Molecular Biochemistry II

Calvin Cycle - Photosynthetic Carbon Reactions

Contents of this page:
Localization of photosynthetic pathways
CO2 Fixation - role of Ribulose Bisphosphate Carboxylase (RuBisCO)
Photorespiration
Rest of the Calvin Cycle (Carbon Reactions) pathway
Regulation of Calvin Cycle

Photosynthesis in plants takes place in chloroplasts. Photosynthesis includes light-dependent reactions and reactions that are not directly energized by light.

The structure of a chloroplast is shown on p. 872 of Biochemistry, by Voet & Voet, 3rd Edition, and schematically represented at right. 

In the photosynthetic light reactions, energy of light is conserved as as "high energy" phosphoanhydride bonds of ATP, and as reducing power of NADPH.  The proteins and pigments responsible for the photosynthetic light reaction are associated with the thylakoid (grana disk) membranes. The light reaction pathways will not be presented here.

The Calvin Cycle, earlier designated the photosynthetic "dark reactions" pathway, is now referred to as the carbon reactions pathway. In this pathway, the free energy of cleavage of ~P bonds of ATP, and reducing power of NADPH, are used to fix and reduce CO2 to form carbohydrate. Enzymes and intermediates of the Calvin Cycle are located in the chloroplast stroma, a compartment somewhat analogous to the mitochondrial matrix

Ribulose Bisphosphate Carboxylase (RuBP Carboxylase) catalyzes CO2 fixation:

ribulose-1,5-bisphosphate + CO2 2 copies of 3-phosphoglycerate

Because it can alternatively catalyze an oxygenase reaction (discussed below), the enzyme is also called RuBP Carboxylase/Oxygenase (RuBisCO). It is the most abundant enzyme on earth. The RuBP Carboxylase reaction mechanism is presented on p. 900.

Extraction of a proton from C3 of ribulose-1,5-bisphosphate (RuBP, below left) promotes formation of an endiolate intermediate.
Nucleophilic attack on CO2 is proposed to yield a b-keto acid intermediate, that reacts with water and cleaves to form 2 molecules of  3-phosphoglycerate.
Transition state analogs of the postulated b-keto acid intermediate bind tightly to the enzyme and inhibit its activity. Examples include 2-carboxyarabinitol-1,5-bisphosphate (CABP, at right) and carboxyarabinitol-1-phosphate (CA1P).

 RuBP Carboxylase in plants is a complex (L8S8) of:

Some bacteria contain only the large subunit, with the smallest functional unit being a homodimer, L2. Roles of the small subunits have not been clearly defined, although there is some evidence that interactions between large and small subunits may regulate catalysis.

At right are 2 views of spinach RuBisCO (RuBP Carboxylase), with large subunits colored blue or cyan, and small subunits colored red.
Large subunits within RuBisCO are arranged as antiparallel dimers, with the N-terminal domain of one monomer adjacent to the C-terminal domain of the other monomer.

Each active site is at an interface between  monomers within an L2 dimer, explaining the minimal requirement for a dimeric structure.

The substrate binding site is at the mouth of an ab-barrel domain of the large subunit. Most active site residues are polar, including some charged amino acids (e.g., Thr, Asn, Glu, Lys).

"Active" RuBP Carboxylase includes a carbamate group, that binds an essential Mg++ at the active site. 

The carbamate forms by reaction of HCO3- with the e-amino group of a lysine residue of RuBP Carboxylase, in the presence of Mg++. HCO3- that reacts to form the carbamate group is distinct from CO2 that binds to RuBP Carboxylase as substrate. 

The active site Mg++ bridges between oxygen atoms of the carbamate and the substrate CO2.

Binding of either the normal substrate ribulose-1,5-bisphosphate or a transition state analog to RuBP Carboxylase causes a conformational change to a "closed" conformation in which access of solvent water to the active site is blocked.

RuBP Carboxylase (RuBisCO) can spontaneously deactivate by decarbamylation. In the absence of the carbamate group, RuBisCO tightly binds ribulose bisphosphate (RuBP) or another sugar phosphate at the active site as a "dead end" complex, with the closed conformation, and is inactive in catalysis. In order for the carbamate to reform, the enzyme must undergo transition to the open conformation.

RuBP Carboxylase Activase, an ATP hydrolyzing (ATPase) enzyme, causes a conformational change in RuBP Carboxylase from closed to open form. This allows release of tightly bound RuBP or other sugar phosphate from the active site, and carbamate formation. Since photosynthetic light reactions produce ATP, the ATP dependence of RuBisCO activation provides a mechanism for light-dependent activation of the enzyme.

RuBP Carboxylase Activase is a member of the AAA family of ATPases, many of which have chaperone-like roles. The activase is a large multimeric protein complex that may surround RuBP Carboxylase while inducing the conformational change to the open state.

Explore at right the bound carbamate and reaction products at the active site of RuBisCO. 


RuBP Carboxylase

Now explore the entire complex of 8 large subunits and 8 small subunits of spinach RuBisCO. (PDB file 1RCX, structure determined by T. C. Taylor & I Andersson in 1996.)

Select protein, display as spacefill, and color chain to distinguish the different subunits.
Note that because of the large number of chains (16), some chains have similar colors.
If necessary, to eliminate water molecules, select residue HOH and select hide

Question: Is the interior of the complex solid or hollow?
Hint: Try selecting option slab mode, and control-drag to view the interior.

View the anti-parallel arrangement of a pair of large subunits.
Turn off slab mode, and then select all, and select hide.
Separately select chains E and H, and change their displays to cartoon, with color chain.
Select hetero-ligand and display spacefill with color CPK to visualize bound substrate ribulose-1,5-bisphosphate. Note the positions of the two active sites within the pair of large subunits.

Now separately select chains E & H and change their displays to spacefill.
Use slab mode  to investigate the extent to which active site residues contact one or both subunits.
Question: How is this structure consistent with the smallest functional complex being a dimer of large subunits?


  C   O   N   S   P


Photorespiration:
O2
can compete with CO2 for binding to RuBisCO, especially when [CO2] is low and [O2] is high. Reaction of O2 with ribulose-1,5-bisphosphate yields one molecule of 3-phosphoglycerate plus the 2-carbon compound 2-phosphoglycolate. See diagram p. 902.

This reaction is the basis for the name RuBP Carboxylase/ Oxygenase (RuBisCO).

The complex pathway that partly salvages carbon from 2-phosphoglycolate, via conversion to 3-phosphoglycerate, involves enzymes of chloroplasts, peroxisomes and mitochondria. This pathway recovers 3/4 of the carbon from 2-phosphoglycolate as 3-phosphoglycerate while the rest is released as CO2. For diagrams see p. 903 and an article by Reumann & Weber.

Photorespiration is a wasteful process, substantially reducing efficiency of CO2 fixation.

C3 vs C4 plants:

Most plants, designated C3, fix CO2 initially via RuBP Carboxylase, yielding the 3-carbon compound 3-phosphoglycerate.

Plants designated C4 have one cell type in which phosphoenolpyruvate (PEP) is carboxylated via the enzyme PEP Carboxylase, to yield the 4-carbon compound oxaloacetate. The oxaloacetate is converted to other 4-carbon intermediates that are transported to cells active in photosynthesis, where CO2 is released by decarboxylation.
See diagram p. 904.
C4 plants maintain a high ratio of CO2/O2 within photosynthetic cells, thus minimizing photorespiration. Some research has been aimed at increasing expression of and/or inserting genes for C4 pathway enzymes, such as PEP Carboxylase, in C3 plants.
 
Continuing with Calvin Cycle:

The normal product of the RuBP Carboxylase reaction, 3-phosphoglycerate, is converted to glyceraldehyde-3-phosphate

A portion of the glyceraldehyde-3-phosphate is converted back to ribulose-1,5-bisphosphate, via reactions catalyzed by Triose Phosphate Isomerase, Aldolase, Fructose Bisphosphatase, Sedoheptulose Bisphosphatase, Transketolase, Epimerase, Ribose Phosphate Isomerase, and Phosphoribulokinase. Many of these enzymes are equivalent to enzymes of the cytosolic Glycolysis, Gluconeogenesis and Pentose Phosphate Pathways, but are separate gene products resident within the chloroplast stroma. (Enzymes of the other pathways listed are located in the cytosol.) The process is similar to the Pentose Phosphate Pathway running backwards.

For three molecules of ribulose-1,5-bisphosphate (total of 15 C) that are carboxylated, cleaved, phosphorylated, reduced, and dephosphorylated, six molecules of glyceraldehyde-3-phosphate are produced (total of 18 C). Of these:

C3 + C3 C6
C3
+ C6
C5 + C4
C3
+ C4
C7
C3
+ C7
C5 + C5

Overall: 5C3 3C

Enzymes in the diagram at right:
TI = Triosephosphate Isomerase
AL = Aldolase
FB = Fructose-1,6-bisphosphatase
SB = Sedoheptulose-1,7-bisphosphatase
TK = Transketolase
EP = Epimerase
IS = Isomerase
PK = Phosphoribulokinase

See also diagram p. 897.

Summary of Calvin Cycle, omitting compounds that are regenerated:

3 CO2 + 9 ATP + 6 NADPH glyceraldehyde-3-phosphate + 9 ADP + 8 Pi + 6 NADP+

Glyceraldehyde-3-phosphate may be converted to other carbohydrates such as metabolites (e.g., fructose-6-phosphate and glucose-1-phosphate), energy stores (e.g., sucrose or starch), or cell wall constituents (e.g., cellulose). Glyceraldehyde-3-phosphate can also be utilized by plant cells as carbon source for synthesis of other compounds such as fatty acids and amino acids.

There is evidence for the existence of multienzyme complexes of Calvin Cycle enzymes within the chloroplast stroma. Positioning of many Calvin Cycle enzymes close to the enzymes that produce their substrates or utilize their reaction products may increase efficiency of the pathway.

Regulation of Calvin Cycle:

Regulation prevents the Calvin Cycle from being active in the dark, when it might function in a futile cycle with Glycolysis and Pentose Phosphate Pathways, wasting ATP and NADPH. 

Light activates, or dark inhibits, the Calvin Cycle (previously called the "dark reaction") in several ways.

Light-activated electron transfer is linked to pumping of H+ into thylakoid disks. The pH in the stroma increases to about 8. The alkaline pH activates stromal Calvin Cycle enzymes RuBP Carboxylase, Fructose-1,6-Bisphosphatase and Sedoheptulose Bisphosphatase.

The light-activated shift of H+ into thylakoid disks is countered by Mg++ release from the thylakoids to the stroma. RuBP Carboxylase (in the stroma) requires Mg++ binding to carbamate at the active site.

Some plants synthesize the transition-state inhibitor carboxyarabinitol-1-phosphate (CA1P) in the dark. RuBP Carboxylase Activase facilitates release of CA1P from RuBP Carboxylase, when it is activated under conditions of light via thioredoxin.

Thioredoxin is a small protein with a disulfide that is reduced in chloroplasts via light-activated electron transfer. Thioredoxin f, from spinach chloroplasts, is shown at right (structure solved by G. Capitani, Z. Markovic-Housley, G. Delval, M. Morris, J. N. Jansonius, & P. Schurmann in 2000).

Select display cartoon and color structure. The twisted 5-stranded b-sheet surrounded by 4 a-helices is typical of members of the thioredoxin protein family.


Select residue-CYS, change display of these residues to ball & stick with color CPK.
Identify the functional disulfide made up of 2 cysteine residues in this oxidized form of thioredoxin.
Look for the thioredoxin consensus sequence that contains the disulfide Trp-Cys-Gly-Pro-Cys.


  C   O   N   S 

During illumination, the thioredoxin disulfide is reduced to a dithiol by ferredoxin, a constituent of the photosynthetic light reaction pathway, via an enzyme Ferredoxin-Thioredoxin Reductase. See also diagram p. 902.

The reduced thioredoxin activates several Calvin Cycle enzymes, including Fructose-1,6-bisphosphatase, Sedoheptulose-1,7-bisphosphatase, and RuBP Carboxylase Activase, by reducing specific disulfides in these enzymes to thiols. 

Copyright 1998-2008 by Joyce J. Diwan. All rights reserved.

Additional material on Calvin Cycle:
Readings, Test Questions & Tutorial

ppicon.gif (2458 bytes)