Biology 2415 Lecture Notes
Chapter 11: Phototrophic energy metabolism: Photosynthesis
- Overview of photosynthesis
- The chloroplast
- Photosynthetic energy transduction
- Accessory pigments
- Photosystems and light harvesting complexes
- Two types of phtotosysntems
- Photoreduction (NADPH synthesis) in oxygenic phototrophs
- Photosystem II transfers electrons from water to plastoquinone
- Cytochrome b6/f complex transfers electrons from a plastoquinol to plastocyanin
- Phtotosytem I transfers electrons from plastocyanin to ferredoxin
- Ferredoxin –NADP+ reductase catalyzes the reduction of NADP+
- ATP synthase comlex couples transport of protons across the thylakoid membrane to ATP sysnthesis
- Cyclic photophosphorylation
- The complete energy transduction system
- Photoreaction center from purple bacteria
- The Calvin cycle: carbon assimilation
- Overall reaction for photosynthesis
This chapter deals with the most important biochemical process on earth, photosynthesis. This is because photosynthesis provides reduced forms of carbon (carbohydrates, fats and proteins) in the biosphere upon which all chemotrophs depend on.
Photosynthesis originated ~ 3.5 billion years ago, probably in a Gram-negative purple bacteria. It was the first to evolve a reaction center, a transmembrane complex of proteins, pigments, and redox cofactors capable of absorbing light energy and initiating an electron transport pathway that pumps H+s across a membrane. Such photosystems have a 40% efficiency at converting light energy into chemical energy (better than photovoltaic cells).
Photoreaction reaction centers can be divided into two basic types:
- Type II:
- Present in purple bacteria, green filamentous bacteria. Have similarity to photosysmtem II of cyanobacteria and chloroplasts.
- Use pheophytin and a quinone as electron acceptor.
- Type I:
- Present in green sulfur bacteria and heliobacteria. Have similarity to photosysmtem I of cyanobacteria and chloroplast.
- Have iron-sulfur centers as electron acceptors
Cyanobacteria are the only photosynthetic prokaryotes that have BOTH types of photosystems. They also have a manganese containing enzyme which splits water to O2. This process has had an enormous impact on the evolution of life on earth.
In Eukaryotes, photosynthesis takes place in chloroplasts which evolved from cyanobacteria. Consequently, the type of photosynthesis carried out in eukaryotes is essentially the same as in cyanobacteria. The chloroplast retains a circular genome containing ~ 250 genes, encoding protein subunits involved in photosynthesis, chloroplast division, ribosomal RNAs and proteins, and a complete set of tRNAs. Over time, many chloroplast genes have moved into the nucleus. Chloroplast proteins encoded in these nuclear genes are synthesized in the cytosol and posttranslationally transported into the chloroplast.
- Photosynthesis = conversion of light energy into chemical energy.
- Phototrophs = organisms that convert solar energy to chemical energy in the form of ATP and NADPH.
- Photoheterotrophs = organisms that use light for energy, but depend on organic sources of reduced carbon.
- Photoautotrophs = organisms that use light energy to drive the synthesis of organic molecules from CO2 and H2O.
- Oxygenic phototrophs = release oxygen as a biproduct of splitting water.
We will focus mostly on how photosynthetic organisms capture sunlight to make ATP and NADPH. Our coverage of carbon assimilation in the Calvin cycle will be brief.
Overview of photosynthesis
|Photosynthesis involves two major processes (Fig 11.1).
- Energy transduction: light energy is captured by chrolophyl and converted into chemical energy in the form of ATP and the reduced coenzyme, NADPH.
- Carbon assimilation: ATP and reducing power in the form of NADPH, used in the Calvin cycle to convert CO2 into sugars.
- Light energy is captured by a family of pigments called chlorophyls, which are present in the green leaves of plants, and in the cells of algae.
- Light excites electrons in chlorophyll, which are ejected and flow energeticaly downhill through an electron transport system. The ETS generates a proton gradient used to drive the synthesis of ATP.
- Photophosphorylation = light-driven ATP synthesis.
- In oxygenic phototrophs, light energy absorbed by chlorophyl drives the movement of electrons from water ( very “+” reduction potential) to ferredoxin (very “-“ reduction potential). From ferredoxin, electrons move exergonically to NADP+ to form NADPH (photoreduction). NAPDH is used as reducing power (ie source of electrons) to make organic molecules in the Calvin cycle.
- Summary equation for photosynthesis:
Light + 3CO2 + 6H2O -----------> C3H6O3 + 3O2 +3H2O
- Immediate product of photosynthesis is triose phosphate (3C), which then enters a variety of biosynthetic pathways, including that of sucrose and starch.
- Sucrose: majort transport carbohydrate in plants; transfer energy from photosynthetic cells to nonphotosynthetic cells in the organism.
- Starch: major storage molecule which accumulates when carbon assimilation exceeds the energy requirements.
- In plants and algae, photosynthesis takes place in chloroplasts (Fig 11.2).
- Typical plant cell contains 20-100. Typical algae, 1 or a few.
- Shapes vary substantially, from flattened spheres to ribbon-shape in Spirogyra.
- Chloroplasts are a kind of plastid and develop from proplastids. Other plastids include: amyloplasts, chromoplasts, proteinoplasts, and elaioplasts.
- Chloroplasts have 3 membrane systems (Fig 11.3):
- Have outer membrane, inner membrane, intermembrane space. Inside inner membrane is the stroma, where carbon, nitrogen and sulfur assimilation takes place.
- Outer membrane contains porins which permit free movement of small organic molecules and ions (anything smaller than 5000 Daltons).
- Inner membrane is a significant permeability barrier. Specific transporters are required to transport virtually all ions and polar solutes. CO2, O2, and H2 diffuse readily across inner and outer membranes.
- Thylakoids = membranous structures on which the light reactions of photosynthesis take place. Made of flat, sac-like structures suspended in stroma. Stacks of thylakoids are called grana. Stacks are interconnected by stroma thylakoids.
- Thylakoid membranes contain the photosynthetic pigments, photosystems, ETS, and ATP synthase, required to perform the light reactions of photosynthesis.
- Thylakoid lumen = single, continuous inner compartment of thylakoids; provides a compartment into which ETS pumps protons to establish a proton motive force (pmf) relative to stroma.
- Prokaryotes do not have chloroplasts. In Cyanobacteria (ancestors of chloroplasts) cell membrane invagination provides photosynthetic membranes (during evolution, pinching off of these membranes resulted in thylakoids).
Photosynthetic energy transduction
- To understand photosynthesis, we must understand the nature of light.
- Light is electromagnetic radiation. Made of photons (light particles) which have wave-like properties. Each photon carries a quantum of energy.
- Energy of light is inversely proportional to the wavelength of the light. Visible light has wavelengths between 380 – 750 nm.
- When a pigment absorbs a photon, the energy of the photon is transferred to an electron, which moves from ground-state to an excited state. This is called photoexcitation, and it is the first stage of photosynthesis.
- Because of different electron configurations, different pigments display unique absorbtion spectra (Fig 11.5).
- Photoexcited electrons in pigmnents are unstable, and must return to ground state or transfer to another molecule.
- When electron returns to ground state in same pigment molecule, excitation energy is lost in either of two ways:
- Fluorescence: light with smaller energy quantum is released as well as heat.
- Resonance energy transfer: photoexcited electrons transfer their energy to an electron in adjacent pigment molecule.
- Photochemical reduction = transfer of photoexcited electrons to a high energy orbital in another molecule.
- Chlorophyl links life to sunlight. Found in almost all photosynthetic organisms; primary energy transduction pigment channeling solar energy into the biosphere.
- Two types: Chlorophyl a and b (Fig 11.6):
- Contains porphyrin ring with conjugated double bonds for absorbing light energy; also contains hydrophobic phytol sidechain used to anchor chlorophyll in the lipid bilayer of the thylakoid membranes.
- Contains Mg2+; affects electron distribution such that more wavelengths of light are absorbed.
- Chlorophyls have a broad absorption spectra (absorb optimally at 420 and 680 nm).
- Plants contain chlorophyls a and b. Together, they increase the spectrum of light absorbed.
- Other organisms supplement chlorophyll a with either chlorophyll c (algae and diatoms), chlorophyll d (red algae), or phycobilin (cyanobacteria).
- Accessory pigments absorb photons that cannot be absorbed by chlorophyll. Enables collection of energy from a larger portion of sunlight.
- Carotenoids: two most common are b-carotene and lutein. When not masked by chlorophyll, confer orange and yellow colors to plants. Absorb photons from broad range of blue wavelengths.
- Phycobilins: found only in red algae and cyanobacteria. Two most common are:
- Phycoerythrin: absorb photons from blue, green, and yellow regions of light, enabling red algae to use dim light under water.
- Phycocyanin: absorbs from orange regions; more characteristic of cyanobacteria living on surface of water.
- Variations in quantity and properties of accessory pigments often reflect adaptations to specific environments.
Light Drives Electron flow in chloroplasts
- Rober Hill (1937): Iluminated leaf extracts containing chlorophyl produce oxygen and reduce an artificial electron acceptor (Dichlorophenolindophenol aka Hill Reagent. (blue when oxydized and colorless in reduced form)). Only occurs in presence of light. Does not occur in the dark.
- This was first evidence that absorbed light energy caused electrons to flow from H2O to an electron acceptor.
Photosystems and light harvesting complexes
- The functional units on the surface of the thylakoid membrane which convert light to chemical energy are called photosystems.
- Each photosystem contains chlorophyl molecules, accessory pigments and associated proteins.
- Chlorophyl-binding proteins stabilize arrangement of chlorophyl within a photosystem. Makes for better absorption spectra.
- Other proteins bind components of ETS or catalize redox reactions.
- Each photosystem contains a reaction center, the business end of photoexcitation. Reaction center contains 2 chlorophyl a molecules. It is this special pair of molecules that catalyzes conversion of solar energy into chemical energy.
- Energy absorbed by other pigments molecules gets transferred to the special pair via resonance energy transfer. The excited electrons move to a high energy orbital, and are then oxidized away.
- Each photosystem is generally associated with ligh-harvesting complex (LHC).
- LHC contains 80 to 250 chlorophyl a and b molecules along with carotenoids and pigment-binding proteins.
- LHC harvests solar energy, which is then transferred to photosystems via resonance energy transfer.
- LHC's do not have a reaction center.
- A photosystem complex refers to a photosystem associated with LHCs.
Two types of phtotosystems in oxygenic phototrophs
- Each electron passing from water to NADP+ must be photoexcited twice, first by photosystem II and then by photosystem I.
- The two photosystems were discovered by Robert Emerson (1940's):
- It was noticed that Chlorella (green algae) underwent a very large drop in O2 production when illuminated with wavelengths greater than 690 nm. When cells were supplemented with shorter wave (650 nm) light, the drop was less severe. The combination of long and short wave length exceeded the sum of activities with either wavelength. This synergistic effect became known as the Emerson enhancement effect, illustrated in Fig 11.8.
- Emerson enhancement effect is due to presence of photosystems I and II.
- Photosystem I (PSI) has reaction center with absorbance max at 700 nm.
- Photosystem II (PSII) has reaction center with absorption max of 680 nm.
- When cells are exposed only to wavlengths above 690, photosystem II does not work and photosynthesis is greatly reduced.
Photoreduction (NADPH synthesis) in oxygenic phototrophs
- Photoreduction (ie NADPH synthesis) in oxygenic phototrophs requires 2 different photosystem complexes to efficiently conserve energy of photoexcited electrons from water to NADP+ to form NADPH.
- Complete photoreduction pathway has several components and are illustrated in Fig 11.9.
- Each photosystem absorbs light and boosts electrons to "top" of an ETS.
- As electrons flow exergonically through ETS, their energy is conserved as a proton gradient, which ATP synthase uses as a source of energy to make ATP.
- Photosystem I passes electrons to ferredoxin (Fd) and then to NADP+.
Photosystem II transfers electrons from water to plastoquinone (Fig 11.9).
- Photoreduction starts at photosystem II which uses electrons from water to reduce plastoquinone to a plastoquinol.
- Photosystem II consists of
- D1 and D2 proteins; bind chlorophyl and componets of ETS.
- Reaction center: made of two chlorophyl a molecules, designated P680.
- 40-50 chlorophyl a and ~ 10 b-carotene molecules; enhance light capture.
- light-harvesting complex: contains ~ 20 molecules of chlorophyl + more catorenoids; further increase ability to capture photons and transfer energy by resonance transfer to the reaction center.
- When energy is absorbed by electrons in reaction center it decreases the reduction potential of the chlorophyl and the electrons pass to phaeophytin (Ph), a modidied chlorophyl (has 2 H+'s instead of Mg2+).
- From phaeophytin, electrons are passed to QA, a plastoquinone associated with D2 protein. Plastoquinone is similar to coenzyme Q.
- From QA, electrons are passed to QB, which is tighly associated with D1 protein.
- When QB receives 2 electrons, it also picks up 2 protons from the stroma to become QBH2, which is soluble in the lipid bilayer.
- To absorb photons continuously, oxidized P680 must be reduced each time electron is lost to plastoquinone.
- PSII contains an oxygen evolving complex (OEC).
- OEC = assembly of proteins and manganese ions that catalyze the oxidation of H2O into O2.
- For every two H2O molecules which are split, 4 electrons move, one at a time, via D1 to oxidized P680. Concomitantly, 4 H+'s and 1 O2 molecule are released within the thylakoid lumen, contributing to the proton gradient.
Cytochrome b6/f complex transfers electrons from a plastoquinol to plastocyanin
Photosytem I transfers electrons from plastocyanin to ferredoxin
- PSI contains a third chlorophyl a called A0 instead of phaeophytin (Ph). Also contains A1( a phylloquinone), and three iron-sulfur centers (FX, FA, and FB) which form an ETS linking A0 to ferredoxin.
- PSI also associated with light harvesting complex I (LHCI), which contains 80-120 chlorophyl and some carotenoid molecules. These antenna pigments capture light energy and transfer it to reaction center , P700. Energy absorbed by PSI lowers the reduction potential (i.e. more negative) of its P700.
- From A0, electrons flow exergonically through the ETS to ferredoxin, a mobile iron-sulfur protein located in the stroma.
- Oxidized P700 is reduced by electrons from plastocyanin in order to transfer electrons continually to ferredoxin.
- Summary equation catalyzed by PSI is:
4 photons + 4Pc(Cu+) + 4Fd(Fe3+) ----> 4Pc(Cu2+) + 4 Fd(Fe2+)
Ferredoxin –NADP+ reductase
- Ferredoxin-NADP+ reductase (FNR) catalyzes the final step in photoreduction, specifically, the transfer of electrons from ferredoxin to NADP+, generating NADPH.
- FNR is a peripheral protein located on the stoma side of the thylakoid membranes.
- For every 2 H2O molecules that are split in PSII, FNR carries out the following reaction:
4 Fd (Fe2+) + 2NADP+ + 2H+stroma ----> 4Fd(Fe3+) + 2 NADPH
- Reaction also contributes to proton gradient because 2H+ are consumed in the stroma, leading to an increase in pH.
- The various components of light reactions as shown in Fig 11.9 provide a continuous, unidirectional flow of electrons from H2O to NADP+. This unidirectional flow is called noncyclic electron flow.
- The net result of the complete light-dependent oxidation of 2 water molecules to O2 is:
4 photons @ PSII + 4 photons @ PSI + 2H2O + 6H+stroma + 2 NADP+ --> 8H+lumen + O2 + 2NADPH.
- Thus, solar energy is captured in two forms: NADPH and a proton gradient.
Photophosphorylation in oxygenic phototrophs
- Thylakoid membranes are impermeable to H+’s. Therefore, when chloroplasts are illuminated, the constant pumping of protons into the thylakoid lumen, coupled with the consumption of protons in the stroma, leads to a pH difference between the lumen and the stroma of about 2 pH units (i.e lumen has [H+] 100x greater than stroma).
- This proton gradient across the thylakoid membrane represents a source of potential energy.
ATP synthase complex
- In chloroplasts, as in mitochondria, ATP synthase couples the movement of protons down their concentration gradient to the phosphorylation of ADP to make ATP.
- ATP synthase in chloroplast is very similar to that of mitochondria and is designated as CF0CF1 complex.
- CF1: hydrophilic assembly of 5 polypeptides that protrudes from the stromal surface of thylakoid membranes; contains ATP synthase activity.
- CF0: assembly of polypeptides embedded in thylakoid membrane; forms hydrophilic channel between the lumen and stroma; proton translocator due to pmf.
- Reaction catalyzed by CF0CF1 complex is:
4H+lumen + ADP + Pi -----> 4H+stroma + ATP
- For every 4 electrons that flow though concyclic pathway, 2 NADPH and 2 ATP molecules are formed. However, the Calcin cycle, like many other metabolic pathaways, does not use ATP and NADPH in a 1:1 ratio. So how do phototrophs balance NADPH and ATP synthesis to meet the needs of the cell?
- When NADPH comsumption is low, phototrophs can divert reducing power generated by PSI into ATP synthesis by cyclic electron flow (Fig 10.10).
- In cyclic electron flow, reduced ferredoxin passes electrons to cytochrome b6/ f complex, instead of donating them to NADP+.
- Movement of electrons from ferredoxin to plastocyanon via cyt b6 / f complex is coupled to proton pumping, contributing to the pmf.
- From plastocyanin, electrons flow to P700 in PSI, completing the circuit. The access ATP generated from cyclic flow is called cyclic photophosphorylation. No water is oxidized and no O2 is generated.
The Calvin cycle: carbon assimilation
- Calvin cycle is the fundamental pathway by which virtually all CO2 is fixed and reduced into 3-carbon carbohydrates. Named after Melvin Calvin (1961 Nobel) who used 14CO2 to show that the primary products of photosynthetic CO2 fixation was triose phosphates.
- In plants and algae, Calvin cycle occurs in the stroma.
- Calvin cycle is shown in Fig 11.12. Divided into three stages:
- 1. Carboxylation of ribulose-1,5-biphosphate followed by immediate hydrolysis to generate 2 molecules of 3-phosphoglycerate.
- Catalyzed by rubisco (ribulose-1,5-biphosphate carboxylase/oxygenase); most abundant protein on the planet (~40 million tons); makes 50% of prtein in chloroplasts, which in turn represents up 10-25% of soluble protein in leaves; found in almost all photosynthetic organisms.
- 2. Reduction of 3-phosphoglycerate to form glyceraldehydes-3-phosphate, a triose sugar.
- 3. Regeneration of acceptor molecule, ribulose-1,5-biphosphate, to allow continued carbon assimilation.
- Overall equation for Calvin cycle is:
3CO2 + 9ATP + 6NADPH + 5H20 -----> G-3-P + 9ADP + 6NADP+ + 8Pi
- Note that Calvin cycle uses 9ATP for every 6NADPH. Extra ATP comes from cyclic photophosphorylation.
- Triose phosphates generated by Calvin cycle are consumed both in the stroma and the cytosol.
- Inner membrane contains phosphate translocator, an antiport system for moving triose phosphates into cytosol in exchange for phosphate.
- In cytosol, triose phosphates may be used to make sucrose, or it can enter glycolysis.
- Triose phosphates left in chloroplast are generally used to make starch.
- Biosynthesis of sucrose and starch from the products of the Calvin cycle is shown in Fig 11.14.
End of Chapter questions: 1, 2, 5, 6, 7, 9
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