PHOTOSYNTHESIS

stages of photosynthesis + where they take place

1. light-dependent reactions

   • takes place in thylakoids

2. light-independent reactions (Calvin cycle)

   • takes place in stroma of chloroplast

chloroplast structure

• two membranes (outer and inner) surrounding a dense fluid called the stroma

• in the stroma is the third membrane, the thylakoid membranes which form interconnected stacks of grana (s. granum)

  • chlorophyll (pigment) is found in thylakoid membranes

• spaced enclosed in thylakoids is thylakoid space

leaf structure + locations (in respect to photosynthesis)

• chloroplasts are found mainly in the cells of the mesophyll

• CO₂ enters and O₂ exits leaves through stomata

• water absorbed by the roots is delivered to the leaves by veins

photons & pigments for capturing light energy

• when light meets matter, it may be reflected, transmitted, or absorbed

• if a pigment is illuminated with white light, the colour we see is the colour most reflected or transmitted by the pigment

• e.g. we see green when we look at a leaf because chlorophyll absorbs violet-blue and red light while transmitting & reflecting green light

pigment

• substances that absorb visible light

• different pigments absorb light of different wavelengths, and the wavelengths that are absorbed disappear

points about light and pigment molecules that are important in photosynthesis (2)

• the absorption of a photon by a pigment molecule excites a single electron, moving it from a low energy (ground) state to a high energy (excited) state

• the difference between the energy level of the ground state and the energy level of the excited state must be equivalent to the energy of the photon of light that was absorbed

  • otherwise, the photon cannot be absorbed by the pigment

  • a compound absorbs only photons corresponding to specific wavelengths; thus, each pigment has a unique absorption spectrum

after light absorption, possible outcomes for the excited electron in a pigment molecule (3)

• the excited electron returns to ground state; energy released as thermal energy or fluorescence

• the energy of the excited electron is transferred to an electron in a neighbouring pigment molecule; the second molecule is now excited, and the original molecule returns to ground state

• the excited-state electron may be transferred to a nearby electron-accepting molecule (primary electron acceptor)

fluorescence

• the emission of light of a longer wavelength (lower energy) than the absorbed light

• emits lower energy because a small amount of energy of the photon that was initially absorbed is always lost as thermal energy

types of chlorophylls

• most dominant types are chlorophyll a (key light-capturing pigment) and chlorophyll b (accessory pigment)

• second major group of photosynthetic pigments is the carotenoids (accessory pigment)

absorption spectrum for chloroplast pigments

• a graph plotting a pigment’s light absorption as a function of wavelength is called an absorption spectrum

• the absorption spectra of chloroplast pigments shows the effectiveness of different wavelengths for driving photosynthesis, since light can perform work in chloroplasts only if it is absorbed

• e.g. chlorophyll a absorbs strongly blue and red light but does not absorb green or yellow light

action spectrum for chloroplast pigments

• photosynthesis depends on the absorption of light by chlorophylls and carotenoids acting in combination

• the effectiveness of light at various wavelengths in driving photosynthesis is plotted as an action spectrum

• graph plots the rate of photosynthesis (amount of O₂ released) at different wavelengths of visible light

why action spectrum of chlorophyll a does not match absorption spectrum

partly due to the absorption of light by accessory pigments, which broaden the spectrum of colours that can be used for photosynthesis

carotenoids for photoprotection

• carotenoids absorb and dissipate excessive light energy that would otherwise damage chlorophyll

• e.g. neutralizing free radicals by donating electrons

photosystems

• pigment molecules are bound very precisely to a number of different proteins (not free in thylakoid membranes)

• these pigment proteins are organized into photosystems

photosystem structure

• composed of the large antenna complex (light-harvesting complex) of proteins and 250-400 pigment molecules surrounding a central reaction centre

• reaction centre composed of some proteins, each bound to a pair of specialized chlorophyll molecules in the reaction centre

actions in the reaction-centre complex

• light energy is absorbed in pigment 1 (P1) which becomes excited

• P1 relaxes to the ground state and the emitted energy excites P2

• repetitive process of excitation followed by relaxation/emission

• energy flows until it excites the chlorophyll a molecules in the reaction-centre complex (P680)

purpose of many pigment molecules in a photosystem

number and variety of pigment molecules enables a photosystem to absorb more light over a larger surface area and a larger portion of the spectrum than any single pigment molecule alone

reaction-centre complex

composed of a special pair of chlorophyll a molecules and the primary electron acceptor

types of photosystems (2)

• photosystem I (PSI): collection of pigment proteins that absorb light at the 700 nm wavelength; found in all autotrophs

• photosystem II (PSII): collection of pigment proteins that absorb light at the 680 nm wavelength; only found in plants

*actions of PSII occur before those of PSI

NADPH (nicotinamide adenine dinucleotide phosphate)

• an energy carrier that stores and donates high-energy electrons

• derivative of vitamins

P680 oxidation

• when a photon of light strikes the antenna complex, it is absorbed and its energy is transferred to the molecule P680, and one of its electrons changes from the ground state to an excited state, resulting in P680*

• the excited electron is transferred to the primary acceptor molecule, which becomes negative charged, while P680 now has a positive charge

• P680⁺ is extremely electronegative and can exert forces strong enough to remove an electron from a molecule of water

  • P680⁺ is the strongest oxidant in biology

P680⁺ reduction

• reduction of P680⁺ to P680 by electrons from water is facilitated by an enzyme subunit of PSII called the water-splitting complex

• driven by the electronegative pull, the water-splitting complex oxidizes a molecule of water into two electrons, two H⁺, and an oxygen atom

• an electron is passed to P680⁺ to make it neutral again

• H⁺ are released into the thylakoid space, and oxygen atoms combine to form O₂

• the process occurs twice for each water molecule that is completely oxidized

linear electron transport steps (6)

1. photoexcitation

2. oxidation of P680

3. oxidation-reduction of plastoquinone

4. oxidation-reduction of P700

5. electron transfer to NADP⁺ by ferredoxin

6. formation of NADPH

linear electron transport: photoexcitation

• a photon of light strikes a pigment molecule in a light-harvesting complex of PSII, boosting an electron to a higher energy level

• process of excitation followed by emission until energy is transferred to the P680 pair of chlorophyll a molecules in the reaction-centre complex

• an electron in this pair is excited to a high energy state

linear electron transport: oxidation of P680

excited P680* is rapidly oxidized and the two electrons are transferred to the primary electron acceptor

electron transport chain for proton gradient

• photo-excited electron is passed from PSII to PSI via an electron transport chain; a series of redox reactions occur

• as electrons flow down the transport chain, released energy is used to pump protons (H⁺) into the thylakoid space, contributing to a proton gradient across the thylakoid membrane

• proton gradient can be used to create ATP through chemiosmosis

components of electron transport chain from PSII to PSI

• electron carrier plastoquinone (PQ)

• a protein cytochrome complex

• a protein plastocyanin (PC)

linear electron transport: oxidation-reduction of plastoquinone

• as PQ accepts electrons from PSII, it also gains protons (H⁺) from the stroma

• when PQ donates electrons to the cytochrome complex, it also releases protons into the lumen, increasing the proton concentration there

• from the cytochrome complex, electrons pass to the mobile carrier plastocyanin, which shuttles electrons from cytochrome complex to PSI

linear electron transport: oxidation-reduction of P700

• when a photon of light is absorbed by the PSI reaction-centre complex, an electron from the pair of chlorophyll a molecules is excited and P700* forms

• P700* chlorophyll transfers its electron to the primary electron acceptor of PSI, forming P700⁺ (oxidation)

• P700⁺ acts as an electron acceptor and is reduced back to P700 by the oxidation of plastocyanin

linear electron transport: electron transfer to NADP⁺ by ferredoxin

• the first electron from P700* is transported down a second electron transport chain with PSI before being transferred to ferredoxin (Fd), an iron-sulfur protein

• the oxidation of Fd results in the transfer of the electron to NADP⁺, reducing it to NADP

linear electron transport: formation of NADPH

• the enzyme NADP⁺ reductase catalyzes the transfer of electrons from ferredoxin to NADP⁺ (oxidation)

• a second electron and a proton (H⁺) from the stroma are added to NADP by NADP⁺ reductase to form NADPH

• NADPH is produced on the side of the membrane facing the stroma, where Calvin cycle reactions take place

in electron transport of photosynthesis, a proton gradient is established across the thylakoid membrane in three ways:

1. protons (H⁺) are taken into the lumen by the reduction and oxidation of plastoquinone as it moves from PSII to the cytochrome complex and back again

2. the concentration of protons inside the lumen is increased by the addition of two H⁺ for each water molecule that is split in the lumen

3. the removal of one H⁺ from the stroma for each NADPH molecule formed decreases the concentration of protons in the stroma outside the thylakoid

chemiosmotic synthesis of ATP

• chemiosmosis: movement of H⁺ across the membrane using ATP synthase

• higher concentration of H⁺ inside the membrane creates a gradient that drives protons out of the lumen, back into the stroma

• thylakoid membrane allows protons to pass out into the stroma through pores in the protein complexes of ATP synthase

• ATP synthase couples the diffusion of H⁺ down its gradient (to stroma) to the phosphorylation of ADP, forming ATP

• ATP is formed in the stroma

driving photosynthetic electron transport

• photosynthesis electron transport begins with low-energy H₂O and ends with high-energy NADPH

• to drive electron transport, low-energy electrons in water must be given enough potential energy to establish a proton gradient and enough energy to form NADPH

• dual function accomplished by the combined actions of PSI and PSII

Z scheme

• by absorbing a photon of light, an electron in a P680 chlorophyll molecule of PSII gets excited and moves farther away from its nucleus

• redox reactions that follow results in a small decrease in the free energy of the electron as oxidizing agents establish an increasingly stronger force of attachment to the electron

• low-energy electron is bound to strongly electronegative P700; primary acceptor of PSI cannot pull it away until PSI absorbs a photon of light

• two photons of light are required to span the energy difference between H₂O and NADPH

• two electrons must be transported for every one molecule of NADPH produced

cyclic electron transport

• PSI functions independently of PSII

• photosynthetic bacteria (e.g. purple sulfur bacteria) use cyclic electron flow as the only mean of generating ATP during photosynthesis

• hypothesized that these bacterial groups descend from ancestral bacteria in which photosynthesis first evolved

• important for overall photosynthesis as CO₂ reduction in Calvin cycle requires more ATP than NADPH, which is provided by cyclic electron transport

  • sometimes called cyclic phosphorylation because light energy is used to drive phosphorylation of ADP to ATP

cyclic electron flow in plants with mutations

• plants with mutations that render them unable to carry out cyclic electron transport are capable of growing well in low light, but do not grow well where light is intense

• this is evidence for the idea that cyclic electron flow is photoprotective

process of cyclic electron transport

• electron transport from PSI to ferredoxin is not followed by electron donation to NADP⁺ reductase complex; instead, to cyt c

• cyt c is continually reduced and oxidized and moves protons across thylakoid membrane without PSII

• energy absorbed form light is converted into chemical energy of ATP without oxidation of water or the reduction of NADP⁺ to NADPH (no NADPH or O₂ made)

Calvin cycle

• uses the chemical energy of ATP and NADPH to reduce CO₂ to sugar

• an anabolic process that builds larger molecules by consuming energy

• spends ATP as an energy source and consumes NADPH for adding high-energy electrons to make the sugar (G3P)

• most dominant method of carbon fixation into carbohydrates

Calvin cycle phases (3)

1. fixation

2. reduction

3. regeneration

Calvin cycle: carbon fixation

• the conversion of carbon from inorganic to organic form

• the enzyme RuBP carboxylase-oxygenase (RuBisCO) catalyzes the attachment of CO₂ to ribulose-1,5-bisphosphate (RuBP), a five-carbon sugar

• the product of the reaction is a short-lived six-carbon intermediate that is so energetically unstable that it is immediately split in half, forming two molecules of 3-phosphoglycerate (3-PGA)

  • called C₃ metabolism due to 3-carbon molecules formed

Calvin cycle: reduction

• each molecule of 3-PGA gets an additional phosphate added from hydrolysis of ATP (phosphorylation) to become 1,3-biphosphoglycerate (1,3-BPG)

• each 1,3-BPG receives two electrons from NADPH and loses one phosphate group

  • electrons reduce a carboxyl on 1,3-BPG to the aldehyde of G3P, which stores more potential energy

• hydride from NADPH is also transferred to contribute to the formation of high energy bonds

• forms two molecules of glyceraldehyde 3-phosphate (G3P), a three-carbon sugar

Calvin cycle: regeneration

• in a complex series of enzyme-catalyzed steps, most of the G3P are combined and rearranged to regenerate RuBP using the phosphorylation of ATP

• allows the cycle to start over again

counting molecules in Calvin cycle

• in each cycle, one molecule of CO₂ is converted into one reduced carbon (CH₂O); it takes three cycles to produce something the cell can actually use

• in three complete turns:

  • 3 CO₂ (3 C) are combined with 3 RuBP (15 C) to produce 6 3-PGA (18 C)

  • this yields 6 G3P (totalling 18 C)

  • 5 of 6 G3P (15 C) are used to regenerate the 3 RuBP (15 C)

• thus, the cycle generates one molecule of G3P (3 C) after three turns

• for the synthesis of six G3P molecules (net 1 G3P), the Calvin cycle requires 9 ATP and 6 NADPH; doubled to 18 ATP and 12 NADPH for a single glucose molecule (from 2 G3P)

RuBisCO quantities

• RuBisCO catalyzes CO₂ fixation in all autotrophs, so provides the source of organic carbon molecules for most of the world’s organisms

• so many RuBisCO molecules in chloroplasts that the enzyme makes up 50%+ of the total protein of plant leaves

• the world’s most abundant protein

what G3P is used to synthesize (5)

• sugars: 2 G3P combine to form glucose

• regeneration of RuBP

• amino acids & proteins: can enter metabolic pathways to contribute carbon skeletons for amino acid synthesis

• fatty acids & lipids: through further steps (e.g. glycolysis), G3P contributes to the formation of acetyl-CoA, a precursor for fatty acid biosynthesis

• nucleotides: G3P can feed into pentose phosphate pathway, which produces ribose sugars for RNA and DNA synthesis

how plants prevent water loss

• to control gas exchange with the atmosphere, leaves have stomata, which are opened or closed by guard cells

• stomata opens during the day, allowing CO₂ to enter; some H₂O is lost through transpiration, but is replaced by H₂O taken by the roots

• at night, photosynthesis stops and stomata close to conserve water

• stomata also close during the day when a plant is at risk of losing water from heat or water shortage

• when stomata (partially) closes, CO₂ concentration decreases and O₂ concentration increases; causes photorespiration

photorespiration

• rubisco’s active site occasionally binds to O₂ instead of CO₂ and catalyzes a reaction between O₂ and RuBP

  • why rubisco is called carboxylase oxygenase

• one product is a 2-carbon molecule not useful to the cell; it is converted into a useful molecule to prevent RuBP from being wasted

• recovery pathway is long and involves reactions within the chloroplast, peroxisomes, and mitochondria

• process consumes ATP and releases one molecule of CO₂

frequency of photorespiration

• when O₂ and CO₂ concentrations are equal, binding of CO₂ happens more frequently as rubisco active site has greater attraction for CO₂ (80x faster)

• in nature, atmosphere contains 21% O₂, 0.04% CO₂; rubisco binds with CO₂ 75% of the time

• 25% of the time, rubisco binds with O₂ and releases (rather than fixes) a molecule of CO₂

why photorespiration is costly

• drains cell resources, but plant can fix enough carbon to meet normal demands

• photorespiration reduces overall efficiency of sugar production

• according to one hypothesis, it is evolutionary baggage, as when the atmosphere had a higher concentration of CO₂ than O₂, the ability of the enzyme’s active site to bind O₂ would have mattered less

issue of photorespiration

• many terrestrial plants, especially those living in hot, dry climates, face the problems of photorespiration and water loss

• they need to open their stomata to let in CO₂ for Calvin cycle, but need to close stomata to conserve water

• when stomata are even partly closed, less CO₂ can enter the leaf; CO₂ concentration drops and photorespiration increases

alternative mechanisms to carbon fixation (2)

• C₄ photosynthesis

• crassulacean acid metabolism (CAM)

photosynthetic cells in C₄ plants

• bundle-sheath cells: arranged into tightly packed sheaths around the veins of the leaf

• between the bundle sheath and the leaf surface are the loosely arranged mesophyll cells

• separation reduces the exposure of rubisco-containing bundle-sheath cells to O₂ and therefore reduces rate of photorespiration

• mesophyll cells also reduce access to CO₂, but not a problem due to C₄ cycle

first step of C₄ photosynthesis (carbon fixation + formation of malate)

• Calvin cycle is preceded by the combination of CO₂ with a 3-carbon molecule, phosphoenolpyryuvate (PEP) to produce the 4-carbon oxaloacetate

• catalyzed by an enzyme only in mesophyll cells PEP carboxylase

• oxaloacetate is then reduced to malate by electrons from NADPH

• PEP carboxylase has much higher affinity for CO₂ than rubisco and no affinity for O₂; thus, can fix carbon more efficiently than rubisco

second step of C₄ photosynthesis (formation of pyruvate)

• after CO₂ is fixed, malate diffuses into bundle sheath cells, where it enters chloroplasts and oxidized to the 3-carbon compound pyruvate, releasing CO₂

• pyruvate is transported to mesophyll cells

• physical arrangement of cells in C₄ pathway establishes a high concentration of CO₂ around rubisco while reducing exposure to oxygen

third step of C₄ photosynthesis (regeneration)

• ATP is used to convert pyruvate to PEP, which can accept another CO₂ and allow reaction cycle to continue

• for each cycle, the double hydrolysis of ATP to AMP (adenosine monophosphate) is required to regenerate PEP from pyruvate

• these cells contain PSI but no PSII, so cyclic electron flow is the only way of generating ATP

• additional energy is required, equivalent to six ATP for each G3P produced by the Calvin cycle

why C₄ plants do well in hot climates

• in hot climates, photorespiration can decrease carbon fixation efficiency by over 50%, so C₄ pathway is worth energy cost

• hot climates receive a lot of sunshine, so additional ATP requirement is easily met by the cyclic light reactions

• in temperate climates, lower temperatures mean that photorespiration is less of a problem, and additional ATP requirement is harder to meet with less sunshine

implications of enhanced efficiency of C₄ plants in hot climates (2)

• C₄ plants can open their stomata less than C₃ plants, enabling them to survive better in arid environments

• C₄ plants require one-third to one-sixth as much rubisco, and so have a much lower nitrogen demand; enables them to survive in more nutrient-poor soil conditions

CAM plants

• run Calvin and C₄ cycle in the same cells, but do so at different times of day

  • vs C₄ plants which run cycles in different locations

• typically live in regions that are hot and dry during the day and cool at night

• cacti and succulent species, with fleshy leaves/stems, have a low surface-to-volume ratio and fewer stomata

• might open their stomata during the night and close them during the day; helps conserve water, but prevents CO₂ from entering leaves

CAM pathway (night)

• stomata open only at night, when they release O₂ that accumulates from photosynthesis during the day and allow CO₂ to enter, which they incorporate into organic acids

• CO₂ that enters is fixed by a C₄ pathway into malate, which accumulates in the night and is stored in the form of malic acid in cell vacuoles

CAM pathway (day)

• as the sun rises and temperature increases, the stomata closes, reducing water loss and cutting off gas exchange

• malic acid diffuses from vacuoles into the cytosol, where malate is oxidized to pyruvate, and a high concentration of CO₂ is released

• CO₂ favours carboxylase activity of rubisco, allow Calvin cycle to proceed efficiently with little loss of CO₂ from photorespiration

• pyruvate produced by malate breakdown accumulates during the day, but is converted back into malate at night; requires ATP expenditure

compare & contrast C₄ and CAM plants

• both incorporate CO₂ into organic intermediates before it enters the Calvin cycle

• in C₄ plants, initial steps of carbon fixation occur in a different cell and are structurally separated from Calvin cycle (“spatial”)

• in CAM plants, both steps occur within the same cell but at different times of day (“temporal”)