Lec 9: Calvin Cycle & CO 2 fixation Reference material Biochemistry 4 th edition, Mathews, Van Holde, Appling, Anthony Cahill. Pearson ISBN:978 0 13 800464 4 Lehninger Principles of Biochemistry 4 th edition, David L. Nelson, Michael M. Cox. W. H. Freeman ISBN:978 0716743392 The Dark Reactions: The Calvin Cycle Carbon rearrangement C5 C5 C5 C5 C1 X 3 C7 C7 C4 C5 C6 C6 國立交通大學生物科技學系蘭宜錚老師 1
The Dark Reactions: The Calvin Cycle Preparation of CO 2 acceptor Ribulose 1,5 bisphosphate (Ru1,5BP) Ribulose 5 phosphate (we saw this previously in pentose phosphate pathway) is activated by ATP to Ribulose 1,5 bisphosphate (Ru1,5BP or RuBP) ATP ADP + Pi 國立交通大學生物科技學系蘭宜錚老師 2
The Dark Reactions: The Calvin Cycle The Dark Reactions: The Calvin Cycle Stage I 1: Carbon Dioxide Fixation using Rubisco The acceptor molecule for CO 2 is ribulose 1,5 bisphosphate (RuBP), which is ATP activated from ribulose 5 phosphate (Ru5P). CO 2 from the air diffuses into the stroma of the chloroplast, where it is added at the carbonyl carbon of RuBP. The reaction is catalyzed by the enzyme ribulose 1,5 bisphosphate carboxylase, also known as ribulose 1,5 bisphosphate carboxylase/oxygenase (or rubisco). This enzyme is one of the most important in the biosphere and certainly the most abundant. It makes up about 15% of all chloroplast proteins, and there are an estimated 40 million tons of it in the world about 20 pounds for every living person. 4 types of rubisco have been identified in nature. The most common type contains large (~50 kda) and small (~15 kda) subunits. 國立交通大學生物科技學系蘭宜錚老師 3
Rubisco reaction Rubisco first deprotonate α Hydrogen of Carbon #3, forming the enediolate intermediate, which is the receptor for CO 2. Rubisco reaction Rubisco catalyzes carboxylation of Ru1,5BP at original carbonyl carbon (Carbon #2), forming a carboxy-β-keto intermediate. 國立交通大學生物科技學系蘭宜錚老師 4
Rubisco reaction Water then hydrates the carbonyl carbon of the carboxy β keto intermediate. Rubisco reaction Cleavage is facilitated by protonation by an active site acid (HB Enz) Net reaction of Rubisco is: 1 Ru1,5BP + 1 CO 2 2 3PG C5 C1 X 2 國立交通大學生物科技學系蘭宜錚老師 5
Active site of rubisco: This model is based on the X ray structure of spinach rubisco complexed with Mg 2+ and the transition state analog 2 carboxy Darabinitol 1,5 bisphosphate (2CABP). Mg2+ is involved in Ru1,5BP binding and activating the H2O that hydrates the carboxy β keto intermediate. The Dark Reactions: The Calvin Cycle 國立交通大學生物科技學系蘭宜錚老師 6
Stage I 2: converting 3PG to G3P 3PG to G3P is the same as gluconeogenesis with one exception. Each molecule of 3PG is phosphorylated by ATP, in a reaction catalyzed by phosphoglycerate kinase. The 1,3 bisphosphoglycerate so produced is then reduced to glyceraldehyde 3 phosphate (GAP), with accompanying loss of one phosphate. EXCEPTION: The reducing agent is NADPH (as opposed to NADH in glycolysis/gluconeogenesis), produced in the light reaction, and the reaction is catalyzed by the enzyme glyceraldehyde 3 phosphate dehydrogenase: The Dark Reactions: The Calvin Cycle KEY concept here: Ru1,5BP + 3 CO 2 2 x 3PG runs 3 times 3PG + 1 ATP + 1 NADPH G3P runs 6 times NET Calvin cycle occurs in these 3 Reactions!! 3 CO 2 + 9 ATP + 6 NADPH 1 G3P *note: G3P = DHAP (triose isomerase) 3PG + 1 ATP + 1 NADPH G3P runs 6 times Because 5 G3P needs to recycle back to make the 3 x Ru1,5BP 國立交通大學生物科技學系蘭宜錚老師 7
The Dark Reactions: The Calvin Cycle The Dark Reactions: The Calvin Cycle Stage II: Regeneration of the Acceptor To complete the Calvin cycle, it is necessary to regenerate enough Ru1,5BP to keep the cycle going. Ru1,5BP is derived from Ru5P. This means that we need to regenerate 3 moles of Ru5P for every 3 moles of CO 2 fixed. This is accomplished by the set of reactions, which constitute the regenerative phase (carbon rearrangement) of the cycle. To regenerate 3 moles of Ru1,5BP. (15 carbons), We need to use 5 moles of G3P (15 carbons) of the 5 moles of, 2 are DHAP 3 are G3P Therefore, Net reaction of this carbon rearrangement: 2 DHAP + 3 G3P 3 Ru5P 2 x 3 x 3 x C5 國立交通大學生物科技學系蘭宜錚老師 8
Regenerative phase Carbon rearrangement aldolase To make 1 glucose, take what we talked about x 2 國立交通大學生物科技學系蘭宜錚老師 9
Combining light and dark reaction Recall that light reaction: NET: 8 photons + 2 H 2 O + 2 NADP + + 3 ADP + 3 Pi O 2 + 2 NADPH + 3 ATP Now we saw dark reaction: X 3 NET: 3 CO 2 + 6 NADPH + 9 ATP G3P So we can say. NET: 24 photons + 3 CO 2 + 6 H 2 O G3P + 3 O 2 Recall that 2x G3P can be used to make 1 glucose How many photons are used to make 1 glucose? Photorespiration side and unwanted reaction of rubisco Rubisco is far a way from being a perfect enzyme. it s relatively slow enzyme with kcat about 2s 1. Under normal environmental conditions, it can behave as an oxygenase as well as a carboxylase. In the oxygenase reaction, it is proposed that the enediolate intermediate nucleophilically attacks O 2 instead of CO 2. This is Photorespiration 2 phosphoglycolate is produced instead of 3PG 國立交通大學生物科技學系蘭宜錚老師 10
Photorespiration consumes extra ATP, NADH, and O 2. If O 2 was used by Rubisco, then cell has to spend extra energy to metabolize this by product! 1. Ru1,5BP is lose from Calvin cycle 2. Fixation of CO 2 is reversed: O 2 is consumed and CO 2 is released 3. Only a part (75%) of the carbon is returned to the chloroplast NADH 4. ATP is expended. *note in this Glycine to Serine step, 2 glycine make 1 serine + CO 2 Fighting photorespiration! C4 plants Malate is used as a shuttle to transport CO 2 into bundle sheath cell, increasing CO 2 concentration there. C4 plants utilize 2 extra ATP for every CO 2 fixed. 國立交通大學生物科技學系蘭宜錚老師 11
Where photo energy are lost in photosynthesis? Xin Guang Zhu, Stephen P Long, Donald R Ort, What is the maximum efficiency with which photosynthesis can convert solar energy into biomass?, Current Opinion in Biotechnology, Volume 19, Issue 2, April 2008, Pages 153 159, ISSN 0958 1669, Photosynthetic efficiency as a function of CO 2 concentration Xin Guang Zhu, Stephen P Long, Donald R Ort, What is the maximum efficiency with which photosynthesis can convert solar energy into biomass?, Current Opinion in Biotechnology, Volume 19, Issue 2, April 2008, Pages 153 159, ISSN 0958 1669, 國立交通大學生物科技學系蘭宜錚老師 12
Cyanobacteria uses protein cage Carboxysome to increase CO 2 concentration around Rubisco Walter Bonacci, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver, and David F. Savage. Modularity of a carbon fixing protein organelle. PNAS 2012 109 (2) 478 483; published ahead of print December 19, 2011, doi:10.1073/pnas.1108557109 Cyanobacteria uses protein cage Carboxysome to increase CO 2 concentration around Rubisco Diameter around 100 nm Tsai Y, Sawaya MR, Cannon GC, Cai F, Williams EB, Heinhorst S, Kerfeld CA, Yeates TO - PLoS biology - Tsai Y, Sawaya MR, Cannon GC, et al (June 2007). "Structural analysis of CsoS1A and the protein shell of the Halothiobacillus neapolitanus carboxysome". PLoS Biol. 5 (6): e144. DOI:10.1371/journal.pbio.0050144. PMID 17518518. PMC: 1872035. 國立交通大學生物科技學系蘭宜錚老師 13