Light Reactions of Photosynthesis

2 H2O + 2 NADP+ + 8 photons →

O2 + 2 NADPH + 2 H+

ANIMATION

Integration of photosystems I and II in chloroplasts. The "Z scheme“ evolved by combining 2 bacterial RCs.

Reaction center chlorophylls lie close to the exiton acceptor preventing internal conversion (fluorescence). This fixed orientation mimics the solid state.

Resembles that of green sSulfur bacteria

Resembles that of purple bacteria.

analogous to cyt c

Cyclic vs. Nonclyclic PS

• animation

Photosystem II of the cyanobacterium Synechococcus elongates

All electron carriers are bound to a

nearly symmetric

dimer. Participants are positioned like the bacterial RC in purple

bacteria.

Light Reactions of Photosynthesis PS II

4 P680 + 4 H+ + 2 PQB + 4 photons →

4 P680+ + 2 PQBH2

The supramolecular complex of PSI and its associated antenna chlorophylls

Light Reactions of Photosynthesis PS I

4 P700 + 2 H+ + 2 NADP+ + 4 photons →

4 P700+ + 2 NADPH

Electron and proton flow through the cytochrome b6f complex

Plastoquinol (PQH2) formed in PSII is oxidized

by the cytochrome b6f complex in a series of

steps like those of the Q cycle in the cytochrome

Complex III of mitochondria. One

electron from PQH2 passes to the Fe-S center of the Rieske protein, the

other to heme bL of cytochrome b6. The net

effect is passage of electrons from PQH2 to

the soluble protein plastocyanin, which carries them to PSI.

Heme groups

f for frons

Localization of PSI and PSII in thylakoid membranes

• Non cyclic electron flow (PSI + PS II) produces a proton gradient + NADPH

• Cyclic electron flow (PSI) produces a proton gradient only

• Calvin cycle requires ATP and NADPH in a ratio of 3:2

• The 2 PS are physically separated to prevent exitons from leaving P680 and transferring to P700. Why would this happen?– (longer wavelength, lower energy)

Localization of PSI and PSII in thylakoid membranes. PSII is present almost exclusively in the appressed regions (granal lamellae [stacks], in which several membranes are in contact) , and PSI almost exclusively in nonappressed (stromal lamellae) regions, exposed to the stroma. LHCII is the "adhesive" that holds appressed lamellae together.

Accumulation of plastoquinol stimulates a protein kinase that phosphorylates a Thr residue in the hydrophobic domain of LHCII, which reduces its affinity for the neighboring thylakoid membrane and converts appressed regions to nonappressed regions (state 2). A specific protein phosphatase reverses this regulatory phosphorylation when the [PQ]/[PQH2] ratio increases.

Balancing of electron flow in PSI and PSII by state transition

noncyclic cyclic

Water-splitting activity of the oxygen-evolving complex

Water splitting complex passes 4 electrons, 1 at a time, back to P680+. The electrons lost from the multinuclear Mn center pass one at a time to an oxidized Tyr residue in a PSII protein, then to P680+

H+ released into lumen.

Light-Induced Redox Reactions and Electron Transfer Cause Acidification of Lumen

Because the volume of the lumen is small, a few hydrogen ions dramatically change the pH: lumen = pH 5, stroma pH = 8!!!The proton-motive force across the thylakoid membrane drives the synthesis of ATP… sound familiar??

Thylakoid membrane is impermeable to hydrogen ions. Reaction centers, electron carriers and ATP synthases are located in this membraneUncouplers decouple light absorption from ATP synthesis.

In vitro ATP synthesis

• Incubate chloroplasts in pH 4 buffer in the dark. Buffer slowly entered thylakoids lowering the pH to 4.

• Add ADP and Pi and suddenly increase the pH to 8. (How would you do this?) IN THE DARK.

• !!!! ATP produced!!!

Proton and electron circuits in thylakoids.

Flow of Protons: Mitochondria, Chloroplasts, Bacteria

• According to endosymbiotic theory, mitochondria and chloroplasts arose from entrapped bacteria

• Bacterial cytosol became mitochondrial matrix and chloroplast stroma

Comparison of the topology of proton movement and ATP synthase orientation in the membranes of mitochondria, chloroplasts, and the bacterium E. coli.

Dual roles of cytochrome b6f and cytochrome c6 in cyanobacteria reflect evolutionary origins. Cyanobacteria use cytochrome b6f, cytochrome c6, and plastoquinone for both oxidative phosphorylation and photophosphorylation.

CHAPTER 20 Carbohydrate Biosynthesis

in Plants and Bacteria

– CO2 assimilation in photosynthetic organisms

– Photorespiration in C3 plants

– Avoiding photorespiration in C4 plants

Key topics:

Introduction to Anabolic Pathways

• Anabolism: how to build biomolecules

• Plants are extremely versatile in biosynthesis

– Can build organic compounds from CO2

– Can use energy of sunlight to support biosynthesis

– Can adopt to a variety of environmental situations

Plant versatility

• Autotrophic

• Nonmobile/motile

• CHO synthesis occurs in plastids

• Plants synthesize thick cell walls exterior to the cell containing the bulk of the cell’s CHO—how do they do this???

Assimilation of CO2 by Plants

Plants and Photosynthetic Microorganisms Support the Life of Animals and Fungi

• Plants capture the energy from the ultimate energy source and make it available via carbohydrates to animals and fungi

Photosynthetic organisms use the energy of sunlight to manufacture glucose and other organic products, which heterotrophic cells use as energy and carbon sources.

Biological reproduction occurs with near-perfect fidelity (although no 2 zebras have exactly the same stripes!).

Zebras are herivores.

CO2 Assimilation Occurs in Plastids

• Self-reproducing organelles found in plants and algae• Enclosed by a double membrane• Have their own small genome• Most plastid proteins are encoded in the nuclear DNA• The inner membrane is impermeable to ions such as

H+, and to polar and charged molecules

Amyloplasts filled with starch (dark granules) are stained with iodine in this section of Ranunculus (buttercups) root cells.

Amyloplasts are pastids without the internal membrane or pigments.

Origin and Differentiation of Plastids

• Plastids were acquired during evolution by early eukaryotes via endosymbiosis of photosynthetic cyanobacteria

• Plastids reproduce asexually via binary fission• The undifferentiated protoplastids in plants can

differentiate into several types, each with a distinct function– Chloroplasts for photosynthesis– Amyloplasts for starch storage– Chromoplasts for pigment storage – Elaioplasts for lipid storage– Proteinoplasts for protein storage

Proplastids in nonphotosynthetic tissues (such as root) give rise to amyloplasts, which contain large quantities of starch. All plant cells have plastids, and these organelles are the site of other important processes, including the synthesis of essential amino acids, thiamine, pyridoxal phosphate, flavins, and vitamins A, C, E, and K.

internal membranes lost

CO2 Assimilation

• The assimilation of carbon dioxide occurs in the stroma of chloroplasts via a cyclic process known as the Calvin cycle

• The key intermediate, ribulose 1,5-bisphosphate is constantly regenerated using energy of ATP

• The key enzyme, ribulose 1,5-bisphosphate carboxylase / oxygenase (Rubisco), is probably the most abundant protein on Earth

• The net result is the reduction of CO2 with NADPH

that was generated in the light reactions of photosynthesis

Early studies of the Calvin cycle

• Design an experiment to discover the pathway for carbon assimilation

• First intermediate recognized was 3-PGA

• Search for a 2 carbon acceptor---FAILURE

• Actual acceptor….

CO2 Assimilation

• The assimilation of carbon dioxide occurs in the stroma of chloroplasts via a cyclic process known as the Calvin cycle

• The key intermediate, ribulose 1,5-bisphosphate is constantly regenerated using energy of ATP

• The key enzyme, ribulose 1,5-bisphosphate carboxylase / oxygenase (Rubisco), is probably the most abundant protein on Earth

• The net result is the reduction of CO2 with NADPH

that was generated in the light reactions of photosynthesis

The Calvin Cycle

The Structure and Function of Rubisco

• Rubisco is a large Mg++-containing enzyme that makes a new carbon-carbon bond using CO2

as a substrate

Structure of ribulose 1,5-bisphosphate carboxylase (rubisco). Ribbon model of form II rubisco from the bacterium Rhodospirillum rubrum. The subunits are in gray and blue. A Lys residue at the active site that is carboxylated to a carbamate in the active enzyme

is shown in red. The substrate, ribulose 1,5-bisphosphate, is yellow; Mg2+ is green.

Central role of Mg2+ in the catalytic mechanism of

rubisco.

Mg2+ is coordinated in a roughly octahedral complex with six

oxygen atoms: one oxygen in the carbamate on Lys201; two in the

carboxyl groups of Glu204 and Asp203; two at C-2 and C-3 of

the substrate, ribulose 1,5-bisphosphate; and one in the

other substrate, CO2.

First stage of CO2 assimilation: rubisco's carboxylase activity.

Another ene-diol intermediate!!!

Catalytic Role of Mg++ in Rubisco’s Carboxylase Activity

• Notice that Mg++ is held by negatively charged side chains of

• glutamate,

• aspartate, and

• carbamoylated lysine

• Mg++ brings together the reactants in a correct orientation, and stabilizes the negative charge that forms upon the nucleophilic attack of enediolate to CO2

Rubisco is Activated via Covalent Modification of the

Active Site Lysine

Synthesis of Glyceraldehyde-3 Phosphate (First Stage)

• Three rounds of the Calvin cycle fix three CO2

molecules and produce one molecule of 3-phosphoglycerate

Fate of Glyceraldehyde 3-phosphate (Second Stage)

• Converted to starch in the chloroplast• Converted to sucrose for export• Recycled to ribulose 1,5-bisphosphate

Interconversion of Triose Phosphates and Pentose

Phosphates

• This is how ribulose 1,5-bisphosphate is regenerated in the third stage of the Calvin cycle

Sugar interconversions

Transketolase Reactions

Transketolase Uses Thiamine Pyrophosphate as the

Cofactor

Stoichiometry and Energy Cost of CO2 Assimilation

• Fixation of three CO2 molecules yields one

glyceraldehyde 3-phosphate

• Nine ATP molecules and six NADPH molecules are consumed

Photosynthesis: From Light and CO2 to Glyceraldehyde 3-

phosphate

• The photosynthesis of one molecule of glyceraldehyde 3-phosphate requires the capture of roughly 24 photons

ATP and NADPH produced by the light reactions are essential substrates for the reduction of CO2

Enzymes in the Calvin Cycle are Regulated by Light

• Target enzymes are – ribulose 5-phosphate kinase, – fructose 1,6-bisphosphatase, – seduloheptose 1,7-bisphosphatase, and– glyceraldehyde 3-phosphate

dehydrogenase

Light activation of several enzymes of the Calvin cycle

Photorespiration• So far, we saw that plants oxidize water to O2

and reduce CO2 to carbohydrates during the

photosynthesis

• Plants also have mitochondria where usual respiration with consumption of O2 occurs in the

dark

• In addition, a wasteful side reaction catalyzed by Rubisco occurs in mitochondria

• This reaction consumes oxygen and is called photorespiration; unlike mitochondrial respiration, this process does not yield energy

Oxygenase Activity of Rubisco

• The reactive nucleophile in the Rubisco reaction is the electron-rich enediol form of ribulose 1,5-bisphosphate

• The active site meant for CO2 also

accommodates O2

• Mg++ also stabilizes the hydroperoxy anion that forms by electron transfer from the enediol to oxygen

Salvage of 2-Phosphoglycerate

• Complex ATP-consuming process for the recovery of C2 fragments from the

photorespiration

• Requires oxidation of glycolate with molecular oxygen in peroxisomes, and formation of H2O2

• Involves a loss of a carbon as CO2 by

mitochondrial decarboxylation of glycine

Glycolate pathway

Rubisco in C3 Plants Cannot

Avoid Oxygen

• Plants that assimilate dissolved CO2 in the

mesophyll of the leaf into three-carbon 3-phosphoglycerate are called the C3 plants

• Our atmosphere contains about 21% of oxygen and 0.038% of carbon dioxide

• The dissolved concentrations in pure water are about 260 M O2 and 11 M CO2 (at the

equilibrium and room temperature)

• The Km of Rubisco for oxygen is about 350 M

Separation of CO2 capture and the

Rubisco Reaction in C4 Plants

• Many tropical plants avoid wasteful photorespiration by a physical separation of CO2 capture and Rubisco

activity

• CO2 is captured into oxaloacetate (C4) in mesophyll

cells

• CO2 is transported to bundle-sheath cells where

Rubisco is located

• The local concentration of CO2 in bundle-sheath cells

is much higher than the concentration of O2

Carbon assimilation in C4 plants

Chapter 20: Summary

• ATP and NADPH from light reactions are needed in order to assimilate

CO2 into carbohydrates

• Assimilations of three CO2 molecules via the Calvin cycle leads to the

formation of one molecule of 3-phosphoglycerate

• 3-Phosphoglycerate is a precursor for the synthesis of larger

carbohydrates such as fructose and starch

• The key enzyme of the Calvin cycle, Rubisco, fixes carbon dioxide into

carbohydrates

• Low selectivity of Rubisco causes a wasteful incorporation of molecular

oxygen in C3 plants; this is avoided in C4 plants by increasing the

concentration of CO2 near Rubisco

In this chapter, we learned that: