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    Oxygenic photophosphorylation

    Generation of NADPH and ATP

    The overall function of light-dependent reactions is to transfer solar energy into chemical compounds, largely the the molecules NADPH and ATP. This energy supports the light-independent reactions and fuels the assembly of sugar molecules. The light-dependent reactions are depicted in Figures 6 and 7. Protein complexes and pigment molecules work together to produce NADPH and ATP.

    Note: possible discussion

    Step back a little. Why is it a reasonable goal to want to make NADPH and ATP? In the discussion of glycolysis and the TCA cycle, the goal was to make ATP and NADH. What is the key difference? Perhaps how those molecules will be used? Something else?


    Figure 6. A photosystem consists of a light-harvesting complex and a reaction center. Pigments in the light-harvesting complex pass light energy to two special chlorophyll a molecules in the reaction center. The light excites an electron from the chlorophyll a pair, which passes to the primary electron acceptor. The excited electron must then be replaced. In (a) photosystem II, the electron comes from the splitting of water, which releases oxygen as a waste product. In (b) photosystem I, the electron comes from the chloroplast electron transport chain discussed below.

    The actual step that transfers light energy into a biomolecule takes place in a multiprotein complex called a photosystem, two types of which are found embedded in the thylakoid membrane, photosystem II (PSII) and photosystem I (PSI). The two complexes differ on the basis of what they oxidize (that is, the source of the low energy electron supply) and what they reduce (the place to which they deliver their energized electrons).

    Both photosystems have the same basic structure; a number of antenna proteins to which the chlorophyll molecules are bound surround the reaction center in which the photochemistry takes place. Each photosystem is serviced by the light-harvesting complex, which passes energy from sunlight to the reaction center; it consists of multiple antenna proteins that contain a mixture of 300–400 chlorophyll a and b molecules as well as other pigments like carotenoids. The absorption of a single photon—a distinct quantity or “packet” of light—by any of the chlorophylls pushes that molecule into an excited state. In short, the light energy has now been captured by biological molecules but is not yet stored in any useful form. The captured energy is transferred from chlorophyll to chlorophyll until, eventually (after about a millionth of a second), it is delivered to the reaction center. Up to this point, only energy has been transferred between molecules, not electrons.

    Figure 7. In the photosystem II (PSII) reaction center, energy from sunlight is used to extract electrons from water. The electrons travel through the chloroplast electron transport chain to photosystem I (PSI), which reduces NADP+ to NADPH. The electron transport chain moves protons across the thylakoid membrane into the lumen. At the same time, splitting of water adds protons to the lumen, and reduction of NADPH removes protons from the stroma. The net result is a low pH in the thylakoid lumen, and a high pH in the stroma. ATP synthase uses this electrochemical gradient to make ATP.

    The reaction center contains a pair of chlorophyll a molecules with a special property. Those two chlorophylls can undergo oxidation upon excitation; they can actually give up an electron in a process called a photoactivation. It is at this step in the reaction center, this step in photophosphorylation, that light energy is transferred into an excited electron. All of the subsequent steps involve getting that electron onto the energy carrier NADPH for delivery to the Calvin cycle where the electron can be deposited onto carbon for long term storage in the form of a carbohydrate. 

    The Z-scheme

    PSII and PSI are two major components of the photosynthetic electron transport chain, which also includes the cytochrome complex. The reaction center of PSII (called P680) delivers its high-energy electrons, one at a time, to a primary electron acceptor called pheophytin (Ph), and then sequentially to two bound plastoquinones QA and QB.  Electrons are then transferred off of PSII onto a pool of mobile plastoquinones (Q pool) which then transfer the electrons to a protein complex called Cytochromeb6f.  The cytochrome complex utilizes the red/ox transfers to pump proton across the thylakoyd membrane establishing a proton-motive force that can be used for the synthesis of ATP.  Electrons leaving the Cytochrome are transferred to a copper-containing protein called plastocyanin (PC) which then transfers electrons to PSI (P700).  P680’s missing electron is replaced by extracting an electron from water; thus, water is split and PSII is re-reduced after every photoactivation step. Just for the sake of sharing some numbers: Splitting one H2O molecule releases two electrons, two hydrogen atoms, and one atom of oxygen. Splitting two molecules of water is required to form one molecule of diatomic O2 gas. In plants, about ten percent of that oxygen is used by mitochondria in the leaf to support oxidative phosphorylation. The remainder escapes to the atmosphere where it is used by aerobic organisms to support respiration.

    As electrons move through the proteins that reside between PSII and PSI, they take part in exergonic red/ox transfers. The free energy associated with the exergonic red/ox reaction is coupled to the endergonic transport of protons from the stromal side of the membrane to the thylakoid lumen by the cytochrome complex. Those hydrogen ions, plus the ones produced by splitting water, accumulate in the thylakoid lumen and create a proton motive force that will be used to drive the synthesis of ATP in a later step. Since the electrons on PSI now have a greater reduction potential than when they started their trek (it is important to note that unexcited PSI has a greater red/ox potential than NADP+/NADPH), they must be re-energized in PSI before getting deposited onto NADP+. Therefore, to complete this process, another photon must be absorbed by the PSI antenna. That energy is transferred to the PSI reaction center (called P700). P700 is then oxidized and sends an electron through several intermediate red/ox steps to NADP+ to form NADPH. Thus, PSII captures the energy in light and couples its transfer via red/ox reactions to the creation of a proton gradient. As already noted, the exergonic and controlled relaxation of this gradient can be coupled to the synthesis of ATP. PSI captures energy in light and couples that, through a series of red/ox reactions, to reduce NADP+ into NADPH. The two photosystems work in concert, in part, to guarantee that the production of NADPH will be in the right proportion to the production of ATP. Other mechanisms exist to fine tune that ratio to exactly match the chloroplast’s constantly changing energy needs.

    Figure 8. A diagram depicting the flow of electrons and the red/ox potentials of their carriers in oxygenic photosynthetic systems expressing both photosystem I (boxed in blue) and photosystem II (boxed in green). Ph = pheophytin; QA = bound plastoquinone, QB = more loosely associated plastoquinone; Q pool = mobile plastoquinone pool; Cyt bf = Cytochrome b6f complex; PC = plastocyanin; Chla0 = special chrolophyl; A1 = vitamin K; Fx and FAB = iron-sulfur centers; Fd = ferredoxin; FNR = ferredoxin-NADP reductase. Attribution: Marc T. Facciotti (own work)



    Light Independent Reactions and Carbon Fixation

    A short introduction

    The general principle of carbon fixation is that some cells under certain conditions can take inorganic carbon, CO2 (also referred to as mineralized carbon), and reduce it to a usable cellular form. Most of us are aware that green plants can take up CO2 and produce O2 in a process known as photosynthesis. We have already discussed photophosphorylation, the ability of a cell to transfer light energy onto chemicals and ultimately to produce the energy carriers ATP and NADPH in a process known as the light reactions. In photosynthesis, the plant cells use the ATP and NADPH formed during photophosphorylation to reduce CO2 to sugar, (as we will see, specifically G3P) in what are called the dark reactions. While we appreciate that this process happens in green plants, photosynthesis had its evolutionary origins in the bacterial world. In this module we will go over the general reactions of the Calvin Cycle, a reductive pathway that incorporates CO2 into cellular material. 

    In photosynthetic bacteria, such as Cyanobacteria and purple non-sulfur bacteria, as well plants, the energy (ATP) and reducing power (NADPH) - a term used to describe electron carriers in their reduced state - obtained from photophosphorylation is coupled to "Carbon Fixation", the incorporation of inorganic carbon (CO2) into organic molecules; initially as glyceraldehyde-3-phosphate (G3P) and eventually into glucose. Organisms that can obtain all of their required carbon from an inorganic source (CO2) are referred to as autotrophs, while those organisms that require organic forms of carbon, such as glucose or amino acids, are referred to as heterotrophs. The biological pathway that leads to carbon fixation is called the Calvin Cycle and is a reductive pathway (consumes energy/uses electrons) which leads to the reduction of CO2 to G3P. 

    The Calvin Cycle: the reduction of CO2 to Glyceraldehyde 3-Phosphate

    Figure 1. Light reactions harness energy from the sun to produce chemical bonds, ATP, and NADPH. These energy-carrying molecules are made in the stroma where carbon fixation takes place.

    In plant cells, the Calvin cycle is located in the chloroplasts. While the process is similar in bacteria, there are no specific organelles that house the Calvin Cycle and the reactions occur in the cytoplasm around a complex membrane system derived from the plasma membrane. This intracellular membrane system can be quite complex and highly regulated. There is strong evidence that supports the hypothesis that the origin of chloroplasts from a symbiosis between cyanobacteria and early plant cells.

    Stage 1: Carbon Fixation

    In the stroma of plant chloroplasts, in addition to CO2, two other components are present to initiate the light-independent reactions: an enzyme called ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and three molecules of ribulose bisphosphate (RuBP), as shown in the figure below. Ribulose-1,5-bisphosphate (RuBP) is composed of five carbon atoms and includes two phosphates.

    Figure 2. The Calvin cycle has three stages. In stage 1, the enzyme RuBisCO incorporates carbon dioxide into an organic molecule, 3-PGA. In stage 2, the organic molecule is reduced using electrons supplied by NADPH. In stage 3, RuBP, the molecule that starts the cycle, is regenerated so that the cycle can continue. Only one carbon dioxide molecule is incorporated at a time, so the cycle must be completed three times to produce a single three-carbon GA3P molecule, and six times to produce a six-carbon glucose molecule.

    RuBisCO catalyzes a reaction between CO2 and RuBP. For each CO2 molecule that reacts with one RuBP, two molecules of another compound (3-PGA) form. PGA has three carbons and one phosphate. Each turn of the cycle involves only one RuBP and one carbon dioxide and forms two molecules of 3-PGA. The number of carbon atoms remains the same, as the atoms move to form new bonds during the reactions (3 atoms from 3CO2 + 15 atoms from 3RuBP = 18 atoms in 3 atoms of 3-PGA). This process is called carbon fixation, because CO2 is “fixed” from an inorganic form into an organic molecule.

    Stage 2: Reduction

    ATP and NADPH are used to convert the six molecules of 3-PGA into six molecules of a chemical called glyceraldehyde 3-phosphate (G3P) - a carbon compound that is also found in glycolysis. Six molecules of both ATP and NADPH are used in the process. The exergonic process of ATP hydrolysis is in effect driving the endergonic redox reactions, creating ADP and NADP+. Both of these "spent" molecules (ADP and NADP+) return to the nearby light-dependent reactions to be recycled back into ATP and NADPH.

    Stage 3: Regeneration

    Interestingly, at this point, only one of the G3P molecules leaves the Calvin cycle to contribute to the formation of other compounds needed by the organism. In plants, because the G3P exported from the Calvin cycle has three carbon atoms, it takes three “turns” of the Calvin cycle to fix enough net carbon to export one G3P. But each turn makes two G3Ps, thus three turns make six G3Ps. One is exported while the remaining five G3P molecules remain in the cycle and are used to regenerate RuBP, which enables the system to prepare for more CO2 to be fixed. Three more molecules of ATP are used in these regeneration reactions.

    Additional Links of Interest

    Khan Academy Links 

    Chemwiki links