The Light Reactions: Electron Transport, Reductions, and Photophosphorylation

The energized electron that leaves the activated chlorophyll in the reaction center needs somewhere to go. It immediately participates in a series of oxidation-reduction (redox) reactions. The energy-rich electron is passed through a chain of electron carriers in the thylakoid membrane in a process termed electron transport. Two energy-rich products of the light reactions, NADPH + H+ and ATP, are the result. The energy-rich NADPH + H+ is a stable, reduced coenzyme. Its oxidized form is NADP+(nicotinamide adenine dinucleotide phosphate). Just as NAD+ couples the metabolic pathways of cellular respiration, NADP+ couples the two photosynthetic pathways. NADP+ is identical to NAD+ except that the former has an additional phosphate group attached to each ribose. Whereas NAD+ participates in catabolism, NADP+ is used in anabolic (synthetic) reactions, such as carbohydrate synthesis from CO₂, that require energy from reducing power. Electron transport in the thylakoid membrane sets up a charge separation, just as electron transport in the inner mitochondrial membrane does .This potential energy is captured by the chemiosmotic synthesis of ATP in a process called photophosphorylation. Both NADPH + H+ and ATP are used in the Calvin–Benson cycle as a source of energy for the endergonic synthesis of carbohydrates. There are two different systems of electron transport in photosynthesis:

Noncyclic electron transportproduces NADPH + H+ and ATP.

Cyclic electron transportproduces only ATP.

Noncyclic electron transport produces ATP and NADPH

In noncyclic electron transport, light energy is used to oxidize water, forming O₂, H+, and electrons. Electrons from water replenish the electrons that chlorophyll molecules lose when they are excited by light. As the electrons are passed from water to chlorophyll, and ultimately to NADP+, they pass through a chain of electron carriers. These redox reactions are exergonic, and some of the free energy released is used ultimately to form ATP by a chemiosmotic mechanism.

Two photosystems are required

Noncyclic electron transport requires the participation of two different photosystems. These photosystems are light-driven molecular units, each of which consists of many chlorophyll molecules and accessory pigments bound to proteins in separate energy-absorbing antenna systems.

Photosystem Iuses light energy to reduce NADP+ to NADPH + H+.

Photosystem IIuses light energy to oxidize water molecules, producing electrons, protons (H+) and O₂.

The reaction center for photosystem I contains a chlorophyll a molecule called P700 because it can best absorb light of wavelength 700 nm. The reaction center for photosystem II contains a chlorophyll a molecule called P680 because it absorbs light maximally at 680 nm. Thus photosystem II requires photons that are somewhat more energetic (i.e., shorter wavelengths) than those required by photosystem I. To keep noncyclic electron transport going, both photosystems I and II must constantly be absorbing light, thereby boosting electrons to higher orbitals from which they may be captured by specific oxidizing agents.

Detalis of the reactions

Photosystem II absorbs photons, sending electrons from P680 to the primary electron acceptor— the first carrier in the redox chain— and causing P680 to become oxidized to P680 +. Electrons from the oxidation of water are passed to P680 +, reducing it once again to P680, which can then absorb more photons. The electron from photosystem II passes through a series of exergonic reactions in the redox chain that are indirectly coupled across the thylakoid membrane to proton pumping. This pumping creates a proton gradient that produces energy for ATP synthesis. In photosystem I, the reaction center containing P700 becomes excited to P700*, which then leads to the reduction of an oxidizing agent called ferredoxin (Fd) and the production of P700 +. Then P700 + returns to the ground state by accepting electrons passed through the redox chain from photosystem II. With this accounting for the source of the electrons entering photosystem II, we can now consider the fate of the electrons from photosystem I. These electrons are used in the last step of noncyclic electron transport, in which two electrons and two protons are used to reduce a molecule of NADP+ to NADPH + H+.

In summary:

Noncyclic electron transport uses a molecule of water, four photons (two each absorbed by photosystems I and II), one molecule each of NADP+ and ADP, and one Pi. Noncyclic electron transport produces NADPH + H+ and ATP and half a molecule of oxygen (1/2 O₂).

Cyclic electron transport produces ATP but no NADPH

Noncyclic electron transport produces ATP and NADPH + H+. However, as we will see, the Calvin–Benson cycle uses more ATP than NADPH + H+. Cyclic electron transport occurs in some organisms when the ratio of NADPH + H+ to NADP+ in the chloroplast is high. This process, which produces only ATP, is called cyclic because an electron passed from an excited chlorophyll molecule at the outset cycles back to the same chlorophyll molecule at the end of the chain of reactions Before cyclic electron transport begins, P700, the reactioncenter chlorophyll of photosystem I, is in the ground state. Itabsorbs a photon and becomes P700*. The P700* then reactswith oxidized ferredoxin (Fdox) to produce reduced ferredoxin(Fdred). The reaction is exergonic, releasing free energy.Reduced ferredoxin (Fdred) passes its added electron to a differentoxidizing agent, plastoquinone (PQ, a small organic molecule),which pumps 2 H+ back across the thylakoid membrane.Thus, Fdred reduces PQ and PQred passes the electronto a cytochrome complex (Cyt). The electron continues downthe electron transport chain until it completes its cycle by returningto P700+, resulting in a restoration of its unchargedform, P700. By the time the electron from P700* travels throughthe redox chain by way of plastocyanin (PC), and comes backto reduce P700+, all the energy from the original photon hasbeen released. This cycle is a series of redox reactions, eachexergonic, and the released energy is stored in the form of aproton gradient that can be used to produce ATP.Having seen how a proton gradient is established acrossthe thylakoid membrane, we’ll now examine in more detailthe role of this gradient in ATP synthesis.

Chemiosmosis is the source of the ATP produced in photophosphorylation

The chemiosmotic mechanism for ATP formation in the mitochondrion. The chemiosmotic mechanism also operates in photophosphorylation (Figure ). In chloroplasts, as in mitochondria, electron transport through the redox chain is coupled to the transport of protons (H+) across the thylakoid membrane, which results in a proton gradient across the membrane. The electron carriers in the thylakoid membranes are oriented so that protons move from the stroma into the interior of the thylakoid. The interior compartment becomes acidic with respect to the stroma. When there is sufficient light, the ratio of H+ inside versus outside a thylakoid is usually 10,000:1, which is a difference of 4 pH units. This difference leads to the diffusion of H+ back out of the thylakoid interior through specific protein channels in the thylakoid membrane. These channels are enzymes—ATP synthases—that couple the diffusion of protons to the formation of ATP, just as in mitochondria. In the chloroplast, the ATP is generated in the stroma, where it will be available to provide the energy for the fixation of CO₂ in the production of carbohydrate by the Calvin-Benson cycle.

Making Carbohydrate from CO₂: The Calvin–Benson Cycle

At the start of this chapter we identified two distinct metabolic pathways operating in photosynthesis. We have now discussed the first pathway: the light reactions, which use light energy to produce ATP and NADPH + H+ in the chloroplasts of green plants. The second pathway, the Calvin–Benson cycle, uses this ATP and NADPH + H+ to incorporate CO₂ into carbohydrates. Most of the enzymes that catalyze the reactions of the Calvin–Benson cycle are dissolved in the chloroplast stroma (the “soup” outside the thylakoids) and that is where those reactions take place. However, these enzymes use the energy in ATP and NADPH, produced in the thylakoids by the light reactions, to reduce CO₂ to carbohydrates. Because there is no stockpiling of these energy-rich coenzymes, these Calvin–Benson cycle reactions take place only in the light, when these coenzymes are being generated.

Isotope labeling experiments revealed the steps of the Calvin–Benson cycle

To identify the sequence of reactions by which CO₂ ends up in carbohydrates, it was necessary to label CO₂ so that it could be followed after being taken up by a photosynthetic cell. In the 1950s, Melvin Calvin, Andrew Benson, and their colleagues used radioactively labeled CO2 in which some of the carbon atoms were not the normal 12C, but its radioisotope 14C. Although 14C is distinguished by its emission of radiation, chemically it behaves virtually identically to nonradioactive 12C. In general, enzymes do not distinguish between isotopes of an element in their substrates, so 14CO₂ is treated the same way by photosynthesizing cells as 12CO₂. Calvin and his colleagues exposed cultures of the unicellular green alga Chlorella to 14CO₂ for 30 seconds. They then rapidly killed the cells, extracted their carbohydrates and separated the different compounds from one another by paper chromatography. Many compounds, including monosaccharides and amino acids, contained 14C (Figure). However, if they stopped the exposure after just 3 seconds, only one compound was labeled—a three-carbon sugar phosphate called 3-phosphoglycerate (3PG):

By tracing the steps in this manner, they soon discovered a cycle that “fixes” CO₂ in a larger molecule, produces a carbohydrate, and regenerates the initial CO₂ acceptor. This cycle was appropriately named the Calvin–Benson cycle. The initial reaction in the Calvin–Benson cycle adds the one-carbon CO₂ to a receptor, the five-carbon compound ribulose 1,5-bisphosphate(RuBP). The product is an intermediate six-carbon compound, which quickly breaks down and forms two three-carbon molecules of 3PG (as Calvin and colleagues observed). The enzyme that catalyzes this fixation reaction, ribulose bisphosphate carboxylase/ oxygenase(rubisco), is the most abundant protein in the world comprising about 20 percent of all the protein in every plant leaf.

The Calvin–Benson cycle is made up of three processes

The Calvin–Benson cycle uses the high-energy coenzymes made in the thylakoids during the light reactions (ATP and NADPH) to reduce CO₂ to a carbohydrate. There are three processes that make up the cycle:

Fixation of CO_2. As we saw, this reaction is catalyzed by rubisco and its product is 3PG.

Reduction of 3PG to form a carbohydrate, glyceraldehydes 3-phosphate (G3P). This series of reactions involves a phosphorylation (using the ATP made in the light reactions) and a reduction (using the NADPH made in the light reactions). Regeneration of the CO₂ acceptor, RuBP. Most of the G3P ends up as RuMP (ribulose monophosphate), and ATP is used to convert this compound to RuBP. So for every “turn” of the cycle, with one CO₂ fixed, the CO₂ acceptor is regenerated. The end product of this cycle is glyceraldehyde 3-phosphate (G3P), which is a three-carbon sugar phosphate, also called triose phosphate. In a typical leaf, there are two fates for the G3P:

One-third of it ends up in the polysaccharide starch, which is stored in the chloroplast.

Two-thirds of it is converted in the cytosol to the disaccharide sucrose, which is transported out of the leaf to other organs in the plant, where it is hydrolyzed to its constituent monosaccharides: glucose and fructose. The G3P produced in photosynthesis is subsequently used by the plant to make other compounds. Its carbon is thus incorporated into amino acids, lipids, and the building blocks of the nucleic acids. The products of the Calvin–Benson cycle are of crucial importance to the entire biosphere, for the covalent bonds of the carbohydrate generated in the cycle represent the total energy yield from the harvesting of light by photosynthetic organisms. Most of this stored energy is released by glycolysis and cellular respiration and used to support plant growth, development, and reproduction. Much plant matter ends up being consumed by animals, supplying them with both raw materials and energy sources. Glycolysis and cellular respiration in the animals release free energy from the plant matter for use in the animal cells.