We now shift our attention to photosynthesis, the second key process for synthesizing ATP. In plants, photosynthesis occurs in chloroplasts, large organelles found mainly in leaf cells. During photosynthesis, chloroplasts capture the energy of sunlight, convert it into chemical energy in the form of ATP and NADPH, and then use this energy to make complex carbohydrates out of carbon dioxide and water. The principal carbohydrates produced are polymers of hexose (six-carbon) sugars: sucrose, a glucose-fructose disaccharide, and starch, a mixture of two types of large, insoluble glucose polymers called amylose and amylopectin. Starch is the primary storage carbohydrate in plants (Figure 1). Starch is synthesized and stored in the chloroplast. Sucrose is synthesized in the leaf cytosol from three-carbon precursors generated in the chloroplast; it is transported to non-photosynthetic (nongreen) plant tissues (e.g., roots and seeds), which metabolize it for energy by the pathways described in the previous sections.
Fig1. Structure of starch. This large glucose polymer and the disaccharide sucrose are the principal end products of photosynthesis. Both are built of six-carbon sugars (hexoses).
Photosynthesis in plants, as well as in eukaryotic single celled algae and in several photosynthetic bacteria (e.g., the cyanobacteria and prochlorophytes), also generates oxygen. The overall reaction of oxygen-generating photosynthesis,
is the reverse of the overall reaction by which carbohydrates are oxidized to CO2 and H2O. In effect, photosynthesis in chloroplasts produces energy-rich sugars that are broken down and harvested for energy by mitochondria using oxidative phosphorylation.
Although green and purple bacteria also carry out photo synthesis, they use a process that does not generate oxygen. detailed analysis of the photo synthetic system in these bacteria has helped elucidate the first stages in the more common process of oxygen-generating photosynthesis. In this section, we provide an overview of the stages in oxygen-generating photosynthesis and introduce the main molecular components of the process, including the chlorophylls, the principal light-absorbing pigments.
Thylakoid Membranes in Chloroplasts Are the Sites of Photosynthesis in Plants
Chloroplasts are lens-shaped organelles with a diameter of approximately 5 μm and a width of approximately 2.5 μm. They contain about 3000 different proteins, 95 percent of which are encoded in the nucleus, made in the cytosol, imported into the organelle, and then transported to their ap propriate membrane or space. Chloroplasts are bounded by two membranes, which do not contain chlorophyll and do not participate directly in the generation of ATP and NADPH driven by light (Figures 2 and 3). Like that of mitochondria, the outer membrane of chloroplasts contains porins and thus is permeable to metabolites of small molecular weight. The inner membrane forms a permeability barrier that contains transport proteins for regulating the movement of metabolites into and out of the organelle.
Fig2. Structure of leaf and chloroplast. Like mitochondria, plant chloroplasts are bounded by two membranes separated by an intermembrane space. Photosynthesis occurs on a third membrane, the thylakoid membrane, which is surrounded by the inner membrane and forms a series of flattened vesicles (thylakoids) that enclose a single interconnected luminal space. The green color of plants is due to the green color of chlorophyll, all of which is located within the thylakoid membrane. A granum is a stack of adjacent thylakoids. The stroma is the space between the inner membrane and the thylakoids. [From Katherine Esau, D-120, Special Collections, University of California Library, Davis.]
Fig3. Overview of the four stages of photosynthesis. In stage 1, light is absorbed by light-harvesting complexes (LHCs) and the reaction center of photosystem II (PSII). The LHCs transfer the absorbed energy to the reaction centers, which use it, or the energy absorbed directly from a photon, to oxidize water to molecular oxygen and generate high-energy electrons (electron paths shown by blue arrows). In stage 2, these electrons move down an electron-transport chain, which uses either lipid-soluble (Q/QH2) or water-soluble (plastocyanin, PC) electron carriers to shuttle electrons between multiple protein complexes. As electrons move down the chain, they release energy that the complexes use to generate a proton-motive force and, after additional energy is introduced by absorption of light in photosystem I (PSI), to synthesize the high-energy electron carrier NADPH. In stage 3, flow of protons down their concentration and voltage gradient through the F0F1 ATP synthase drives ATP synthesis. Stages 1–3 in plants take place in the thylakoid membrane of the chloroplast. In stage 4, in the chloroplast stroma, the energy stored in NADPH and ATP is used to incorporate CO2 into the three-carbon molecule glyceraldehyde 3-phosphate, the first step in a process known as carbon fixation. These molecules are then transported to the cytosol of the cell for conversion to hexose sugars in the form of sucrose. Glyceraldehyde 3-phosphate is also used to make starch within the chloroplast. Inset: Three-dimensional reconstruction from cryoelectron tomography of a chloroplast in the unicellular green alga Chlamydomonas reinhardtii, showing thylakoid membranes (dark green), thylakoid lumen (light green), inner and outer membranes (blue), and one small starch granule (tan). [Inset from Engel, B. D., et al., “Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography,” eLIFE, 2015; 4: e04889.]
Unlike mitochondria, chloroplasts contain a third membrane—the thylakoid membrane—on which the light driven generation of ATP and NADPH occurs. The chloroplast thylakoid membrane is believed to constitute a single sheet that forms numerous small, interconnected flattened structures, the thylakoids, which are commonly arranged in stacks termed grana (see Figure 2). The spaces within all the thylakoids constitute a single continuous compartment, the thylakoid lumen (see Figure 3). The thylakoid membrane contains a number of integral membrane proteins to which are bound several important prosthetic groups and light-absorbing pigments, most notably chlorophylls. Starch synthesis and storage occurs in the stroma, the aqueous compartment between the thylakoid membrane and the inner membrane. In photosynthetic bacteria, extensive invaginations of the plasma membrane form a set of internal membranes, also termed thylakoid membranes, where photosynthesis occurs.
Chloroplasts Contain Large DNAs Often Encoding More Than a Hundred Proteins
Like mitochondria, chloroplasts are thought to have evolved from an ancestral endosymbiotic photosynthetic bacterium. However, the endosymbiotic event that gave rise to chloroplasts occurred more recently (1.2 billion–1.5 billion years ago) than the event that led to the evolution of mitochondria (1.5 billion– 2.2 billion years ago). Consequently, contemporary chloroplast DNAs show less structural diversity than do mtDNAs. Also like mitochondria, chloroplasts contain multiple copies of the organelle DNA as well as ribosomes, which synthesize some chloroplast DNA–encoded proteins using the standard genetic code. Like plant mtDNA, chloroplast DNA is inherited exclusively in a uniparental fashion through the female parent (egg). Other chloroplast proteins are encoded by nu clear genes, synthesized on cytosolic ribosomes, and then incorporated into the organelle.
In higher plants, chloroplast DNA molecules are 120–160 kb long, depending on the species. Plant chloroplast DNAs are long head-to-tail linear concatemers plus recombination inter mediates between these long linear molecules. They contain 120–135 genes, 130 in the important model plant Arabidopsis thaliana. A. thaliana chloroplast DNA encodes 76 protein-coding genes and 54 genes with RNA products such as rRNAs and tRNAs. Chloroplast DNAs encode the subunits of a bacteria like RNA polymerase, and they express many of their genes from polycistronic operons, as in bacteria. Some chloroplast genes contain introns, but these introns are similar to the specialized introns found in some bacterial genes and in mitochondrial genes from fungi and protozoans, rather than the introns of nuclear genes. Many genes essential for chloroplast function have been transferred to the nuclear genome of plants over evolutionary time. Recent estimates from sequence analysis of the A. thaliana and cyanobacterial genomes indicate that somewhat less than 4500 genes have been transferred from the original endosymbiont to the nuclear genome.
Methods similar to those used for the transformation of yeast cells have been developed for stably introducing foreign DNA into the chloroplasts of higher plants. The large number of chloroplast DNA molecules per cell permits the introduction of thousands of copies of an engineered gene into each cell, resulting in extraordinarily high levels of foreign protein production, comparable with that achieved with engineered bacteria. Chloroplast transformation has led to the engineering of plants that are resistant to bacterial and fungal infections, drought, and herbicides as well as to plants that can be used to make human pharmaceutical drugs (called pharming). The first such pharming drug, approved in the United States for use in adults in 2012 and children in 2014, is an enzyme to treat Gaucher’s disease, a genetic disorder. This approach might also be used for the engineering of food crops containing high levels of all the amino acids essential to humans.
Three of the Four Stages in Photosynthesis Occur Only During Illumination
The photosynthetic process in plants can be divided into four stages (see Figure 3), each localized to a defined area of the chloroplast: (1) absorption of light, generation of high energy electrons, and formation of O2 from H2O; (2) electron transport leading to reduction of NADP+ to NADPH, and generation of a proton-motive force; (3) synthesis of ATP; and (4) conversion of CO2 into carbohydrates, commonly referred to as carbon fixation. The enzymes that incorporate CO2 into chemical intermediates and then convert them to starch are soluble constituents of the chloroplast stroma; the enzymes that form sucrose from three-carbon intermediates are in the cytosol. All four stages of photosynthesis are tightly coupled and controlled so as to produce the amount of carbohydrate required by the plant. All the reactions in stages 1–3 are catalyzed by multiprotein complexes in the thylakoid membrane. The generation of a proton-motive force and the use of that proton-motive force to synthesize ATP resemble stages III and IV of mitochondrial oxidative phosphorylation.
Stage 1: Absorption of Light Energy, Generation of High- Energy Electrons, and O2 Formation The initial step in photosynthesis is the absorption of light by chlorophylls attached to proteins in the thylakoid membranes. Like the heme component of cytochromes, chlorophylls consist of a porphyrin ring attached to a long hydrocarbon side chain (Figure 4). In contrast to the hemes, chlorophylls contain a central Mg2+ ion (rather than Fe2+) and have an additional five-member ring. The energy of the absorbed light is ultimately used to remove electrons from a donor (water in the case of green plants), forming oxygen:
The electrons are transferred to a primary electron acceptor, a quinone designated Q, which is similar to CoQ in mitochondria. In plants, the oxidation of water takes place in a multiprotein complex called photosystem II (PSII).
Fig4. Structure of chlorophyll a, the principal pigment that traps light energy. Electrons are delocalized among three of chlorophyll a’s four central rings (yellow) and the atoms that interconnect them. In chlorophyll, a Mg2+ ion, rather than the Fe2+ ion found in heme, sits at the center of the porphyrin ring, and an additional five member ring (blue) is present; otherwise, the structure of chlorophyll is similar to that of heme, found in molecules such as hemoglobin and cytochromes. The hydrocarbon phytol “tail” facilitates the binding of chlorophyll to hydrophobic regions of chlorophyll binding proteins. The CH3 group (green) is replaced by a formaldehyde (CHO) group in chlorophyll b.
Quantum mechanics established that light, a form of electromagnetic radiation, has properties of both waves and particles. When light interacts with matter, it behaves as discrete packets of energy (quanta) called photons. The energy of a photon is proportional to the frequency of the light wave, and thus inversely proportional to its wavelength. Thus pho tons of shorter wavelengths have higher energies. The energy of visible light is considerable. Light with a wavelength of 550 nm (550 × 10−7 cm), typical of sunlight, has about 52 kcal of energy per mole of photons. This is enough energy to synthesize several moles of ATP from ADP and Pi if all the energy were used for this purpose.
Stage 2: Electron Transport and Generation of a Proton Motive Force Electrons move from the quinone primary electron acceptor through a series of electron carriers until they reach the ultimate electron acceptor, usually the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+), reducing it to NADPH. The structure of NADP+ is identical to that of NAD+ except for the presence of an additional phosphate group. Both molecules gain and lose electrons in the same way. In plants, the reduction of NADP+ takes place in a complex called photosystem I (PSI) (Figure 3). The transport of electrons in the thylakoid membrane is coupled to the movement of protons from the stroma to the thylakoid lumen, forming a pH gradient across the membrane (pHlumen < pHstroma). This process is analogous to the generation of a proton-motive force across the inner mitochondrial membrane and in bacterial membranes during electron transport.
Thus the overall reaction of stages 1 and 2 can be summarized as
Stage 3: Synthesis of ATP Protons move down their con centration gradient from the thylakoid lumen to the stroma through the chloroplast F0F1 complex (ATP synthase), which couples proton movement to the synthesis of ATP from ADP and Pi, as we have seen for the ATP synthases in mitochondria and bacteria.
Stage 4: Carbon Fixation The NADPH and ATP generated by stages 2 and 3 of photosynthesis provide the energy and the electrons to drive the synthesis of polymers of six-carbon sugars from CO2 and H2O. The overall chemical equation is written as
The reactions that generate the ATP and NADPH used in carbon fixation are directly dependent on light energy; thus stages 1–3 are called the light reactions of photosynthesis. The reactions in stage 4 are indirectly dependent on light energy; they are sometimes called the dark reactions of photosynthesis because they can occur in the dark, using the supplies of ATP and NADPH generated by light energy (see Figure 3). However, the reactions in stage 4 are not confined to the dark; in fact, they occur primarily during illumination.
Photosystems Comprise a Reaction Center and Associated Light-Harvesting Complexes
The absorption of light energy and its conversion into chemical energy occurs in multiprotein complexes called photosystems. Found in all photosynthetic organisms, both eukaryotic and prokaryotic, photosystems consist of two closely linked components: a reaction center, where the primary events of photosynthesis—light absorption and generation of high-energy electrons—occur; and an antenna complex consisting of numerous protein complexes, including internal antenna proteins. Each photosystem is also associated with external antenna complexes termed light-harvesting complexes (LHCs), made up of specialized proteins that capture light energy and efficiently transmit it to the reaction center to generate high-energy electrons (see Figure 3).
Both reaction centers and antennas contain tightly bound light-absorbing pigment molecules. Chlorophyll a, the principal pigment involved in photosynthesis, is present in both reaction centers and antennas. In addition to chlorophyll a, antennas contain other light-absorbing pigments: chlorophyll b in vascular plants and carotenoids in both plants and photosynthetic bacteria. Carotenoids con sist of long branched hydrocarbon chains with alternating single and double bonds; they are similar in structure to the visual pigment retinal, which absorbs light in the eye. The presence of various antenna pigments, which absorb light at different wavelengths, greatly ex tends the range of light that can be absorbed and used for photosynthesis.
One of the strongest pieces of evidence for the involvement of chlorophylls and carotenoids in photosynthesis is that the absorption spectrum of these pigments is similar to the action spectrum of photosynthesis (Figure 5). The latter is a measure of the relative ability of light of different wavelengths to support photosynthesis.
Fig5. The rate of photosynthesis is greatest at the wavelengths of light absorbed by three plant pigments. The action spectrum of photosynthesis in plants (the relative ability of light of different wavelengths to support photosynthesis) is shown in black. The energy from light can be converted into ATP only if it can be absorbed by pigments in the chloroplast. Absorption spectra (showing how well light of different wavelengths is absorbed) for three photosynthetic pigments present in the antennas of plant photosystems are shown in color. Comparison of the action spectrum of photosynthesis with the individual absorption spectra of these pigments suggests that photosynthesis at 680 nm is primarily due to light absorbed by chlorophyll a; at 650 nm, to light absorbed by chlorophyll b; and at shorter wavelengths, to light absorbed by chlorophylls a and b and by carotenoid pigments, including β-carotene.
When chlorophyll a (or any other molecule) absorbs visible light, the absorbed light energy raises electrons in the chlorophyll a to a higher-energy (excited) state. This state differs from the ground (unexcited) state largely in the distribution of the electrons around the C and N atoms of the porphyrin ring. Excited states are unstable, and the electrons return to the ground state by one of several competing processes. For chlorophyll a molecules dissolved in organic solvents such as ethanol, the principal reactions that dissipate the excited-state energy are the emission of light (fluorescence and phosphorescence) and thermal emission (heat). However, when the same chlorophyll a is bound in the unique protein environment of the reaction center, dissipation of excited-state energy occurs by a different process, which is the key to photosynthesis.
Photoelectron Transport from Energized Reaction-Center Chlorophyll a Produces a Charge Separation
Within the reaction center, two adjacent chlorophyll a molecules, referred to as the special-pair chlorophylls, lie close to the luminal face of the thylakoid membrane (Figure 6).
Fig6. Photoelectron transport, the primary event in photosynthesis. After absorption of a photon of light, one of the excited special-pair chlorophyll a molecules in the reaction center (left) donates, via several intermediates (not shown), an electron to a loosely bound acceptor molecule, the quinone Q, on the stromal surface of the thylakoid membrane, creating an essentially irreversible charge separation across the membrane (right). Subsequent transfers of this electron release energy that is used to generate ATP and NADPH. The positively charged chlorophyll a+ generated when the light-excited electron moves to Q is eventually neutralized by the transfer to the chlorophyll a+ of another electron. In plants, the oxidation of H2O to O2 provides this neutralizing electron and takes place in a multiprotein complex called photosystem II. Photosystem I uses a similar photoelectron transport pathway, but instead of oxidizing water, it receives an electron from a protein carrier called plastocyanin to neutralize the positive charge on chlorophyll a+.
When a photon of light with a wavelength of about 680 nm is absorbed by one of these two chlorophyll a molecules, the energy of that chlorophyll a molecule increases by 42 kcal/ mol (the first excited state). This energized molecule rapidly donates an electron to the adjacent chlorophyll, which passes it on to a series of intermediate acceptors. In this manner, the electron is rapidly passed on to the primary electron acceptor, quinone Q, near the stromal surface of the thylakoid membrane. This light-driven electron transfer, called photoelectron transport, depends on the unique environment of both the chlorophylls and the acceptor within the reaction center. Photoelectron transport, which occurs nearly every time a photon is absorbed, leaves a positive charge on the chlorophyll a close to the luminal surface of the thylakoid membrane (on the opposite side from the stroma) and generates a reduced, negatively charged acceptor (Q−) near the stromal surface.
The Q− produced by photoelectron transport is a powerful reducing agent with a strong tendency to transfer an electron to another molecule, ultimately to NADP+. The positively charged chlorophyll a+, a strong oxidizing agent, attracts an electron from an electron donor on the luminal surface to regenerate the original chlorophyll a. In plants, the oxidizing power of four chlorophyll a+ molecules is used, by way of intermediates, to remove four electrons from two H2O molecules bound to a site on the luminal surface to form O2:
These potent biological reductants and oxidants provide all the energy needed to drive all subsequent reactions of photosynthesis: electron transport (stage 2), ATP synthesis (stage 3), and CO2 fixation (stage 4).
Chlorophyll a also absorbs light at discrete wavelengths shorter (and therefore of higher energy) than 680 nm (see Figure 5). Such absorption raises the molecule into one of several excited states whose energies are higher than that of the first excited state described above, and which decay by releasing energy within 2 × 10−12 seconds (2 picoseconds, ps) to the lower-energy first excited state, with loss of the extra energy as heat. Because photoelectron transport and the resulting charge separation occur only from the first ex cited state of the reaction-center chlorophyll a, the quantum yield—the amount of photosynthesis per absorbed photon— is the same for all wavelengths of visible light shorter than 680 nm. How closely the wavelength of light matches the absorption spectrum of the pigment determines how likely it is that the photon will be absorbed. Once absorbed, the photon’s exact wavelength is not critical, provided it is at least energetic enough to push the chlorophyll a into the first excited state.
Internal Antennas and Light-Harvesting Complexes Increase the Efficiency of Photosynthesis
Although the special-pair chlorophyll a molecules within the reaction center that are involved directly in charge separation and electron transfer are capable of directly absorbing light and initiating photosynthesis, they are most commonly energized indirectly by energy transferred to them from other light-absorbing and energy-transferring pigments. These other pigments, which include many other chlorophylls, absorb photons and pass the energy to the special pair chlorophylls (Figure 7). Some of these pigments are bound to protein subunits that are considered to be intrinsic components of the photosystem, which is made up of several distinct protein chains, and thus are called internal antennas. Others are incorporated into protein complexes that bind to, but are distinct from, the photosystem core proteins and are called light-harvesting complexes (LHCs). Even at the maximum light intensity encountered by photosynthetic organisms (tropical noontime sunlight), each reaction-center chlorophyll a molecule absorbs only about one photon per second, which is not enough to support photosynthesis sufficient for the needs of the plant. The involvement of internal antennas and LHCs greatly increases the efficiency of photosynthesis, especially at more typical light intensities, by increasing absorption of 680-nm light and by extending the range of wavelengths of light that can be absorbed by other antenna pigments.
Fig7. Light-harvesting complexes and photosystems in cyanobacteria and plants. (a) Diagram of the membrane of a cyanobacterium, in which each multiprotein light-harvesting complex (LHC) contains 90 chlorophyll molecules and 31 other small molecules, all held in a specific geometric arrangement for optimal light absorption and energy transfer. Of the six chlorophyll molecules in the reaction center, two constitute the special-pair chlorophylls that can initiate photoelectron transport (blue arrow) when excited. Resonance transfer of energy (red arrows) rapidly funnels energy from absorbed light to one of two “bridging” chlorophylls and thence to the special-pair chlorophylls in the reaction center. (b) Three-dimensional organization of photosystem I (PSI) and its associated LHCs from Pisum sativum (garden pea), as determined by x-ray crystallography, seen from the plane of the membrane. Only the chlorophylls and the reaction-center electron carriers are shown. (c) Expanded view of the reaction center from (b), rotated 90° about a vertical axis. See W. Kühlbrandt, 2001, Nature 411:896, and P. Jordan et al., 2001, Nature 411:909. [Parts (b) and (c) data from A. Ben-Sham et al., 2003, Nature 426:630, PDB ID 1qvz; and Y. Mazor, A. Borovikova, and N. Nelson, 2015, Elife 4:e07433, PDB ID 4y28.]
Photosystem core proteins and LHC proteins maintain the pigment molecules in the precise orientations and positions that are optimal for light absorption and rapid ( <10−9 seconds) energy transfer, called resonance transfer, to one of the special-pair chlorophyll a molecules in the associated reaction center. Resonance energy transfer does not involve the transfer of an electron. Studies on one of the two pho tosystems in cyanobacteria, which are similar to those in multicellular, seed-bearing plants, suggest that energy from absorbed light is funneled first to a “bridging” chlorophyll in each LHC and then to the special-pair chlorophylls in the reaction center (see Figure 7a). Surprisingly, however, the molecular structures of LHCs from plants and cyanobacteria are completely different from those from green and purple bacteria, even though both types contain carotenoids and chlorophylls. Figure 7b shows the distribution of the chlorophyll pigments in photosystem I from Pisum sati vum (garden pea) together with those from peripheral LHC antennas. The large number of internal and LHC antenna chlorophylls that surround the reaction center permit efficient transfer of absorbed light energy to the special-pair chlorophylls in the reaction center.
Although LHC antenna chlorophylls can transfer light energy absorbed from a photon, they cannot release an electron. As we’ve seen already, this function resides in the two reaction-center chlorophylls. To understand their electron releasing ability, we examine the structure and function of the reaction center in bacterial and plant photosystems in the next section.
