EFB530 Plant Physiology

Carbon fixation-Photosynthetic carbon reduction (PCR) or C3 cycle

Mechanism by which plants can take up CO₂ from the atmosphere and incorporate it into complex organic molecules (biomass)

• 200 billion tons of CO₂ converted to biomass annually
• 40% of that by marine phytoplankton

Light reactions generated ATP and NADPH-those will be utilized in the reactions of the C3 cycle

Breakthrough experiments in the discovery of the C3 cycle

• Melvin Calvin, Andy Benson, and James Bassham
• UC-Berkeley in the late 40's and early 50's
• Calvin won the Nobel prize in 1961 (Calvin cycle)

They used carbon-14 in the form of ¹⁴CO₂ fed to algal cells to radioactively label the compounds formed early on in photosynthesis

• added ¹⁴CO₂, then poured the algae into hot ethanol to stop the reactions
• extracted the radioactive compounds
• separated them by paper chromatography
• compared those spots to known standards

They saw the ¹⁴C label appear earliest in a 3-carbon sugar phosphate (3-phosphoglycerate)

• later learned it occurred by the addition of CO₂ to a 5-carbon sugar-phosphate
• (ribulose-1,5-bisphosphate) that split into 2 molecules of 3-PGA

Shortly after that, the enzyme that catalyzes this reaction was identified

• ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)
• Rubisco=most abundant protein on Earth=10,000,000 tons of Rubisco
• represents ~15-25% of all of the protein in a leaf, so we eat alot of it

Active Rubisco consists of

• 8 large subunits (catalytic site, chloroplast gene)
• and 8 small subunits (assists assembly, nuclear gene)

1) Carboxylation

2) Reduction

3) Regeneration

Carboxylation

Reaction mechanism: 2-step reaction, note position of ¹⁴C atom

See reaction mechanism in Figure 8.4 (page 149)

1) The equilibrium constant for the carboxylation reaction strongly favors the forward reaction

2) Rubisco has strong affinity for CO₂

• these combined allow carboxylation reaction to proceed to completion even in very low CO₂ concentrations

12 PGA + 12 ATP -> 12 bisPGA + 12 NADPH + 12 H⁺ -> 12 GAP + 12 NADP⁺ + 12 P_i

See reactions diagrammed in Table 8.1-reactions 2 and 3

• the reduction reaction is accomplished by the NADPH (provides reducing equivalents)
• ATP provides chemical energy

the carboxylation reaction requires RuBP (5 C sugar-phosphate)

• so this process of carbon fixation needs to regenerate RuBP to allow fixation to continue (that makes this a cycle)

combinations of different length sugar-phosphates to eventually regenerate RuBP, also yields sugar-phosphate for sucrose/starch synthesis

• 3-GAP (3 C) -> DHAP (3 C)
• DHAP (3C) + GAP (3 C) -> fructose-1,6-bisphosphate (6C)

• fru-1,6-bisP (6C) + H₂O -> fru-6-P (6C) + Pi
• fru-6-P (6 C) + GAP (3 C) -> Xylulose-5-phosphate (5 C) + erythrose-4-phosphate (4 C)

• erythrose 4-P (4 C) + DHAP (3 C) -> sedoheptulose 1,7-bisphosphate (7 C)
   
• sedoheptulose 1,7-bisP + H₂O -> sedoheptulose 7-P + P_i
• sedoheptulose 7-P (7 C) + GAP (3 C) -> xylulose 5-phosphate (5 C) + ribose 5-phosphate (5 C)

• xylulose 5-P or ribose 5-P -> ribulose 5-P
• ribulose 5-P + ATP -> RuBP

for 6 CO₂:

• 12 ATP and 12 NADPH during reduction steps
• 6 ATP to regenerate RuBP

=18 ATP + 12 NADPH

• this generates 2 triose-phosphates for sucrose/starch synthesis

• per CO₂ the C3 cycle uses 3 ATP and 2 NADPH
• to reduce 2 NADP⁺ to 2 NADPH,  4 electrons must be driven through the electron transport chain
• need 8 photons (minimum to drive 4 electrons through PSII and PSI) per CO₂
• 680 nm light = 175 kJ per quantum
• minimum light energy to fix a molecule of hexose=6 x 8 x 175 kJ = 8400 kJ
• energy in fructose=2804 kJ
• overall efficiency =  ~30%
   
• most of that energy is lost in the "light reactions", because . . .
• ATP=29 kJ; NADPH=217 kJ (products of light reactions)
• 18 ATP=(18x29) + 12 NADPH=(12x217)=3126 kJ
• 2804 kJ (in fructose) / 3126 kJ = ~90% efficiency of C3 cycle

Many of the enzymes in the C3 cycle are regulated at the level of enzyme activity by light

• they are active in the light

1) Mg²⁺ concentration increases in the stroma in the light

• Mg²⁺ exchanges for H⁺ pumped into the lumen during electron transport to maintain charge balance
• Mg²⁺ activates Rubisco

2) pH rises in the stroma during electron transport (H⁺ pumped into lumen) pH 7 -> 8

• Rubisco and others more active at pH 8

3) covalent modification related to the redox state

• during electron transport, ferredoxin is reduced
• some of the Fd reduces thioredoxin
• thioredoxin can reduce some of the C3 cycle enzymes, making them active
• (S-S bonds reduced to -SH HS-)
• oxidation occurs in the dark to inactivate these enzymes

4) many of these proteins are encoded by genes that are regulated at the level of transcription by light

besides, ATP and NADPH are only produced when photosynthetic electron transport is active

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