We have just seen how we can transduce the chemical potential energy stored in carbohydrates into chemical potential energy of ATP - namely through coupling the energy released during the thermodynamically favored oxidation of carbon molecules through intermediaries (high energy mixed anhydride in glycolysis or a proton gradient in aerobic metabolism) to the thermodynamically uphill synthesis of ATP. There is a situation that occurs when we wish to actually reverse the entire process and take CO2 + H2O to carbohydrate + O2. This process is of course photosynthesis which occurs in plants and certain photosynthetic bacteria and algae. Given that this process must by nature be an uphill thermodynamic battle, let us consider the major requirements that must be in place for this process to occur:
- A strong oxidizing agent must be formed which can take water and oxidize it to dioxygen. We know that redox reactions occur in the direction of stronger to weaker oxidizing agent (just as acid base reactions are thermodynamically favored in the direction of strong to weak acid). Somehow we must generate a stronger oxidizing agent than dioxygen, which often has the most positive standard reduction potential in tables.
- Plants must have high concentrations of a reducing agent for the reductive biosynthesis of glucose from CO2. The reducing agent used for most biosynthetic reactions in nature is NADPH, which differs from NADH only by the addition of a phosphate to the ribose ring. This phosphate differentiates the pool of nucleotides in the cells used for reductive biosynthesis (NADPH/NADP+) from those used for oxidative catabolism (NADH/NAD+)
- Finally, plants need an abundant source of ATP which will be required for reductive biosynthesis.
We will discuss only the light reaction of photosynthesis which produces these three types of molecules. The dark reaction , which as the name implies can occur in the dark, involves that actual fixation of carbon dioxide into carbohydrate using the ATP and NADPH produced in the light reaction.
The Light Reaction
Obviously, the energy to power the light reactions comes directly from sunlight. Clue two is that plants have an organelle that animal cells don't - the chloroplast. Its structure is in many ways similar to a mitochondria in that it has internal membranes (thylacoid membranes) surrounding enclosed compartments.
Plants have many pigments (chlorophyll, phycoerthryins, carotenoids, etc.) whose absorption spectra overlap that of the solar spectra. The main pigment, chlorophyll, has a protophorphryin IX ring (same as in heme groups) with Mg instead of Fe. When the chlorophyll absorbs light, the excited electrons must relax eventually to their ground state. It can do this by either radiative or nonradiative decay. In radiative decay, a photon of lower energy is emitted (after some energy has already been lost by vibrational transitions) in a process of either fluorescence or phosphorescence. In nonradiative decay, the energy of an excited electron can be transferred to another similar molecule (in this case other chlorophyll molecules) in a process which excites the energy of an electron in the second molecule to the same excited state. (It is as if a photon is released by the first excited molecule which then is absorbed by an electron in a second molecule to excite it to the same exited state, although the excitation occurs without photon production). In this fashion, energy is transferred from one chlorophyll to another. This type of energy transfer is called resonance energy transfer or exciton transfer.
Figure: resonance energy transfer
One type of chlorophyll has slightly different characteristics, however. Because of its unique environment, the energy level of the excited state electron is lower than in the rest of the chlorophyll molecules, in much the same way that pKa's of amino acid side chains differ with environment, and the standard reduction potential of FADs that are tightly bound to enzymes differ due to the different environment of FAD/FADH2 These unique chlorophyll centers are called reaction centers.
Figure: reaction centers
The rest of the chlorophyll molecules act as antennas which transfer energy to the reaction centers. An electron in an adjacent excited state chlorophyll (which is at a higher level than the excited state energy of the reaction center) can then be transferred to this lower energy state level in the reaction center, in a process which forms a positively charged ion from the first excited state molecule and an anion from the recipient reaction center. This process of energy transfer is called electron transfer.
Figure: electron transfer
Photoexcitation of the non-reaction center chlorophyll turns that molecule into a good reducing agent, which transfers its electron to the nearest excited state level of the reaction center chlorophyll. If you count both step together, the non-reaction center chlorophyll gets "photooxidized", in the process producing the "strong" oxidizing agent which is the positively charged chlorophyll derivative. The extra electron passed onto the second molecule will eventually be passed on to NADP+ to produce NADPH. The light reaction of photosynthesis in green plants is shown below. In this process, in a scheme that is reminiscent of electron transport in mitochondria, water is oxidized by photosystem II. Electrons from water are moved through PSII to a mobile, hydrophobic molecule, plastaquinone (PQ) to form its reduced form, PQH2. PSII is a complicated structure with many polypeptide chains, lots of chlorophylls, and Mn, Ca, and Fe ions. A Mn cluster, called the oxygen evolving complex, OEC, is directly involved in the oxidation of wate. Two key homologous 32 KD protein subunits, D1 and D2, in PSII are transmembrane proteins and are at the heart of the PSII complex. Another photosystem, PS1, is also found further "downstream" in the electron transport pathway. It takes electrons from another reduced mobile carrier of electrons, plastocyanin (PCred) to ferredoxin, which becomes a strong reducing agent. Ferrodoxin is a protein with an Fe-S cluster (Fe-S-Fe-S in a 4 membered ring, with 2 additions Cys residues coordinating each Fe). It ultimately passes its electrons along to NADP+ to form NADPH. A summary of the light reaction in plants and standard reduction potentials of the participants, are shown below. Note that many of the complexes produce a transmembrane proton gradient. In contrast to mitochondria, the lumen (as compared to the mitochondrial matrix) becomes more acidic that the other stroma. Protons then can move down a concentration gradient through the C0C1ATPase to produce ATP required for reductive biosythesis of glucose.
The net reaction carried out by PS2 is the oxidation of water and reduction of plastoquinone.
PQ + H2O → PQH2 + O2 (g)
Note that water is not converted to 2H2 + O2 , as in the electrolysis of water. Rather the Hs are removed from water as protons in the lumen of the cholorplast, since the part of PSII which oxides water is near the lumenal end of the transmembrane complex. Protons required to protonated the reduced (anionic) form of plastaquinone to form PQH2, an activity of PSII found closer to the stroma, derive from the stroma. which then can be used to protonated the "anionic" form of reduced PQ to form PQH2.
A quick look at standard reduction potentials shows that the passing of electrons from water (dioxygen SRP = +0.816 V) to plastoquinone (approx SRP of 0.11 ) is not thermodynamically favored. The process is driven thermodynamically by the energy of the absorbed photons.
Recently the crystal structure of PSII from a photosynthetic cyanobacterium was determined. It consists of 17 polypeptide subunits with metal and pigment cofactors and over 45,000 atoms (Zouni, Nature, 409, 739, 2001). Of particular interest is the P680 chlorophyll reaction center, which consists of four monomeric chlorphylls adjacent to a cationic Tyr-D side chain which destabilizes the chlorophyll molecules. When H2O gets oxidized to form dioxygen, 4 electrons must be remove by photoactivated P680. In PSII, this process occurs in four single-electron steps, with the electrons first being transferred to a Mn4 cluster cofactor (of composition Mn4Ca1Cl1-2(HCO3)y. This inorganic Mn cluster is often called OEC (oxygen evolving complex) or WOC (water oxidizing complex). The electrons passed through the Mn complex are delivered to P680 by a photoactive Tyr free radical (Tyr Z). The actual structure of the OEC could not be resolved, but other structural and spectroscopic data support the structure below (Chem. Rev., 2001, 101, 21-35), which also shows a possible mechanism for electron and proton transfer from water to form dioxygen. Five discrete intermediates of the OEC, S0-S4, are suggested from the experimental data (Kok cycle). These were postulated from experiments in which spinach chloroplast were illuminated with short light pulses. A pattern of dioxygen release was noted that repeated after 4 flashes. Ultimately, light absorption by P680 forms excited state P680* which donates an electron to pheophytin (which passes them to quinones) to form P680+, which receives electrons from the OEC, specifically the TyrZ radical.
Investigators have made non-peptide mimetics of superoxide dismutase to facilitate therapeutic removal of excess superoxide formed in brain and heart tissue. These may arise after an oxidative burst from reperfusion of these tissues after heart or brain attacks. Likewise, scientist are trying to build synthetic PSII-OEC complexes which could be used to form dioxygen or hydrogen gas for fuels.
For PSII in plants
- a pair of chlorophylls (P680) in the D subunits absorb light (maximum absorbance around 680 nm) and reach an excited state
- electron transfer from P680 to a nearby chlorophyll with a lower energy level for the excited state electron occurs. This chlorophyll has 2 H+ ions in the chlorophyll instead of Mg2+occurs. The P680 now becomes cationic, P680+.
- This "anionic" chlorophyll transfers an electron to oxidized plastoquinone.
- The P680+, a strong oxidizing agent, removes one electron from H2O-OEC complex.
Steps 1-4 repeat three more times, each requiring another photon and each cycle producing another electron which passes on through the system. Remember that when O2 acts as an oxiding agent, it gains four electrons. The first produces superoxide, the next peroxide, and two more produce oxide which when protonated is water. Hence two waters and four cycles are required to remove the four electrons required to produce dioxygen.
A similar mechanism is found in PSI, except plastacyanin, not dioxygen is oxidized, with electrons moved to ferrodoxin. This is likewise a difficult process since the reduction potential for oxidized plastocyanin (the form that can act as a reducing agent) is +0.37 while for ferrodoxin it is -0.75. This transfer of electrons is an uphill thermodynamic battle since the more positive the standard reduction potential, the better the oxidizing agent and the more likely the agent becomes reduced. What drives this uphill flow of electrons. Of course, it is the energy input from the photon.
Plants have evolved a great ability to absorb light over the entire visible range of the spectra. Can they absorb to much energy? The answer is yes, so plants have developed many ways to protect themselves. IF too much light is absorbed, the pH gradient developed across the thylacoid membranes becomes greater. This is sensed by a protein, PsbS, and through subsequent conformational changes transmitted through the light-harvesting antennae, the excess light energy is dissipated as thermal energy. Mutants lacking PsbS showed decreased seed yield, a sign that it became less adaptable under conditions of stress (such as exposure to rapidly fluctuating light levels). Molecules called xanthophylls (synthesized from carotenes - vit A precursors) such as zeaxanthin are also important in excess energy dissipation. These molecules appear to cause excited state chlorophyll (a singlet like excited state dioxygen) to become deexcited. Without the xanthophylls, the excited state chlorophyll could deexcite by transfer of energy to ground state triplet dioxygen, promoting it to the singlet, reactive state, which through electron acquisition, could also be converted to superoxide. These reactive oxygen species (ROS) can lead to oxidative damage to proteins, lipids and nucleic acids, alteration in gene transcription, and even programmed cell death. Carotenoids can also acts as ROS scavengers. Hence both heat dissipation and inhibition of formation of ROS (by such molecules as vitamin E) are both mechanism of defense of excessive solar energy
Given that both plants and animals must be protected from ROS, antioxidant molecules made by plants may prove to protect humans from diseases such as cancer, cardiovascular disease, and general inflammatory diseases, all of which have been shown to involve oxidative damage to biological molecules. Humans, who can't synthesize the variety and amounts of antioxidants that are found in plants, appear to be healther when they consume large amounts of plant products. These phytomolecule also have other properties, including regulation of gene transcription which can also have a significant effect on disease propensity.
Production of Hydrogen: Hydrogenases
Our world desperately needs an energy efficient way to produce H2 for energy production without producing waste pollutants. Catalytic cracking of molecules and newly developed fuel cells offer two possibilities. Wouldn't it be great if a reactant like water could be used for H2 production (without the use of electrolysis) or expensive metal catalysts? Nature may show the way. Bacteria (even E. Coli found in our GI system) can use simple metals like iron to produce H2 from H+ with electrons for the reduction of H+ coming from a donor (such as a reduced heme in proteins):
Dred + H+ ↔ Dox + H2
The reaction is also reversible in the presence of an acceptor of electrons from H2 as it gets oxidized:
Aox + H2 ↔ Ared + H+
The enzymes that catalyze hydrogen production are hydrogenases (not dehydrogenases). Note that the name hydrogenases best reflects the reverse reaction when a molecule (P) in an oxidized state gets reduced (to S) and H2 gets oxided to H+.
Crystal structures of hydrogenases show them to be unique among metal-containing enzymes. They contain two metals bonded to each other. The metal centers can either be both iron or one each of iron and nickel. The ligands interacting with the metals are two classical metabolic poisons, carbon monoxide and cyanide. Passages for flow of electrons and H2 connect the buried metals and the remaining enzymes. The metals are also bound to sulfhydryl groups of cysteine side chains. It appears that two electrons are added to a single proton making a hydride anion which accepts a proton to form H2. In the two Fe hydrogenases, the geometry of the coordinating ligands distorts the bond between the two iron centers, leading to irons with different oxidation numbers. Electrons appear to flow from one center to the other, as does carbon monoxide as well. Ultimately, hydrogenases or small inorganic mimetics of the active site could be coated on electrodes and used to general H2 when placed in water in electrolytic experiments.
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- Demming-Adams, B. & Adams, W. Antioxidants in photosynthesis and human nutrition. Science. 298, pg 2149 (2002)
- Jordan et al. 3D Structure of Cyanobacterial Photosystem I (shows how chlorophylls use solar energy to transport electrons) Nature. 411. pg 896, 909 ((2001)
- Balabin and Onuchic. Exploiting thermal motion in electron transfer (combined molecular dynamics, quantum mechanics to study e transfer in photosynthetic reaction center) Science. 290. pg 61, 114 (2000)
- Dismukes. Photosynthesis: Splitting Water. Science. 292. pg 447 (2001)