Photosynthesis: Light Reactions

 

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Photosynthesis

Photosynthesis is a fundamental process in which photosynthetic organisms, primarily plants and algae, convert light energy into chemical energy. This energy is then stored in the form of complex carbon compounds, such as carbohydrates, and results in the release of oxygen from carbon dioxide and water.

Thylakoid Reactions (Light-Dependent Reactions)

The thylakoid reactions, also known as the light reactions, are the initial stage of photosynthesis. They take place specifically within the thylakoids of the chloroplast. The primary function of these reactions is to convert light energy into chemical energy, which is stored in two high-energy compounds: ATP and NADPH.

• ATP (Adenosine triphosphate): This molecule serves as the primary energy currency for the cell. In the thylakoid reactions, ATP is produced through a process called photophosphorylation, which is driven by the movement of protons across the thylakoid membrane.

• NADPH (Nicotinamide adenine dinucleotide phosphate): This is a crucial electron carrier that provides the reducing power necessary for the synthesis of sugars.

The ATP and NADPH generated during the thylakoid reactions are then used in the next stage of photosynthesis, the carbon fixation reactions (also known as the Calvin cycle), which occur in the stroma of the chloroplast. These reactions use the stored chemical energy to synthesize sugars from carbon dioxide.

The Nature of Light

Light exhibits characteristics of both a wave and a particle.

Wave Properties

• A wave is defined by its wavelength (λ), which is the distance between consecutive wave crests.

• The frequency (ν) is the number of wave crests that pass a given point in a specific time.

• The relationship between these is given by the equation

  c = λν

                where c is the speed of the wave. For light, this speed is approximately 3.0 × 10⁸ m s⁻¹.

• Light is a transverse electromagnetic wave.

Wave properties of light

Particle Properties

• Light can also be considered a stream of particles called photons.

• Each photon contains a discrete amount of energy, known as a quantum.

• The energy (E) of a photon is directly proportional to the light's frequency, as described by Planck's law:

                                                                        E = hν

                                            where h is Planck's constant (6.626 × 10⁻³⁴ J s).

• The human eye can only perceive a small range of frequencies within the electromagnetic spectrum, known as the visible-light region.

Electromagnetic spectrum

Light Absorption and Pigments

Absorption and Action Spectra

• An absorption spectrum measures how much light energy a substance absorbs at different wavelengths. For example, the absorption spectrum of chlorophyll a show the portion of solar output that plants utilize.

• Chlorophyll appears green because it primarily absorbs light in the red and blue parts of the spectrum, while reflecting green wavelengths (around 550 nm).

• An action spectrum measures the effectiveness of different wavelengths of light in driving a biological response, such as oxygen evolution during photosynthesis.

• If the pigment responsible for a response is the same as the one whose absorption is being measured, the absorption and action spectra will be similar. 

• However, a slight mismatch occurs in the carotenoid absorption region (450–550 nm), suggesting that energy transfer from carotenoids to chlorophylls is less efficient than between chlorophylls themselves.

The solar spectrum and its relation to the absorption spectrum of chlorophyll 

Absorption spectrum and action spectrum for photosynthesis does not overlap in the 450-550 nm region

• Chlorophyll has high absorption peaks in the blue (430 nm) and red (662 nm) regions of the spectrum, while carotenoids absorb light in the blue-green region (450-550 nm).

• The overall photosynthetic activity, measured by the action spectrum, is lower in the region of carotenoids absorption compared to the peaks of chlorophyll absorption. 

• This is because the energy absorbed by the carotenoids must be transferred to chlorophylls to be used in photosynthesis. This energy transfer is not 100% efficient, meaning some of the absorbed light energy is lost during the transfer process as heat. 

• As a result, the action spectrum shows a less pronounced effect in the 450-550 nm range, even though significant absorption is occurring in that same range.

Absorption and action spectra of photosynthesis

The Red Drop and Emerson's Enhancement Effect

• The Red Drop: When measuring the quantum yield of photosynthesis (the rate of a photochemical event relative to the rate of photon absorption), Emerson observed a sharp decrease in efficiency in the far-red region (wavelengths greater than 680 nm). This indicates that light with a wavelength longer than 680 nm is far less effective at driving photosynthesis compared to shorter wavelengths.

• The Enhancement Effect: When Emerson provided red and far-red light simultaneously, the rate of photosynthesis was greater than the sum of the rates from each light source individually. This discovery led to the understanding that two distinct photochemical complexes, Photosystem I (PSI) and Photosystem II (PSII), work in tandem. PSII absorbs red light (680 nm), while PSI preferentially absorbs far-red light (greater than 680 nm).

                                                    where ΔO2 is the rate of oxygen evolution

Red drop

Photochemical Reactions and Energy Transfer

When a chlorophyll molecule absorbs a photon, it becomes energized, transitioning from its stable "ground state" to an unstable "excited state." This high-energy state is fleeting and must quickly release its energy. Absorption of blue light transitions chlorophyl to higher excited state than red light. In the higher excited state, it quickly emits some energy as heat and enters lower excited state. This excited state has four possible pathways to release its energy:

1. Fluorescence: Re-emitting a photon and returning to the ground state. The emitted photon has a longer wavelength and lower energy than the absorbed one. Chlorophyll fluoresces in the red region of the spectrum. 

2. Heat Dissipation: Converting the excitation energy directly into heat without emitting a photon.

3. Energy Transfer: Transferring the energy to an adjacent molecule. This is how antenna pigments transfer energy to the reaction center.

4. Photochemistry: Using the absorbed energy to drive a chemical reaction. The excited chlorophyll molecule transfers an electron to an electron-acceptor molecule, initiating a chain of redox (reduction-oxidation) reactions. This is the primary event that converts light energy into chemical energy.

Photon absorption by chlorophyll and its fate

Photosynthetic Pigments

Chlorophyll

• Chlorophyll molecules have a porphyrin-like ring with a central magnesium ($M{g}^{2+}$) ion. This ring is the site of electron changes during excitation, oxidation, and reduction.

• Chlorophylls differ in the substituents on their rings, which gives them different properties. For example, chlorophyll a has a -CH₃ group, while chlorophyll b has a -CHO group.

• The molecule has a long hydrocarbon tail that anchors it in the photosynthetic membrane.

Chlorophyll a structure

Carotenoids

• Carotenoids are linear molecules with multiple conjugated double bonds found in photosynthetic organisms. They absorb light in the 400-500 nm range, which gives them their characteristic orange color.

• They act as accessory pigments by absorbing light and transferring the energy to chlorophyll for photosynthesis.

• Carotenoids also provide photoprotection. They act as a safety valve, dissipating excess energy as heat before it can damage the photosynthetic machinery. If unquenched, excited chlorophyll can react with oxygen to form highly reactive singlet oxygen (¹O₂*), which can harm cellular components.

Structure of beta-carotene

Photoprotection by Carotenoids

• Carotenoids are essential accessory pigments in photosynthesis that provide crucial protection against the damaging effects of excess light. Their primary photoprotective function is to dissipate surplus light energy as heat, a process known as non-photochemical quenching (NPQ).
• When light intensity is high, the light-harvesting complex can absorb more energy than the photosystems can use. This excess energy can lead to the formation of a highly reactive and toxic molecule called singlet oxygen (¹O₂)*. Singlet oxygen can cause oxidative damage to cellular components, particularly lipids in the thylakoid membranes, leading to a condition called photoinhibition.
• Carotenoids, such as zeaxanthin, antheraxanthin, and violaxanthin, act as the plant's first line of defense. They are involved in the xanthophyll cycle, a process that regulates the dissipation of excess light energy. In high light conditions, violaxanthin is converted to zeaxanthin. Zeaxanthin binds to proteins in the light-harvesting antenna complex, inducing a conformational change that promotes the safe release of excess energy as heat.

• Furthermore, carotenoids can also directly quench the excited state of chlorophyll molecules, preventing them from transferring energy to oxygen and forming damaging singlet oxygen. The excited state of carotenoids has a lower energy level than that of singlet oxygen, so it cannot transfer energy to form it. Instead, the carotenoid returns to its ground state by releasing the energy as harmless heat, thus protecting the photosynthetic apparatus from damage.

The Photosynthetic Apparatus and Units

Location

• Photosynthesis takes place in chloroplasts.

• The essential proteins for photosynthesis are embedded in the thylakoid membranes.

• PSII is primarily located in the grana lamellae, while PSI and ATP synthase are mostly in the stroma lamellae and at the edges of the grana.

• The cytochrome b₆f complex is distributed between both the stroma and grana lamellae.

Antenna and Reaction Centers

• Pigments like chlorophylls and carotenoids form an antenna complex that collects light and transfers the energy to the reaction center.
• The antenna system is a physical phenomenon where energy is transferred via fluorescence resonance energy transfer (FRET). This transfer is highly efficient (95-99%).

• The reaction center, which contains special chlorophylls (P700 and P680), is where the photochemical redox reactions occur, converting light energy into chemical energy.

• The reaction center includes 4-6 chlorophyll a molecules (P700 and P680), and associated proteins and cofactors.

• The reaction center chlorophyll is an energy sink that absorbs light of longest wavelength and lowest energy in the complex.

• Energy transfer that occurs in the antenna complex is a physical phenomenon while electron transfer (redox reactions) at the reaction center is a chemical phenomenon.

Energy transfer through antenna complex

The energy trapping mechanism is directional and irreversible

• The directionality of energy transfer in the antenna complex is maintained by a carefully arranged sequence of pigments.

• Energy Gradient: The pigments within the antenna complex are organized in a way that creates an "energy gradient." The pigments that are further away from the reaction center absorb light at shorter wavelengths, which corresponds to higher energy.

• Progressive Energy Loss: As the excitation energy is transferred from one pigment molecule to the next, closer to the reaction center, it moves to pigments that absorb at progressively longer, redder wavelengths and therefore have lower energy excited states.

• Irreversibility: The small difference in energy between each consecutive excited chlorophyll is lost to the surroundings as heat. This minor energy loss makes the transfer process energetically favorable in one direction (towards the reaction center) and largely irreversible, ensuring that the collected light energy is efficiently funneled to the reaction center.

Energy funneling through antenna complex to reaction center

Photosynthetic Unit (Quantasome)

• A photosynthetic unit is the minimum number of collaborating pigment molecules required for a photochemical event.

• The evolution of one oxygen molecule requires 8 quanta of light and approximately 2500 chlorophyll molecules. This means about 300 chlorophyll molecules are needed to process each light quantum.

• Within this unit, only one specialized chlorophyll molecule participates in the photochemical reaction, while the others serve to absorb and transfer light to it.

Photosystem I (PSI) and Photosystem II (PSII) Reaction Centers

The reaction centers of PSI and PSII are crucial pigment-protein complexes embedded within the thylakoid membranes of chloroplasts that convert light energy into chemical energy during photosynthesis. These two photosystems work in series to drive the light reactions.

Photosystem II (PSII) Reaction Center

• It is designated as P680 as its core chlorophyll pigment absorbs light most efficiently at a wavelength of 680 nm.

• Location: Primarily found in the grana lamellae, which are the stacked regions of the thylakoid membranes.

• Structure: The core of the reaction center consists of two membrane proteins, D1 and D2. It also contains a special pair of chlorophyll-a molecules (P680), bound one to each of these two proteins, along with additional chlorophylls, pheophytins (colorless chlorophyll lacking Mg2+), carotenoids, and electron acceptors like plastoquinones. Additionally, the oxygen evolving complex is also a part of PSII.

• Function: PSII acts as a powerful oxidant. Its primary role is to split water molecules (photolysis) to replace the electron it loses. This process releases oxygen as a byproduct. The electrons from the water are used to re-reduce the oxidized P680. The splitting of water and subsequent electron transfer also pumps protons into the thylakoid lumen, contributing to the proton motive force needed for ATP synthesis.

Photosystem I (PSI) Reaction Center

• It is designated as P700 as its core chlorophyll pigment absorbs light most effectively at a wavelength of 700 nm.

• Location: Predominantly located in the stroma lamellae and at the edges of the grana lamellae.

• Structure: It is a large, multi-subunit complex. Its core consists of a heterodimer of two membrane proteins, PsaA and PsaB, each binding a reaction center chlorophyll molecule (P700). Another pair of chlorophyll a molecules (A0) also remains associated with the heterodimer and accepts electron from P700. There are additional proteins PsaC to PsaN and a pair of vitamin K1 molecules called phylloquinone bound to the heterodimer, one per subunit. It also contains a core antenna of about 100 chlorophyll molecules and a series of 3 membrane associated iron sulfur clusters - FeSX, FeSA and FeSB. A soluble iron sulfur protein, ferredoxin (Fd) is also a part of this large complex.

• Function: PSI produces a strong reductant. The electron it receives from the electron transport chain (via plastocyanin) is excited by light and transferred to acceptors, eventually being used to reduce NADP+ to NADPH. NADPH is a high-energy compound used in the Calvin cycle.

Feature	Photosystem I (PSI)	Photosystem II (PSII)
Primary Pigment	P700	P680
Location	Stroma lamellae and edges of grana lamellae	Grana lamellae (stacked thylakoid membranes)
Wavelength of Absorption Maxima	700 nm	680 nm
Primary Function	Reduces NADP+ to NADPH (produces a strong reductant that can reduce NADP+ and a weak oxidant)	Splits water and releases oxygen (generates a strong oxidant that can oxidise water and a weak reductant)
Electron Source	Receives electrons from the electron transport chain (via plastocyanin)	Receives electrons from the splitting of water (photolysis)
Product	NADPH	Oxygen, protons, and electrons for the electron transport chain

Electron Transport and Photophosphorylation

The Z-Scheme (Non-cyclic Electron Transport)

• The Z-scheme illustrates how PSI and PSII work in series. The entire chemical reaction is carried out by four integral membrane complexes that are vectorially arranged in the thylakoid membrane:
• PSII
• Cytochrome b₆f complex
• PSI
• ATP Synthase

• PSII: P680 in PSII absorbs a photon, gets excited (P680*), and transfers an electron to pheophytin. The electron-deficient P680+ is then re-reduced by electrons from water oxidation. This process releases protons into the thylakoid lumen, and the reduced product is plastohydroquinone (PQH₂).

• Cytochrome b₆f: This complex oxidizes PQH₂ and transfers electrons to plastocyanin (PC). This process also pumps protons from the stroma into the lumen, creating a proton motive force.

• PSI: Plastocyanin then reduces the oxidized P700+ in PSI. P700 absorbs a photon, gets excited, and transfers an electron down a series of acceptors, ultimately reducing NADP⁺ to NADPH by the action of ferredoxin and flavoprotein-ferredoxin NADP+ reductase (FNR).

• ATP Synthase: It produces ATP as protons diffuse back through it from lumen to stroma.

Components of photosynthetic electron transport chain

Mechanism of non-cyclic electron transport

Light Absorption and Water Splitting at PSII: 

• The process begins when light energy is absorbed by the antenna pigments of Photosystem II (PSII) and funneled to its reaction center, P680. This energy excites an electron in P680 to a higher energy state, and the electron is then transferred to an electron acceptor molecule, pheophytin (primary photochemical act). Now the P680 is in oxidized state (electron deficient) and pheophytin is in reduced state (electron rich). 

• This primary electron acceptor will now donate its extra electron to a secondary eelectron acceptor and so on down the ETC. On the other hand, the oxidized reaction center chlorophyll will accept the missing electron from a secondary electron donor, which in turn will regain alectron from a tertiary electron donor and so on. 

• The ultimate electron donor is water and ultimate electron acceptor is NADP+.

• To replace the lost electron, PSII uses a powerful oxidant to split a water molecule (photolysis). This reaction releases oxygen, protons (H+), and electrons. The electrons from water are donated to P680, allowing it to return to its ground state and participate in another photochemical event.

Electron Transfer and Proton Pumping: 

The electron from PSII is transferred to a mobile electron carrier, plastoquinone (PQ), which becomes reduced to plastohydroquinone (PQH2​).

PQH2​ then moves to the cytochrome $b_{6}f$ complex, where it is oxidized. The cytochrome b6​f complex acts as a proton pump, using the energy from the electron transfer to move protons from the stroma into the thylakoid lumen, which contributes to the proton motive force for ATP synthesis.

Electron Transfer to PSI: 

After passing through the cytochrome b6​f complex, the electron is transferred to another mobile carrier, a soluble copper protein called plastocyanin (PC). Plastocyanin carries the electron to the second photosystem, Photosystem I (PSI), and reduces its oxidized reaction center, P700.

Light Absorption and NADPH Formation at PSI: 

When PSI's reaction center, P700, absorbs light, its electron is re-excited to a very high energy level. This energized electron is transferred to a series of acceptors, including a quinone and iron-sulfur proteins. The electron is then passed to the soluble protein ferredoxin (Fd). Finally, the enzyme ferredoxin-NADP+ reductase (FNR) uses the electron from ferredoxin to reduce NADP+ to NADPH.

Z scheme of non-cyclic electron transport chain

Cyclic Electron Transport

• This mode of electron transport involves only PSI. It occurs when NADP⁺ is not available.

• Electrons ejected from P700 are shuttled back to the cytochrome b₆ complex and then return to the electron hole of PSI.

• This process does not produce NADPH or oxygen but is coupled to the phosphorylation of ADP to ATP.

Photophosphorylation (ATP Synthesis)

• ATP synthesis in chloroplasts is driven by a chemiosmotic mechanism.

• The vectorial electron flow results in a proton gradient across the thylakoid membrane, making the lumen acidic and the stroma alkaline.

• The energy of this proton gradient, known as the proton motive force (∆p), is used by the ATP synthase (CF₀-CF₁ complex) to synthesize ATP.

• The CF₀ part of the enzyme acts as a proton channel, while the CF₁ part, which sticks into the stroma, contains the catalytic sites for ATP synthesis. This enzyme functions like a tiny molecular motor, rotating as protons pass through it to synthesize ATP.

Nitrogen Metabolism

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Nitrogen Cycle

Nitrogen metabolism is the complex process of how organisms convert and utilize nitrogen. The nitrogen cycle describes the exchange of nitrogen between three main pools: the atmosphere, soil and groundwater, and biomass.

The nitrogen cycle involves several key processes:

Nitrogen Fixation: This is the conversion of atmospheric nitrogen gas (N2​) into a usable form like ammonia (NH3​). It can occur through biological, industrial, or electrical means.

Ammonification: In this decomposition process, microorganisms convert organic nitrogen from decaying biomass into ammonia (NH3​) and ammonium ions (NH4+​). Example: Bacillus vulgaris and Bacillus ramosus

Nitrification: This two-step process converts ammonia to nitrate (NO3−​). First, bacteria like Nitrosomonas or Nitrococcus oxidize ammonia to nitrite (NO2−​). Next, Nitrobacter oxidizes the nitrite to nitrate. These bacteria are chemoautotrophs and are known as nitrifying bacteria.

Denitrification: This process converts nitrates and nitrites back into dinitrogen gas (N2​) and is carried out by microorganisms called denitrifiers, such as Thiobacillus denitrificans.

Nitrate Assimilation: Plants actively absorb nitrate from the soil and convert it into organic nitrogen compounds.

Nitrate Assimilation in Plants

Nitrate assimilation is a two-step process in plants:

Step 1: Reduction of Nitrate to Nitrite 

This reaction occurs in the cytosol and is catalyzed by the enzyme nitrate reductase (NR). It involves the transfer of two electrons from an electron donor, typically NADH in most cases or NADPH in nongreen tissues.

Nitrate reductases in higher plants are composed of two identical subunits, each containing three prosthetic groups:

• flavin adenine dinucleotide (FAD)
• heme
• a molybdenum ion complexed to a pterin molecule

FAD accepts two electrons from NAD(P)H, which are then passed to the heme domain and finally to the molybdenum complex to be transferred to nitrate. Molybdenum is crucial for this enzyme's function, and a deficiency in it can lead to nitrate accumulation.

Regulation of Nitrate Reductase

NR synthesis is regulated at transcriptional and translational levels by several factors, including nitrate, light, and carbohydrate accumulation. The enzyme is also subject to posttranslational modification through reversible phosphorylation.

• Activation: Light, carbohydrates, and other environmental factors stimulate a protein phosphatase that dephosphorylates a key serine residue on the hinge 1 region of NR enzyme, activating it.

• Deactivation: In darkness, a protein kinase stimulated by Mg2+ phosphorylates the same serine residue. This phosphorylation allows the enzyme to interact with a 14-3-3 inhibitor protein, rendering it inactive.

This phosphorylation-based regulation provides a rapid way to control NR activity, happening in minutes compared to the hours it takes for enzyme synthesis or degradation.

Step 2: Reduction of Nitrite to Ammonium 

Nitrite is a highly reactive and potentially toxic ion, so it's immediately transported from the cytosol to chloroplasts or plastids where it is reduced to ammonium (NH4+​) by the enzyme nitrite reductase (NiR). This process requires the transfer of six electrons. The electrons are donated by reduced ferredoxin (Fred​). 

NiR consists of a single polypeptide with two prosthetic groups: 

• an iron-sulfur cluster (Fe4​S4​) 
• a specialized heme

Electrons flow from ferredoxin, through the iron-sulfur cluster and heme, to nitrite. Reduced ferredoxin is derived from the photosynthetic electron transport chain or from NADPH generated in oxidative pentose phosphate pathway in nongreen tissue.

Regulation of Nitrite Reductase

• Activation: High nitrate levels or exposure to light induce the transcription of NiR mRNA.

• Deactivation: The accumulation of end products, such as asparagine and glutamine, represses this induction.

Ammonia Assimilation

Plant cells quickly convert the ammonium generated from nitrate assimilation into amino acids to prevent toxicity. The main pathway for this conversion is the GS-GOGAT pathway, which involves two key enzymes: 

• glutamine synthetase (GS) 
• glutamate synthase (GOGAT)

Step 1: Glutamine Synthetase (GS) 

GS combines ammonium with glutamate to form glutamine. This reaction requires ATP and a divalent cation like Mg2+, Mn2+,or Co2+ as a cofactor.

Plants have three classes of GS with different functions and locations:

• Cytosolic GS: Found in germinating seeds and vascular bundles, producing glutamine for intercellular nitrogen transport. Its expression isn't regulated by light or carbohydrates.

• Plastidal GS: Located in roots, generating amide nitrogen for local consumption. Its expression is regulated by light and carbohydrate levels.

• Chloroplastic GS: Found in shoots, where it reassimilates photorespiratory ammonium (NH4+​). Its expression is also regulated by light and carbohydrate levels.

Step 2: Glutamate Synthase (GOGAT) 

GOGAT transfers the amide group from glutamine to 2-oxoglutarate, producing two molecules of glutamate. There are two types of GOGAT in plants:

• NADH-GOGAT: Accepts electrons from NADH and is found in the plastids of non-photosynthetic tissues like roots or vascular bundles. It assimilates ammonium absorbed from the soil or glutamine translocated from roots or senescing leaves.

• Fd-GOGAT: Accepts electrons from ferredoxin and is found in chloroplasts, where it functions in photorespiratory nitrogen metabolism.

Overall reaction in GS-GOGAT pathway

Alternate Ammonia Assimilation Pathways

Plants can also assimilate ammonia through several alternative pathways.

Glutamate Dehydrogenase (GDH)

This enzyme catalyzes a reversible reaction that can either synthesize or deaminate glutamate. An NADH-dependent form is found in mitochondria, while an NADPH-dependent form is in the chloroplasts of photosynthetic organs.

Transamination

This process incorporates nitrogen from glutamine and glutamate into other amino acids. It involves the transfer of an amino group from one amino acid to a keto acid, producing a new amino acid and a new keto acid. This is a reversible process requiring pyridoxal phosphate (vitamin B6) as a cofactor. 

Examples:

Asparagine Synthetase (AS)

AS transfers the amide nitrogen from glutamine to aspartate, producing asparagine and glutamate. It is found in the cytosol of leaves, roots, and nitrogen-fixing nodules.

Regulation of Ammonium Metabolism

• The regulation of ammonium metabolism is vital for maintaining the nitrogen-to-carbon (N:C) ratio in plants.

• High light and carbohydrate levels: Under these conditions, AS is inhibited, and plastid GS and Fd-GOGAT are activated. This favors the production of carbon-rich compounds like glutamine and glutamate, which are used for synthesizing new plant materials.

• Energy-limited conditions: In these situations, the expression of plastid GS and Fd-GOGAT is reduced, and AS is activated. This leads to more asparagine, an amide that helps in long-distance transport or long-term nitrogen storage.

Amino acid biosynthesis

Humans and most animals cannot synthesize certain essential amino acids and must obtain them from their diet. In contrast, plants can synthesize all 20 standard amino acids. The nitrogen-containing amino group is derived from transamination reactions with glutamine or glutamate. The carbon skeletons for amino acids are derived from intermediates of glycolysis (3-phosphoglycerate, phosphoenolpyruvate, or pyruvate) or the citric acid cycle (2-oxoglutarate or oxaloacetate).