THE PHOTOSYNTHETIC PROCESS

In: "Concepts in Photobiology: Photosynthesis and Photomorphogenesis", Edited by GS Singhal, G Renger, SK Sopory, K-D Irrgang and Govindjee, Narosa Publishers/New Delhi; and Kluwer Academic/Dordrecht, pp. 11-51.

John Whitmarsh
Photosynthesis Research Unit, Agricultural Research Service/USDA
Department of Plant Biology and Center of Biophysics and Computational Biology,
University of Illinois at Urbana-Champaign
 

Govindjee
Department of Plant Biology and Center of Biophysics and Computational Biology
University of Illinois at Urbana-Champaign
 

Summary

1. Introduction

Photosynthesis is the physico-chemical process by which plants, algae and photosynthetic bacteria use light energy to drive the synthesis of organic compounds. In plants, algae and certain types of bacteria, the photosynthetic process results in the release of molecular oxygen and the removal of carbon dioxide from the atmosphere that is used to synthesize carbohydrates (oxygenic photosynthesis). Other types of bacteria use light energy to create organic compounds but do not produce oxygen (anoxygenic photosynthesis). Photosynthesis provides the energy and reduced carbon required for the survival of virtually all life on our planet, as well as the molecular oxygen necessary for the survival of oxygen consuming organisms1 . In addition, the fossil fuels currently being burned to provide energy for human activity were produced by ancient photosynthetic organisms. Although photosynthesis occurs in cells or organelles that are typically only a few microns across, the process has a profound impact on the earth's atmosphere and climate. Each year more than 10% of the total atmospheric carbon dioxide is reduced to carbohydrate by photosynthetic organisms. Most, if not all, of the reduced carbon is returned to the atmosphere as carbon dioxide by microbial, plant and animal metabolism, and by biomass combustion. In turn, the performance of photosynthetic organisms depends on the earth's atmosphere and climate. Over the next century, the large increase in the amount of atmospheric carbon dioxide created by human activity is certain to have a profound impact on the performance and competition of photosynthetic organisms. Knowledge of the physico-chemical process of photosynthesis is essential for understanding the relationship between living organisms and the atmosphere and the balance of life on earth. Several books on photosynthesis are available for the uninitiated (Hall and Rao, 1994; Lawlor, 1993; and Walker, 1992) or advanced student (Govindjee, 1982; Amesz, 1987; Briggs, 1989; Barber, 1992; Scheer, 1991; Bryant, 1994; Blankenship et al. 1995; Amesz and Hoff, 1996, Baker, 1996; and Ort and Yocum, 1996). Taiz and Zeiger (1991) place the photosynthetic process in the context of over all plant physiology, and Cramer and Knaff (1991) describe the bioenergetic foundation of photosynthesis.

All life can be divided into three domains, Archaea, Bacteria and Eucarya, which originated from a common ancestor (Woese et al., 1990). Historically, the term photosynthesis has been applied to organisms that depend on chlorophyll (or bacteriochlorophyll) for the conversion of light energy into chemical free energy (Gest , 1993). These include organisms in the domains Bacteria (photosynthetic bacteria) and Eucarya (algae and higher plants). The most primitive domain, Archaea, includes organisms known as halobacteria, that convert light energy into chemical free energy. However, the mechanism by which halobacteria convert light is fundamentally different from that of higher organisms because there is no oxidation/reduction chemistry and halobacteria cannot use CO2 as their carbon source. Consequently some biologists do not consider halobacteria as photosynthetic (Gest 1993). This chapter will follow the historical definition of photosynthesis and omit halobacteria.

3.1 Oxygenic Photosynthetic Organisms
The photosynthetic process in all plants and algae as well as in certain types of photosynthetic bacteria involves the reduction of CO2 to carbohydrate and removal of electrons from H20, which results in the release of O2. In this process, known as oxygenic photosynthesis, water is oxidized by the photosystem II reaction center, a multisubunit protein located in the photosynthetic membrane. Years of research have shown that the structure and function of photosystem II is similar in plants, algae and certain bacteria, so that knowledge gained in one species can be applied to others. This homology is a common feature of proteins that perform the same reaction in different species. This homology at the molecular level is important because there are estimated to be 300,000-500,000 species of plants. If different species had evolved diverse mechanisms for oxidizing water, research aimed at a general understanding of photosynthetic water oxidation would be hopeless.

The energy that drives photosynthesis originates in the center of the sun, where mass is converted to heat by the fusion of hydrogen. Over time, the heat energy reaches the sun's surface, where some of it is converted to light by black body radiation that reaches the earth. A small fraction of the visible light incident on the earth is absorbed by plants. Through a series of energy transducing reactions, photosynthetic organisms are able to transform light energy into chemical free energy in a stable form that can last for hundreds of millions of years (e.g., fossil fuels). A simplified scheme describing how energy is transformed in the photosynthetic process is presented in this section. The focus is on the structural and functional features essential for the energy transforming reactions. For clarity, mechanistic and structural details are omitted. A more highly resolved description of oxygenic and anoxygenic photosynthesis is given in the remaining sections.

The photosynthetic process in plants and algae occurs in small organelles known as chloroplasts that are located inside cells. The more primitive photosynthetic organisms, for example oxygenic cyanobacteria, prochlorophytes and anoxygenic photosynthetic bacteria, lack organelles. The photosynthetic reactions are traditionally divided into two stages - the "light reactions," which consist of electron and proton transfer reactions and the "dark reactions," which consist of the biosynthesis of carbohydrates from CO2. The light reactions occur in a complex membrane system (the photosynthetic membrane) that is made up of protein complexes, electron carriers, and lipid molecules. The photosynthetic membrane is surrounded by water and can be thought of as a two-dimensional surface that defines a closed space, with an inner and outer water phase. A molecule or ion must pass through the photosynthetic membrane to go from the inner space to the outer space. The protein complexes embedded in the photosynthetic membrane have a unique orientation with respect to the inner and outer phase. The asymmetrical arrangement of the protein complexes allows some of the energy released during electron transport to create an electrochemical gradient of protons across the photosynthetic membrane.

Photosynthetic electron transport consists of a series of individual electron transfer steps from one electron carrier to another. The electron carriers are metal ion complexes and aromatic groups. The metal ion complexes and most of the aromatic groups are bound within proteins. Most of the proteins involved in photosynthetic electron transport are composed of numerous polypeptide chains that lace through the membrane, providing a scaffolding for metal ions and aromatic groups. An electron enters a protein complex at a specific site, is transferred within the protein from one carrier to another, and exits the protein at a different site. The protein controls the pathway of electrons between the carriers by determining the location and environment of the metal ion complexes and aromatic groups. By setting the distance between electron carriers and controlling the electronic environment surrounding a metal ion complex or aromatic group, the protein controls pairwise electron transfer reactions. Between proteins, electron transfer is controlled by distance and free energy, as for intraprotein transfer, and by the probability that the two proteins are in close contact. Protein association is controlled by a number of factors, including the structure of the two proteins, their surface electrical and chemical properties and the probability that they collide with one another. Not all electron carriers are bound to proteins. The reduced forms of plastoquinone or ubiquinone and nicotinamide adenine dinucleotide phosphate (NADPH) or NADH act as mobile electron carriers operating between protein complexes. For electron transfer to occur, these small molecules must bind to special pockets in the proteins known as binding sites. The binding sites are highly specific and are a critical factor in controlling the rate and pathway of electron transfer.

The light reactions convert energy into several forms (Fig. 1). The first step is the conversion of a photon to an excited electronic state of an antenna pigment molecule located in the antenna system. The antenna system consists of hundreds of pigment molecules (mainly chlorophyll or bacteriochlorophyll and carotenoids) that are anchored to proteins within the photosynthetic membrane and serve a specialized protein complex known as a reaction center. The electronic excited state is transferred over the antenna molecules as an exciton. Some excitons are converted back into photons and emitted as fluorescence, some are converted to heat, and some are trapped by a reaction center protein. (For a discussion of the use of fluorescence as a probe of photosynthesis, see e.g., Govindjee et al., 1986 and Krause and Weis, 1991.) Excitons trapped by a reaction center provide the energy for the primary photochemical reaction of photosynthesis - the transfer of an electron from a donor molecule to an acceptor molecule. Both the donor and acceptor molecules are attached to the reaction center protein complex. Once primary charge separation occurs, the subsequent electron transfer reactions are energetically downhill.

In oxygenic photosynthetic organisms (see section 5), two different reaction centers, known as photosystem II and photosystem I, work concurrently but in series. In the light photosystem II feeds electrons to photosystem I. The electrons are transferred from photosystem II to the photosystem I by intermediate carriers. The net reaction is the transfer of electrons from a water molecule to NADP+, producing the reduced form, NADPH. In the photosynthetic process, much of the energy initially provided by light energy is stored as redox free energy (a form of chemical free energy) in NADPH, to be used later in the reduction of carbon. In addition, the electron transfer reactions concentrate protons inside the membrane vesicle and create an electric field across the photosynthetic membrane. In this process the electron transfer reactions convert redox free energy into an electrochemical potential of protons. The energy stored in the proton electrochemical potential is used by a membrane bound protein complex (ATP-Synthase) to covalently attach a phosphate group to adenosine diphosphate (ADP), forming adenosine triphosphate (ATP). Protons pass through the ATP-Synthase protein complex that transforms electrochemical free energy into a type of chemical free energy known as phosphate group-transfer potential (or a high-energy phosphate bond) (Klotz, 1967). The energy stored in ATP can be transferred to another molecule by transferring the phosphate group. The net effect of the light reactions is to convert radiant energy into redox free energy in the form of NADPH and phosphate group-transfer energy in the form of ATP. In the light reactions, the transfer of a single electron from water to NADP+ involves about 30 metal ions and 7 aromatic groups. The metal ions include 19 Fe, 5 Mg, 4 Mn, and 1 Cu. The aromatics include quinones, pheophytin, NADPH, tyrosine and a flavoprotein. The NADPH and ATP formed by the light reactions provide the energy for the dark reactions of photosynthesis, known as the Calvin cycle or the photosynthetic carbon reduction cycle. The reduction of atmospheric CO2 to carbohydrate occurs in the aqueous phase of the chloroplast and involves a series of enzymatic reactions. The first step is catalyzed by the protein Rubisco (D-ribulose 1,5-bisphosphate carboxylase/oxygenase), which attaches CO2 to a five-carbon compound. The reaction produces two molecules of a three-carbon compound. Subsequent biochemical reactions involve several enzymes that reduce carbon by hydrogen transfer and rearrange the carbon compounds to synthesize carbohydrates. The carbon reduction cycle involves the transfer and rearrangement of chemical bond energy.

5.1 Chloroplasts - Structure and Organization
In plants the photosynthetic process occurs inside chloroplasts, which are organelles found in certain cells. Chloroplasts provide the energy and reduced carbon needed for plant growth and development, while the plant provides the chloroplast with CO2, water, nitrogen, organic molecules and minerals necessary for the chloroplast biogenesis. Most chloroplasts are located in specialized leaf cells, which often contain 50 or more chloroplasts per cell. Each chloroplast is defined by an inner and an outer envelope membrane and is shaped like a meniscus convex lens that is 5-10 microns in diameter (Fig. 2), although many different shapes and sizes can be found in plants. For details of chloroplast structure, see Staehlin (1986). The inner envelope membrane acts as a barrier, controlling the flux of organic and charged molecules in and out of the chloroplast. Water passes freely through the envelope membranes, as do other small neutral molecules like CO2 and O2. There is evidence that chloroplasts were once free living bacteria that invaded a non-photosynthetic cell long ago. They have retained some of the DNA necessary for their assembly, but much of the DNA necessary for their biosynthesis is located in the cell nucleus. This enables a cell to control the biosynthesis of chloroplasts within its domain.

Inside the chloroplast is a complicated membrane system, known as the photosynthetic membrane (or thylakoid membrane), that contains most of the proteins required for the light reactions. The proteins required for the fixation and reduction of CO2 are located outside the photosynthetic membrane in the surrounding aqueous phase. The photosynthetic membrane is composed mainly of glycerol lipids and protein. The glycerol lipids are a family of molecules characterized by a polar head group that is hydrophilic and two fatty acid side chains that are hydrophobic. In membranes, the lipid molecules arrange themselves in a bilayer, with the polar head toward the water phase and the fatty acid chains aligned inside the membrane forming a hydrophobic core (Fig. 3). The photosynthetic membrane is vesicular, defining a closed space with an outer water space (stromal phase) and an inner water space (lumen). The organization of the photosynthetic membrane can be described as groups of stacked membranes (like stacks of pita or chapati bread with the inner pocket representing the inner aqueous space), interconnected by non-stacked membranes that protrude from the edges of the stacks (Fig. 2). Experiments indicate that the inner aqueous space of the photosynthetic membrane is likely continuous inside of the chloroplast. It is not known why the photosynthetic membrane forms such a convoluted structure. To understand the energetics of photosynthesis the complicated structure can be ignored and the photosynthetic membrane can be viewed as a simple vesicle.

5.2 Light Absorption - The Antenna System
Plant photosynthesis is driven primarily by visible light (wavelengths from 400 to 700 nm) that is absorbed by pigment molecules (mainly chlorophyll a and b and carotenoids). The chemical structure of chlorophyll a molecule is shown in Fig. 4. In chlorophyll b, CH3 in ring II is replaced by CHO group. Plants appear green because of chlorophyll, which is so plentiful that regions of the earth appear green from space. The absorption spectrum of chloroplast chlorophyll a and b and carotenoids along with the action spectrum of photosynthesis of a chloroplast is shown in Fig. 5. Light is collected by 200-300 pigment molecules that are bound to light- harvesting protein complexes located in the photosynthetic membrane. The light-harvesting complexes surround the reaction centers that serve as an antenna. The three-dimensional structure of the light-harvesting complex (Kühlbrandt et al., 1994) shows that the protein determines the position and orientation of the antenna pigments. Photosynthesis is initiated by the absorption of a photon by an antenna molecule, which occurs in about a femtosecond (10-15 s) and causes a transition from the electronic ground state to an excited state. Within 10-13 s the excited state decays by vibrational relaxation to the first excited singlet state. The fate of the excited state energy is guided by the structure of the protein. Because of the proximity of other antenna molecules with the same or similar energy states, the excited state energy has a high probability of being transferred by resonance energy transfer to a near neighbor. Exciton energy transfer between antenna molecules is due to the interaction of the transition dipole moment of the molecules. The probability of transfer is dependent on the distance between the transition dipoles of the donor and acceptor molecules (1/R6), the relative orientation of the transition dipoles, and the overlap of the emission spectrum of the donor molecule with the absorption spectrum of the acceptor molecule (see van Grondelle and Amesz, 1986). Photosynthetic antenna systems are very efficient at this transfer process. Under optimum conditions over 90% of the absorbed quanta are transferred within a few hundred picoseconds from the antenna system to the reaction center which acts as a trap for the exciton. A simple model of the antenna and its reaction center is shown in Fig. 6.

5.3 Primary Photochemistry - Photosystem II and Photosystem I Reaction Centers
Photosystem II uses light energy to drive two chemical reactions - the oxidation of water and the reduction of plastoquinone. The photosystem II complex is composed of more than fifteen polypeptides and at least nine different redox components (chlorophyll, pheophytin, plastoquinone, tyrosine, Mn, Fe, cytochrome b559, carotenoid and histidine) have been shown to undergo light-induced electron transfer (Debus, 1992). However, only five of these redox components are known to be involved in transferring electrons from H2O to the plastoquinone pool - the water oxidizing manganese cluster (Mn)4, the amino acid tyrosine, the reaction center chlorophyll (P680), pheophytin, and the plastoquinone molecules, QA and QB. Of these essential redox components, tyrosine, P680, pheophytin, QA and QB have been shown to be bound to two key polypeptides that form the heterodimeric reaction center core of photosystem II (D1 and D2). Recent work indicates that the D1 and D2 polypeptides also provide ligands for the (Mn)4 cluster. The three-dimensional structure of photosystem II is not known. Our knowledge of its structure is guided by the known structure of the reaction center in purple bacteria and biochemical and spectroscopic data. Fig. 7 shows a schematic view of photosystem II that is consistent with current data.

Photochemistry in photosystem II is initiated by charge separation between P680 and pheophytin, creating P680+/Pheo-. Primary charge separation takes about a few picoseconds (Fig. 8). Subsequent electron transfer steps have been designed through evolution to prevent the primary charge separation from recombining. This is accomplished by transferring the electron within 200 picoseconds from pheophytin to a plastoquinone molecule (QA) that is permanently bound to photosystem II. Although plastoquinone normally acts as a two-electron acceptor, it works as a one-electron acceptor at the QA-site. The electron on QA- is then transferred to another plastoquinone molecule that is loosely bound at the QB-site. Plastoquinone at the QB-site differs from QA in that it works as a two-electron acceptor, becoming fully reduced and protonated after two photochemical turnovers of the reaction center. The full reduction of plastoquinone requires the addition of two electrons and two protons, i.e., the addition of two hydrogen atoms. The reduced plastoquinone (Fig. 9) then debinds from the reaction center and diffuses into the hydrophobic core of the membrane. After which, an oxidized plastoquinone molecule finds its way to the QB-binding site and the process is repeated. Because the QB-site is near the outer aqueous phase, the protons added to plastoquinone during its reduction are taken from the outside of the membrane.

Photosystem II is the only known protein complex that can oxidize water, resulting in the release of O2 into the atmosphere. Despite years of research, little is known about the molecular events that lead to water oxidation. Energetically, water is a poor electron donor. The oxidation- reduction midpoint potential (Em,7) of water is +0.82 V (pH 7). In photosystem II this reaction is driven by the oxidized reaction center, P680+ (the midpoint potential of P680/P680+ is estimated to be +1.2 V at pH 7). How electrons are transferred from water to P680+ remains a mystery (Govindjee and Coleman, 1990). It is known that P680+ oxidizes a tyrosine on the D1 protein and that Mn plays a key role in water oxidation. Four Mn ions are present in the water oxidizing complex. X-ray absorption spectroscopy shows that Mn undergoes light-induced oxidation. Water oxidation requires two molecules of water and involves four sequential turnovers of the reaction center. This was shown by an experiment demonstrating that oxygen release by photosystem II occurs with a four flash dependence (Fig. 10; Joliot et al., 1969; Joliot and Kok, 1975). Each photochemical reaction creates an oxidant that removes one electron. The net reaction results in the release of one O2 molecule, the deposition of four protons into the inner water phase, and the transfer of four electrons to the QB-site (producing two reduced plastoquinone molecules) (reviewed by Renger, 1993; Klein et al., 1993; and Lavergne and Junge , 1993).

Photosystem II reaction centers contain a number of redox components with no known function. An example is cytochrome b559, a heme protein, that is an essential component of all photosystem II reaction centers (discussed by Whitmarsh and Pakrasi, 1996). If the cytochrome is not present in the membrane, a stable PS II reaction center cannot be formed. Although the structure and function of Cyt b559 remain to be discovered, it is known that the cytochrome is not involved in the primary enzymatic activity of PS II, which is the transfer of electrons from water to plastoquinone. Why PS II reaction centers contain redox components that are not involved in the primary enzymatic reactions is a puzzling question. The answer may be found in the unusual chemical reactions occurring in PS II and the fact that the reaction center operates at a very high power level. Photosystem II is an energy transforming enzyme that must switch between various high energy states that involve the creation of the powerful oxidants required for removing electrons from water and the complex chemistry of plastoquinone reduction which is strongly influenced by protons. In saturating light a single reaction center can have an energy throughput of 600 eV/s (equivalent to 60,000 kW per mole of PS II). Operating at such a high power level results in damage to the reaction center. It may be that some of the "extra" redox components in photosystem II may serve to protect the reaction center.

Photosystem II has another perplexing feature. Many plants and algae have been shown to have a significant number of photosystem II reaction centers that do not contribute to photosynthetic electron transport (e.g., Chylla and Whitmarsh, 1989). Why plants devote resources for the synthesis of reaction centers that apparently do not contribute to energy conversion is unknown (for reviews of photosystem II heterogeneity see Ort and Whitmarsh, 1990; Guenther and Melis, 1990; Govindjee, 1990; Melis, 1991; Whitmarsh et al., 1996; Lavergne and Briantais, 1996)

The photosystem I complex catalyzes the oxidation of plastocyanin, a small soluble Cu- protein, and the reduction of ferredoxin, a small FeS protein (Fig. 11). Photosystem I is composed of a heterodimer of proteins that act as ligands for most of the electron carriers (Krauss et al., 1993). The reaction center is served by an antenna system that consists of about two hundred chlorophyll molecules (mainly chlorophyll a) and primary photochemistry is initiated by a chlorophyll a dimer, P700. In contrast to photosystem II, many of the antenna chlorophyll molecules in photosystem I are bound to the reaction center proteins. Also, FeS centers serve as electron carriers in photosystem I and, so far as is known, photosystem I electron transfer is not coupled to proton translocation. Primary charge separation occurs between a primary donor, P700, a chlorophyll dimer, and a chlorophyll monomer (Ao). The subsequent electron transfer events and rates are shown in Fig. 12 (see Golbeck, 1994).

5.4 Electron Transport
Electron transport from water to NADP+ requires three membrane bound protein complexes operating in series - photosystem II, the cytochrome bf complex and photosystem I (Fig. 3). Electrons are transferred between these large protein complexes by small mobile molecules (plastoquinone and plastocyanin in plants). Because these small molecules carry electrons (or hydrogen atoms) over relatively long distances, they play a unique role in photosynthetic energy conversion. This is illustrated by plastoquinone (PQ), which serves two key functions. Plastoquinone transfers electrons from the photosystem II reaction center to the cytochrome bf complex and carries protons across the photosynthetic membrane (see Kallas, 1994). It does this by shuttling hydrogen atoms across the membrane from photosystem II to the cytochrome bf complex. Because plastoquinone is hydrophobic its movement is restricted to the hydrophobic core of the photosynthetic membrane. Plastoquinone operates by diffusing through the membrane until, due to random collisions, it becomes bound to a specific site on the photosystem II complex. The photosystem II reaction center reduces plastoquinone at the QB-site by adding two electrons and two protons creating PQH2. The reduced plastoquinone molecule debinds from photosystem II and diffuses randomly in the photosynthetic membrane until it encounters a specific binding site on the cytochrome bf complex. The cytochrome bf complex is a membrane bound protein complex that contains four electron carriers, three cytochromes and an FeS center. The crystal structure has been solved for cytochrome f from turnip (Martinez et al., 1994) and the FeS center from bovine heart mitochondria (Iwata et al., 1996). In a complicated reaction sequence that is not fully understood, the cytochrome bf complex removes the electrons from reduced plastoquinone and facilitates the release of the protons into the inner aqueous space. The electrons are eventually transferred to the photosystem I reaction center. The protons released into the inner aqueous space contribute to the proton chemical free energy across the membrane.

Electron transfer from the cytochrome bf complex to photosystem I is mediated by a small Cu-protein, plastocyanin (PC). Plastocyanin is water soluble and operates in the inner water space of the photosynthetic membrane. Electron transfer from photosystem I to NADP+ requires ferredoxin, a small FeS protein, and ferredoxin-NADP oxidoreductase, a peripheral flavoprotein that operates on the outer surface of the photosynthetic membrane. Ferredoxin and NADP+ are water soluble and are found in the outer aqueous phase.

The pathway of electrons is largely determined by the energetics of the reaction and the distance between the carriers. The electron affinity of the carriers is represented in Fig. 13 by their midpoint potentials, which show the free energy available for electron transfer reactions under equilibrium conditions. (It should be kept in mind that reaction conditions during photosynthesis are not in equilibrium.) Subsequent to primary charge separation, electron transport is energetically downhill (from a lower (more negative) to a higher ( more positive) redox potential). It is the downhill flow of electrons that provides free energy for the creation of a proton chemical gradient.

Photosynthetic membranes effectively limit electron transport to two dimensions. For mobile electron carriers, limiting diffusion to two dimensions increases the number of random encounters (Whitmarsh, 1986). Furthermore, because plastocyanin is mobile, any one cytochrome bf complex can interact with a number of photosystem I complexes. The same is true for plastoquinone, which commonly operates at a stoichiometry of about six molecules per photosystem II complex.

5.5 Creation of a Proton Electrochemical Potential
Electron transport creates the proton electrochemical potential of the photosynthetic membrane by two types of reactions. (1) The release of protons during the oxidation of water by photosystem II and the translocation of protons from the outer aqueous phase to the inner aqueous phase by the coupled reactions of photosystem II and the cytochrome bf complex in reducing and oxidizing plastoquinone on opposite sides of the membrane. This creates a concentration difference of protons across the membranes (DpH = pHin - pHout). (2) Primary charge separation at the reaction center drives an electron across the photosynthetic membrane, which creates an electric potential across the membrane (DY = Yin - Yout). Together, these two forms of energy make up the proton electrochemical potential across the photosynthetic membrane (DmH+) which is related to the pH difference across the membrane and the electrical potential difference across the membrane by the following equation:

DmH+ = F DY - 2.3 RT DpH, (4)

where F is the Faraday constant, R is the gas constant, and T the temperature in Kelvin. Although the value of DY across the photosynthetic membrane in chloroplasts can be as large as 100 mV, under normal conditions the proton gradient dominates. For example, during photosynthesis the outer pH is typically near 8 and the inner pH is typically near 6, giving a pH difference of 2 across the membrane that is equivalent to 120 mV. Under these conditions the free energy for proton transfer from the inner to the outer aqueous phase is -12 kJ/mol of protons.

5.6 Synthesis of ATP by the ATP Synthase Enzyme
The conversion of proton electrochemical energy into chemical free energy is accomplished by a single protein complex known as ATP synthase. This enzyme catalyzes a phosphorylation reaction, which is the formation of ATP by the addition of inorganic phosphate (Pi) to ADP

ADP-3 + Pi-2 + H+ _____> ATP-4 + H2O. (5)

The reaction is energetically uphill (DG = +32 kJ/mol) and is driven by proton transfer through the ATP synthase protein. The ATP Synthase complex is composed of two major subunits, CF0 and CF1 (Fig. 14). The CF0 subunit spans the photosynthetic membrane and forms a proton channel through the membrane. The CF1 subunit is attached to the top of the CF0 on the outside of the membrane and is located in the aqueous space. CF1 is composed of several different protein subunits, referred to as a, b, g, d and e. The top portion of the CF1 subunit is composed of three ab-dimers that contain the catalytic sites for ATP synthesis. A recent major breakthrough has been the elucidation of the structure of ATPase of beef heart mitochondria by Abrahams et al. (1994). The molecular processes that couple proton transfer through the protein to the chemical addition of phosphate to ADP are poorly understood. It is known that phosphorylation can be driven by a pH gradient, a transmembrane electric field, or a combination of the two. Experiments indicate that three protons must pass through the ATP synthase complex for the synthesis of one molecule of ATP. However, the protons are not involved in the chemistry of adding phosphate to ADP. Paul Boyer and coworkers have proposed an alternating binding site mechanism for ATP synthesis (Boyer, 1993). One model based on their proposal is that there are three catalytic sites on each CF1 that cycle among three different states (Fig. 15). The states differ in their affinity for ADP, Pi and ATP. At any one time, each site is in a different state. This model is supported by the structure of ATPase elucidated by Abrahams et al. (1994). Initially, one catalytic site on CF1 binds one ADP and one inorganic phosphate molecule relatively loosely. Due to a conformational change of the protein, the site becomes a tight binding site, that stabilizes ATP. Next, proton transfer induces an alteration in protein conformation that causes the site to release the ATP molecule into the aqueous phase. In this model, the energy from the proton electrochemical gradient is used to lower the affinity of the site for ATP, allowing its release to the water phase. The three sites on CF1 act cooperatively, i.e., the conformational states of the sites are linked. It has been proposed that protons affect the conformational change by driving the rotation of the top part (the three ab-dimers) of CF1. Such a rotating model has recently been supported by recording of a rotation of the gamma subunit relative to the alpha-beta subunits by Sabbert et al. (1996). This revolving site mechanism would require rates as high as 100 revolutions per second. It is worth noting that flagella that propel some bacteria are driven by a proton pump and can rotate at 60 revolutions per second.

5.7 Synthesis of Carbohydrates
All plants and algae remove CO2 from the environment and reduce it to carbohydrate by the Calvin cycle. The process is a sequence of biochemical reactions that reduce carbon and rearrange bonds to produce carbohydrate from CO2 molecules. The first step is the addition of CO2 to a five-carbon compound (ribulose 1,5-bisphosphate) (Fig. 16). The six-carbon compound is split, giving two molecules of a three-carbon compound (3-phosphoglycerate). This key reaction is catalyzed by Rubisco, a large water soluble protein complex. The 3-dimensional structure has been determined by X-ray analysis for Rubisco isolated from tobacco (Schreuder et al. 1993) from a cyanobacterium (Synechococcus) (Newman and Gutteridge, 1993) and from a purple bacterium (Rhodospirillum rubrum) (Schneider et al. 1990). The carboxylation reaction is energetically downhill. The main energy input in the Calvin cycle is the phosphorylation by ATP and subsequent reduction by NADPH of the initial three-carbon compound forming a three-carbon sugar, triosephosphate. Some of the triosephosphate is exported from the chloroplast and provides the building block for synthesizing more complex molecules. In a process known as regeneration, the Calvin cycle uses some of the triosephosphate molecules to synthesize the energy rich ribulose 1,5-bisphosphate needed for the initial carboxylation reaction. This reaction requires the input of energy in the form of one ATP. Overall, thirteen enzymes are required to catalyze the reactions in the Calvin cycle. The energy conversion efficiency of the Calvin cycle is approximately 90%. The reactions do not involve energy transduction, but rather the rearrangement of chemical energy. Each molecule of CO2 reduced to a sugar [CH2O]n requires 2 molecules of NADPH and 3 molecules of ATP.

Rubisco is a bifunctional enzyme that, in addition to binding CO2 to ribulose bisphosphate, can also bind O2. This oxygenation reaction produces the 3-phosphoglycerate that is used in the Calvin cycle and a two-carbon compound (2-phosphoglycolate) that is not useful for the plant. In response, a complicated set of reactions (known as photorespiration) are initiated that serve to recover reduced carbon and to remove phosphoglycolate. The Rubisco oxygenation reaction appears to serve no useful purpose for the plant. Some plants have evolved specialized structures and biochemical pathways that concentrate CO2 near Rubisco. These pathways (C4 and CAM), serve to decrease the fraction of oxygenation reactions (see Chapter this volume on carbon reduction).

5.8 Photosynthetic Quantum Yield and Energy Conversion Efficiency
The theoretical minimum quantum requirement for photosynthesis is 8 quanta for each molecule of oxygen evolved (four quanta required by photosystem II and four by photosystem I). Measurements in algal cells and leaves under optimal conditions (e.g., low light) give quantum requirements of 8-10 photons per oxygen molecule released (see Emerson, 1958). These quantum yield measurements show that the quantum yields of photosystem II and photosystem I reaction centers under optimal conditions are near 100%. These values can be used to calculate the theoretical energy conversion efficiency of photosynthesis (free energy stored as carbohydrate/light energy absorbed). If 8 red quanta are absorbed (8 mol of red photons are equivalent to 1,400 kJ) for each CO2 molecule reduced (480 kJ/mol), the theoretical maximum energy efficiency for carbon reduction is 34%. Under optimal conditions, plants can achieve energy conversion efficiencies within 90% of the theoretical maximum. However, under normal growing conditions the actual performance of the plant is far below these theoretical values. The factors that conspire to lower the quantum yield of photosynthesis include limitations imposed by biochemical reactions in the plant and environmental conditions that limit photosynthetic performance. One of the most efficient crop plants is sugar cane, which has been shown to store up to 1% of the incident visible radiation over a period of one year. However, most crops are less productive. The annual conversion efficiency of corn, wheat, rice, potatoes, and soybeans typically ranges from 0.1% to 0.4% (Odum, 1971).

5.9 Oxygenic Photosynthesis in Algae
Algae are photosynthetic eukaryotic organisms that, like plants, evolve O2 and reduce CO2. They represent a diverse group that include the dinoflagellates, the euglenoids, yellow-green algae, golden-brown algae, diatoms, red algae, brown algae, and green algae. The photosynthetic apparatus and biochemical pathways of carbon reduction of algae are similar to plants. Photosynthesis occurs in chloroplasts that contain photosystems II and I, the cytochrome bf complex, the Calvin cycle enzymes and pigment-protein complexes containing chlorophyll a, and other antenna pigments (e.g., chlorophyll b in green algae, chlorophyll c and fucaxanthol in brown algae and diatoms, and phycobilins in red algae). Green algae are thought to be the ancestral group from which land plants evolved (see Douglas, 1994). Algae are abundant and widespread on the earth, living mainly in fresh and sea water. Some algae live as single celled organisms, while others form multicellular organisms some of which can grow quite large, like kelp and seaweed. Phytoplankton in the ocean is made up of algae and oxygenic photosynthetic bacteria. Most photosynthesis in the ocean is due to phytoplankton, which is an important source of food for marine life.

5.10 Oxygenic Photosynthesis in Bacteria
Cyanobacteria are photosynthetic prokaryotic organisms that evolve O2 (Bryant, 1994). Fossil evidence indicates that cyanobacteria existed over 3 billion years ago and it is thought that they were the first oxygen evolving organisms on earth (Wilmotte, 1994). Cyanobacteria are presumed to have evolved in water in an atmosphere that lacked O2. Initially, the O2 released by cyanobacteria reacted with ferrous iron in the oceans and was not released into the atmosphere. Geological evidence indicates that the ferrous Fe was depleted around 2 billion years ago, and earth's atmosphere became aerobic. The release of O2 into the atmosphere by cyanobacteria has had a profound affect on the evolution of life.

The photosynthetic apparatus of cyanobacteria is similar to that of chloroplasts. The main difference is in the antenna system. Cyanobacteria depend on chlorophyll a and specialized protein complexes (phycobilisomes) to gather light energy (Sidler, 1994). They do not contain chlorophyll b. As in chloroplasts, the chlorophyll a is located in membrane bound proteins. The phycobilisomes are bound to the outer side of the photosynthetic membrane and act to funnel exciton energy to the photosystem II reaction center. They are composed of phycobiliproteins, protein subunits that contain covalently attached open ring structures known as bilins that are the light absorbing pigments. Primary photochemistry, electron transport, phosphorylation and carbon reduction occur much as they do in chloroplasts. Cyanobacteria have a simpler genetic system than plants and algae that enable them to be easily modified genetically. Because of this cyanobacteria have been used as a model to understand photosynthesis in plants. By genetically altering photosynthetic proteins, researchers can investigate the relationship between molecular structure and mechanism (Barry et al., 1994).

Anoxygenic photosynthetic bacteria differ from oxygenic organisms in that each species has only one type of reaction center (Blankenship et al., 1995). In some photosynthetic bacteria the reaction center is similar to photosystem II and in others it is similar to photosystem I. However, neither of these two types of bacterial reaction center is capable of extracting electrons from water, so they do not evolve O2. Many species can only survive in environments that have a low concentration of O2. To provide electrons for the reduction of CO2, anoxygenic photosynthetic bacteria must oxidize inorganic or organic molecules available in their environment. For example, the purple bacterium Rhodobacter sphaeroides can use succinate to reduce NAD+ by a membrane-linked reverse electron transfer that is driven by a transmembrane electrochemical potential. Although many photosynthetic bacteria depend on Rubisco and the Calvin cycle for the reduction of CO2, some are able to fix atmospheric CO2 by other biochemical pathways.

Despite these differences, the general principles of energy transduction are the same in anoxygenic and oxygenic photosynthesis. Anoxygenic photosynthetic bacteria depend on bacteriochlorophyll, a family of molecules that are similar to the chlorophyll, that absorb strongly in the infrared between 700 and 1000 nm. The antenna system consists of bacteriochlorophyll and carotenoids that serve a reaction center where primary charge separation occurs. The electron carriers include quinone (e.g., ubiquinone, menaquinone) and the cytochrome bc complex, which is similar to the cytochrome bf complex of oxygenic photosynthetic apparatus. As in oxygenic photosynthesis, electron transfer is coupled to the generation of an electrochemical potential that drives phosphorylation by ATP synthase and the energy required for the reduction of CO2 is provided by and ATP and NADH, a molecule similar to NADPH.

6.1 Purple Bacteria
There are two divisions of photosynthetic purple bacteria, the non-sulfur purple bacteria (e.g., Rhodobacter sphaeroides and Rhodospeudomonas viridis) and the sulfur purple bacteria (e.g., Chromatium vinosum) (Blankenship et al., 1995). Non-sulfur purple bacteria typically use an organic electron donor, such as succinate or malate, but they can also use hydrogen gas. The sulfur bacteria use an inorganic sulfur compound, such as hydrogen sulfide as the electron donor. The only pathway for carbon fixation by purple bacteria is the Calvin cycle. Sulfur purple bacteria must fix CO2 to live, whereas non-sulfur purple bacteria can grow aerobically in the dark by respiration on an organic carbon source.

The determination of the three-dimensional structures of the reaction center of the non- sulfur purple bacteria, Rhodopseudomonas viridis and Rhodobacter sphaeroides, has provided an unprecedented opportunity to understand the structure and function of photosynthetic reaction centers (Deisenhofer et al., 1984, 1985; Feher et al., 1989; Lancaster et al., 1995). The positions of the electron transfer components in the reaction center of Rhodobacter sphaeroides are shown in Fig. 17 (Norris and van Brakel, 1986), and those of the three protein subunits L, M, and H, in Fig. 18. The reaction center contains four bacteriochlorophyll and two bacteriopheophytin molecules. Two of the bacteriochlorophyll molecules form the primary donor (P870). At present, there is controversy over whether a bacteriochlorophyll molecule is an intermediate in electron transfer from the P870 to bacteriopheophytin. However, there is agreement that the remaining steps involve two quinone molecules (QA and QB) and that two turnovers of the reaction center results in the release of reduced quinone (QH2) into the photosynthetic membrane. Although there is a non-heme Fe between the two quinone molecules, there is convincing evidence that this Fe is not involved directly in transferring an electron from QA to QB. Because the primary donor (P870), bacteriopheophytin and quinone acceptors of the purple bacterial reaction center are similar to the photosystem II reaction center, the bacterial reaction center is used as guide to understand the structure and function of photosystem II.

Light driven electron transfer is cyclic in Rhodobacter sphaeroides and other purple bacteria (Fig. 19). The reaction center produces reduced quinone, which is oxidized by the cytochrome bc complex. Electrons from the cytochrome bc complex are transferred to a soluble electron carrier, cytochrome c2, which reduces the oxidized primary donor P870+. The product of the light driven electron transfer reactions is ATP. The electrons for the reduction of carbon are extracted from an organic donor, such as succinate or malate or from hydrogen gas, but not by the reaction center. The energy needed to reduce NAD+ is provided by light driven cyclic electron transport in the form of ATP. The energy transformation pathway is complicated. Succinate is oxidized by a membrane bound enzyme (succinate dehydrogenase) that transfers the electrons to quinone, which is the source of electrons for the reduction of NAD+. However, electron transfer from reduced quinone to NAD+ is energetically uphill. By a mechanism that is poorly understood, a membrane bound enzyme is able to use energy stored in the proton electrochemical potential to drive electrons from reduced quinone to NAD+.

6.2 Green Sulfur Bacteria
Green sulfur bacteria (e.g., Chlorobium thiosulfatophilum and Chlorobium vibrioforme) can use sulfur compounds as the electron donor as well as organic hydrogen donors (Blankenship et al., 1995). As shown in Fig. 19 the reaction center of green sulfur bacteria is similar to the photosystem I reaction center of oxygenic organisms (Feiler and Hauska, 1995). The FeS centers in the reaction center can reduce NAD+ (or NADP+) by ferredoxin and the ferredoxin-NAD(P)+ oxidoreductase enzyme, therefore green sulfur bacteria are not necessarily dependent on reverse electron flow for carbon reduction. The antenna system of the green sulfur bacteria is composed of bacteriochlorophyll and carotenoids and is contained in complexes known as a chlorosomes that are attached to the surface of the photosynthetic membrane. This antenna arrangement is similar to the phycobilisomes of cyanobacteria. Green sulfur bacteria can fix CO2 without Rubisco. It has been proposed that they accomplish this by using the respiratory chain that normally oxidizes carbon (known as the Krebs cycle), resulting in the release of CO2. With the input of energy this process can be run in the reverse direction, resulting the uptake and reduction of CO2.

6.3 Green Gliding Bacteria
Green gliding bacteria (e.g., Chloroflexus aurantiacus), also known as green filamentous bacteria, can grow photosynthetically under anaerobic conditions or in the dark by respiration under aerobic conditions. Like the green sulfur bacteria, green gliding bacteria harvest light using chlorosomes. The green gliding bacteria appear to have reaction centers similar to those of the purple bacteria (Fig. 19), but there are several notable differences. For example, instead of two monomer bacteriochlorophyll molecules, C. aurantiacus has one bacteriochlorophyll and one bacteriopheophytin and the metal between the two quinones is Mn rather than Fe (Feick et al., 1995). C. aurantiacus appears to fix CO2 by a scheme that does not involve the Calvin cycle or the reverse Krebs cycle (Ivanovsky et al., 1993).

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