ADP ribulose-5-pi kinase H ribulose-1,5-bpi C 5 7
A review of synthesis: Start with? Using C x notation, summarize the rubisco reaction: C 5 + C 1 C 3 How is made from C 3? Reverse of glycolysis starting from 3-phosphoglycerate! 3,4 C H,5 C - 1,6 CH Pi 3-phosphoglycerate (3PG) A review of synthesis: Continue with? CH C 1,6CH Pi glyceraldehyde-3-ph osphate (GAP) H 3, 4,5 1,6 3,4 H,5 C C 6 CH Pi Pi + NADP + NADPH + H + 1,3-bisph osp hoglycerate (1,3-B PG) H,5 1,6 3,4 C C P - CH Pi - - 3-phosphoglycerate (3PG) 7 ADP ATP 6 - glyceraldehyde-3-phosphate dehydrogenase D GB N 7 Start here = + 6.3 kj/mol 7 - phosphoglycerate kinase DG B NN = - 18.5 kj/mol 1 st energy conservation step 8
A review of synthesis: End with? Pi Yeah! Pi glyceraldehyde-3-phosphate (GAP) 6 fructose-1,6-bisphosphate (F6P) 5 4 6 5 4 4 5 3 1 Pi 3 1 Pi + dihydroxyacetone phosphate (DHAP) 4 - aldolase DG B NN = + 4.0 kj/mol Cleavage of into x C 3 fragments 5 - triosephosphate isomerase DG B NN = - 7.6 kj/mol Isomerization of C 3 to interconvert forms For C 3, how many ATP required? NADPH? Note: 1 ATP and 1 NADPH 3PG (C 3 ) How is C 5 regenerated? Rubisco reaction: C 5 + C 1 C 3 C Multiply by 6 6 ops! 6 C 5 + 6 C 1 1 C 3 C 5 consumed? C 5 regenerated using: 30 6 36 10 10 C 3 1) C transfers (transketolase) ) Condensations (transaldolase)! 30 6 C 3 9
C Fixation is called Calvin Cycle 6 C 5 + 6 C 1 1 C 3 30 6 36 10 10 C 3 30 6 C 3 http://www.nobel.se/chemistry/laureates/1961/calvin-bio.html Photorespiration: xygenase Activity of Rubisco competes with C for enol form of RuBPi K m 350 m M nly 1 x 3PG formed K m C 9 m M ther product, Pi-glycolate, is salvaged through a complex series of reactions involving both peroxizomes and mitochondria! Fig. 0-38 10
Salvage Pathway of Pi-glycolate Photorespiration is a wasteful process, and is especially problematic at higher temperatures (above 8 C) At 5 C, the carboxylase activity is four times the oxygenase activity) Fig. 0-39 Function of Photorespiration? xygenase Activity of Rubisco? K m C 9 mm K m 350 mm Rubisco most likely evolved before atmosphere had! retains ability to react with? Why? Photorespiration dominates if light levels are high high production scavenges to help prevent oxidative damage to cells under intense light conditions 11
C Fixation by C4 Plants (Tropical) Tropical grasses such as sugar cane use a different pathway for C fixation Short-time C fixation with 14 C leds to 14 C in C4 intermediates C is fixed as follows: PEP + C XAL (PEP carboxylase) Good summaries: http://methanogens.pdx.edu/boone/courses/bi336/bi336lectures/0001/bi336lec16.html See also Fig 0-40 http://138.19.68.68/bio/courses/biochem /Photosynthesis/Photosynthesis.html C fixation: photorespiration Depends upon C : ratio Air has ~ 3 x 10-4 atm Advantage of C4 Plants (300 ppm) of C biotin http://138.19.68.68/bio/courses/biochem/photosynthesis/photosynthesis.html C4 plants use C at P C down to 1 to x 10-6 atm (1 to ppm). In C3 plants, C fixation stops when P C is 5 x 10-5 atm (50 ppm). At 5 x 10-5 atm, C fixation rate = photorespiration rate. However, plants living in hot climates need to conserve water, which requires them to use low C concentration (water is used in rubisco reaction!) The disadvantage of C4 plants is extra ATP used and a more complex pathway http://methanogens.pdx.edu/boone/courses/bi336/bi336lectures/0001/bi336lec16.html 1
Photoinhibition Photoinhibition: Too much light can cause problems for photosynthetic cells Destruction of RXN CTR occurs by side reactions of the Chla +. The P +. 680 (reaction center of photosystem II) is strongly oxidizing RXN CTR proteins are oxidatively damaged by Chla +. At high hv intensity, RXN CTR proteins turn over rapidly http://methanogens.pdx.edu/boone/courses/bi336/bi336lectures/0001/bi336lec16.html 13