Spring.wmf (18300 bytes) Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

Photosynthesis: Light Dependent Reactions
(or, Life is a photochemical phenomenon)

 I. Overview of photosynthesis:
    Photosynthesis can be defined as the light-driven synthesis of carbohydrate. The equation for this reaction, that you’ve seen many times is:

CO2 + H2O + light + chloroplast → (CH20)n + O2

From this simple equation we can make some elegant conclusions:

A. Photosynthesis is a redox reaction.

  1. Some definitions: (a) Reduction - gain of electrons; (b) Oxidation - loss of electrons; (c) helpful mnemonics to remember: "oil rig" - oxidation is loss, reduction is gain, or "Leo says grrrr" - loss equals oxidation, gain reduction; (d) Redox reaction - reaction in which one component is oxidized and the other is reduced. Obviously, electrons must come from somewhere and go somewhere.
  2. The reduction sequence of carbon: carbon dioxide (most oxidized form of carbon) → carboxyl (organic acid) → carbonyl (aldehydes, ketones) → hydroxyl (alcohols) → methyl → methane (most reduced form of carbon). Note: each step requires the addition (or removal) of two electrons and two protons for reduction (oxidation). Two steps also require the addition/removal of water.
  3. How can you tell if a molecule has been oxidized or reduced? (1) look for a change in valence (i.e., Fe2+ → Fe3+ represents an oxidation because an electron was lost, increasing the total positive charge); (2) In many biological redox reactions, oxidation is usually accompanied by a loss of protons (hydrogen ions) and reduction is accompanied by a gain of protons; and (3) look for a decrease in the number of oxygen atoms.
  4. Biological redox reactions may require electron donors and/or acceptors. These include: (1) NAD+; (2) NADP+; and (3) FAD; which are coenzymes (organic compounds, other than the substrate, required by an enzyme for activity):
  5. NAD(P) + (ox) + 2e- + 2H+ → NAD(P)H (red) + H+
    FAD(ox) + 2e- + 2H+ → FADH2 (red)

  6. Reducing Potential - potential for components to participate in a redox reaction; to predict the direction and tendency of electrons to flow between two electron carriers. The take-home-lessons are: (1) the more negative the reducing potential the better the electron donor; (2) the more positive the reducing potential the better the electron acceptor; (3) spontaneous electron passage occurs from a carrier with more negative reducing potential to one with a more positive reducing potential.

B.  CO2 is reduced to a carbohydrate.

C.  Water is oxidized (to oxygen).

D.  Water supplies the electrons for the reduction; water is cleaved in the process yielding oxygen as a byproduct.

E. Light provides the energy for the reduction.

F.  Photosynthesis is an energy conversion process that ultimately converts light energy to chemical energy (carbohydrate). In a broad sense, it is an example of the 1st Law of Thermodynamics - energy cannot be created nor destroyed, but it can be changed from one form to another.

G. BLACK BOX summary model for photosynthesis. Diagram in class that shows two boxes (light dependent & light independent reactions). This model further shows that during the light-dependent and light-independent reactions that there are three major types of energy conversions during photosynthesis:

Conversion 1:  Radiant energy (sunlight) → electrical energy (passage of electrons via a series of carrier). This reaction series is part of the light-dependent reactions (z-scheme, non-cyclic electron flow)

Conversion 2:  Electrical energy → "Labile" chemical energy (ATP, NADPH; unstable, not readily stored). During this step, ATP and NADPH are produced as the end result of non-cyclic electron flow.

Conversion 3:  "Labile" chemical energy → Stable chemical energy (carbohydrate). This last step is the light-independent reactions or Calvin-Benson cycle. This process requires ATP and NADPH.

II. Chloroplasts - specialized organelles that carry out the process of photosynthesis

A. Structure.
    Remember the cell unit?  To jog your memory, reread Chapter 1. Terms that you should know are thylakoid (or lamellae), lumen (intermembrane space), envelope, double membrane, stroma, granum, granal thylakoids (or lamellae), stromal thylakoids (lamellae), and starch grains.  Chloroplasts may contain fat globules (plastoglobuli). Stacked (or appressed) regions - portion of granum in which thylakoids are adjacent to one another. Non-stacked (non-appressed) regions - regions of the chloroplast where the thylakoids are not adjacent to another.

B. Ontogeny and phylogeny - recall the cell unit?

C. Chemistry - Chloroplasts contain:

    1. DNA - circular loop; 120-160 kilobases that code for about 120 proteins;
    2. RNA;
    3. ribosomes;
    4. proteins - some are coded by the nuclear genome, others by the chloroplastic genome. For example, rubisco, an important enzyme, has 2 different subunits, one from each source. The nuclear genes are essential for chloroplast function;
    5. pigments - make up about 7% of the chloroplast. These are molecules with a color that absorb light. Two major groups of pigments in higher plants, chlorophylls and carotenoids/xanthophylls. These occur in the thylakoids because they are highly hydrophobic (fat soluble)

D. Pigments

1.  Chlorophylls
    These molecules look like a tennis racket. The head of the racket is a porphyrin ring system, made of four pyrolle units linked together (tetrapyrolle). It has a long hydrocarbon tail, called phytol (C-20), that is derived from the terpene pathway (diterpene), built from the isoprene skeleton.  Magnesium is chelated in the ring. The tail is important for orienting the molecule in the membrane. The interaction of the chlorophyll with the membrane is non-covalent and is important because it ultimately determines the physical properties of the chlorophyll.

  • chlorophyll a - methyl group
  • chlorophyll b - formyl group
  • phaeophytin - chlorophyll without the magnesium
  • chlorophyllide - chlorophyll without the tail

2. Carotene/xanthophylls
    Both are terpenoid pigments, tetraterpenoids (C-40).  Carotenes are hydrocarbons, xanthophylls are oxygenated. These pigments are orange and yellow in color.

3. Chlorophyll biosynthesis - Some take-home-lessons:

    1. ALA (Δ-aminolevulinic acid) is the first well-established precursor
    2. ALA is derived from α-ketoglutarate (or glutamate) (a Kreb's cycle intermediate, from the mitochondrion)
    3. 2 ALA condense to form a unit of pyrolle
    4. 4 pyrolles condense to form porphyrin (tetrapyrolle)
    5. Magnesium is inserted
    6. A photoreduction step occurs (converts protochlorophyllide → chlorophyllide)
    7. the tail is added

4. Light and the Greening Process
    Recall that etiolated plants (grown in the dark) are yellowish but turn green rapidly when placed in the light.  Light is required, among reasons, to:

  1. convert etioplasts → chloroplasts;
  2. photo-reduce protochlorophyllide to chlorophyllide; and
  3. activate enzymes for ALA synthesis.

III. Conversion 1: Photons to electrons

A. Nature of light
    Light is part of the electromagnetic spectrum - radiation emitted by sun. Acts as discrete particles (called photons) traveling as waves. Wavelength - distance between any two crests (or troughs). Symbolized by lambda
(λ); frequency - number of waves passing a point in one second (υ). Frequency is inversely related to wavelength υ = c/λ where c = speed of light (3 x 1010 cm sec-1). The energy of a photon is a quantum. 

B. Which photons are important in photosynthesis?
    Run an action spectrum (plot of a physiological process vs. wavelength).

insert action spectrum of photosynthesis here

    Conclusion: radiations between 400-700 nm are photosynthetically active (termed PAR). Specifically, red (600’s) and blue (400’s) light are important.

C. Photons must be absorbed to be used in a photochemical reaction.
    In other words, only those molecules that absorb quanta participate in photosynthesis. So, which molecules absorb the red and blue light? Run an absorption spectrum of potential pigment candidates (plot of light absorption vs. wavelength) and compare it to the action spectrum.

insert absorption spectrum of photosynthetic pigments here

    Chlorophyll a & b absorb light in the red and blue regions of the visible spectrum. Note that the absorption spectra match the action spectrum of photosynthesis and hence, implicates (though doesn’t prove) that they are involved in the process. (Subsequent work has shown the chlorophylls to be the major photosynthetic pigments).

D. Quantity vs. Quality

    1. Light quality - refers to the wavelengths of light that are important. Photosynthetically active radiations (PAR) range from 400 - 700 nm with peaks in the red and blue.
    2. Light quantity - refers to the amount of light (PAR) received; units of mol m-2 s-1 , called the photon fluence rate; or units of energy, J m-2 s-1.

E. What happens when chlorophyll absorbs light?
    The chlorophyll molecule becomes excited (this takes only 10-15 sec = femptosec) and an electron moves to an outer energy level. This is diagrammed:

CHL (ground state) → CHL* (excited state)

    Blue light excites an electron to a higher energy level than red light. Imagine the "bell ringer" at a carnival. The electrons change spin at the first (S1) and second (S2) excited singlet states. Electrons don’t stay excited long (10-9 sec), because they either:

    1. return to the ground state and release their absorbed energy as heat (thermal deactivation);
    2. return to ground state and release their extra energy as light (fluorescence);
    3. transfer their energy to another molecule; kind of like hitting pool balls (resonance transfer); or
    4. change spin and revert to a triplet state (same spin as ground state) and be used in a photochemical reaction (photochemistry).

F. Why excite electrons?
    The ultimate purpose of exciting electrons from chlorophyll is to provide the energy needed to transfer electrons from water to NADP+.  Recall that spontaneous electron transfers proceed from a carrier with a more negative redox potential to a more positive one. The redox potential of water/oxygen = +0.82 eV while for NADP/H = -0.32 eV.  Thus, photosynthetic electron flow is not a spontaneous process and requires an energy.

G. How much energy is required to transfer electrons from water to NADP+?
    First, let's calculate the actual redox difference (
ΔEm) between water and NADPH:

    ΔEm = Em (acceptor) - Em (donor). Or, ΔEm = -0.320 - (0.820) = -1.14 = ca. -1.2 eV.

The actual amount of energy involved is calculated from the equation:

ΔG = -n F Em

where F = Faraday constant = 96,000 J/coulombs, and n = number of electrons involved in the reaction (which equals one for each photon). Substituting in the equation:

ΔG = - (1) x 96000 x (-1.14) = 109440 J mol-1  (=109.4 kJ mol-1 )

To summarize, approx. 110 kJ mol-1 is required to reduce NADPH from water.

H. Do red and blue photons have enough energy?
    Let's calculate the energy in red photons. Assume red photons have a wavelength of 660 nm = 6.6x10-5 cm.

The energy of a photon is expressed by the following equation:

E = hυ

where h = Planck’s constant which relates energy to frequency of oscillation and is 6.6255 x 10-34 J sec photon-1; and υ = pulses sec-1.

Since υ  = c/λ (see A above), we can substitute back in original equation:

E = hc/λ

Take home lesson #1: the energy of a photon is inversely proportional to its wavelength. Thus, blue light has more energy than red light.

E = ((6.625x10-34 j sec photon-1)(3x1010 cm sec-1))/6.6x10-5  cm

= 3.01x10-19 j photon-1

multiply by Avogadro’s number

= 3.01x10-19 j photon-1 x 6.02 x 1023 photon mol-1

= 181,000 j mol-1

= 181 kj mol-1

Take home lesson #2: Red light has more than enough energy to do the job.

IV. Chloroplast complexes:
    There are four major complexes in the chloroplast. These are physically distinct from one another and can be isolated from the chloroplast by electrophoresis and ultracentrifugation.

A. Photosystem II (PSII) Complex

    1. large multi-subunit protein complex
    2. occurs in the stacked regions of the granal thylakoids
    3. integral proteins - coded by the chloroplast genome; including D1 (33 k) and D2 (31 KD)
    4. peripheral proteins - coded by nuclear genome; bind Ca2+ and Cl-
    5. P680 reaction center - a unique chlorophyll a, maximum red light absorption at 680nm; maybe two chlorophyll a molecules; this is the chlorophyll that "looses" electrons
    6. manganese ions (Mn2+
    7. )
    8. phaeophytin, plastoquinone
    9. LHCII - Light harvesting pigment complex associated with PSII. It is comprised of (a) 250 chlorophyll a & b, in approximately equal amounts; (b) several carotenoids; (c) proteins - each pigment is associated with protein (ca. 15 pigments/protein); the protein is coded by the nuclear genome

B. Cytochrome b/f Complex

1. occurs in stacked and non-stacked regions
2. cytochrome b (b-type cytochrome, not associated with protein)
3. cytochrome f (c-type cytochrome, associated with protein)
4. non heme iron-sulfur protein (Fe-SR)

C. Photosystem I (PSI) Complex

    1. occurs in non-stacked regions (stromal thylakoids)
    2. about 11 polypeptides - including 1a & 1b that are coded by a single operon in the chloroplast genome, bind p700
    3. 50-100 chl a
    4. electron carriers
    5. LHCI - contains about 100 chlorophylls; 4:1 ratio of chl a: chl b.; the protein is encoded by nuclear genome
    6. P700 reaction center chlorophyll a

D. ATP synthase/Coupling Factor Complex

1. occurs in non-stacked regions
2. stalk - CFo (4 polypeptides)
3. head - CF1 (5 polypeptides)
4. nine polypeptides, some nuclear, some chloroplastic

V. The Z-Scheme (Or, the Light-Dependent Reactions; Or, Non-cyclic photophosphorylation).

A. Overview
    During the light-dependent reactions of photosynthesis, electrons are transferred from water to NADP+. This reaction is depicted as follows:


    As the electrons move from water to NADP+, they pass through three of the four complexes described     above - Photosystem II (PSII), a cytochrome b/f complex (cyt b/f), and Photosystem I (PSI). After electrons are removed from water, they are sequentially shuttled from PSII to the cyto b-f complex to PSI and then finally to NADP+. Thus:

H2O → PSII → Cytb/f → PSI → NADP+

    Since PSII, cyt b/f, and PSI are physically separated from one another, there must be a means to transfer electrons between the complexes. A mobile form of plastoquinone (PQ) transfers electrons from PSII to cyt b-f. A copper-containing protein, plastocyanin (PC), transfers electrons from the cytochrome b-f complex to PSI. Thus, the reaction sequence is modified as follows:

H2O → PSII → PQ → Cytb/f → PC → PSI → NADP+

The transfer of electrons from PSI to NADP+ is mediated by a soluble complex found in the stroma, ferredoxin (Fd). Thus our revised equation:

H2O → PSII → PQ → Cytb/f → PC → PSI → Fd → NADP+

The transfer of electrons from water to PSII involves an "oxygen evolving complex" (OEC), part of PSII, that is rich in chloride and manganese ions. Thus,

H2O → OEC → PSII → PQ → Cytb/f → PC → PSI → Fd → NADP+

B. Origin of the name
    Derived from the zig-zag arrangement of components with regard to redox potential. But, why don’t we call it the N-scheme?

C. Oxygen evolving complex
    The energy of a single photon is not sufficient to split water. Experiments suggest that 4 photons are required to split two water molecules. Since only one electron can be excited at a time (Einstein Law of Photochemical equivalents), this presents a minor problem.

    The solution – a water oxidizing "clock". Single electrons are transferred through a series of intermediate stages sequentially increasing the electron deficit to a total of four. At this point the original oxidation state is restored by extracting four electrons from water.

Diagram of the water-oxidizing clock - in class


    1. A series of five intermediate states, S0 - S4 are postulated;
    2. Initially the clock is in the So state, and may be associated with Mn II
    3. S1, which may be associated with Mn III, is the most stable form;
    4. S2 may be associated with Mn IV;
    5. S3 may be associated with a histidine (one of the amino acids in the D1 protein);
    6. The nature of S4 isn’t clear;
    7. Conversion from one state to the next requires one photon and results in the loss of one electron to P680; and
    8. the loss of 4 total electrons generates a strong enough potential to split water.


    1. after a dark equilibration period, oxygen is released after the third light flash and then after every fourth flash;
    2. explains occurrence of Mn in photosystem II.

D. PQ Shuttle (Q cycle)

    1. PQH2 is reduced on the stromal side of the thylakoid in PSII
    2. PQH2 shuttles over to the lumen side of thylakoid and gets oxidized when it transfers its electrons to the cyt b/f complex
    3. One electron is given to an Fe-S protein, which in turn, passes it to cyt f and then to PC. The other electron is given to cyt b which then partially reduces another PQ.
    4. The "leftover" protons are dumped into the lumen
    5. A second PQH2 from PSII shuttles to the cyto b/f complex and essentially repeats step 3 - one electron is passed to Fe-S then to cyt f and to PC. The other electron from PQH2 is given to cyt b and then to PQ to fully reduce it to PQH2 which must also grab two protons from the stroma.

Take-Home-Lessons: for every two electrons shuttled to PSI, four protons are moved across the membrane! The stoichiometry requires more than 2 protons per electron pair to account for ATP synthesis.

E. Herbicides and electron transport

    1. Urea derivatives - DCMU (diuron) blocks electron flow at a point after Q. They bind to the Qb binding site, preventing PQ from doing so. Interestingly, there are resistant varieties, that have a single amino acid substitution in the D1 binding protein.
    2. Viologen dyes - paraquat/diquat - accept electrons from the reducing side of PSI - thus, they interrupt electron flow and also convert oxygen to superoxide - which causes damage to membranes, etc.

VI. Photophosphorylation

  • Literal translation - "the light driven synthesis of ATP"
  • Occurs by the same mechanism, Chemiosmotic hypothesis, that occurs during ATP synthesis in the mitochondria

A. Non-cyclic
    Electrons are passed in a single direction, one-way, no backtracking, from water to NADP+.

B. Cyclic
    Electrons cycle through PSI. This occurs when carriers get backed up with electrons but still want to get rid of electrons. This is a mechanism for generating additional ATP.


    1. asymmetric distribution of electron carriers in the thylakoids;
    2. some carriers require only electrons (i.e., cytochromes, chlorophyll), others both (i.e., PQ);
    3. at the transitions, protons will be extracted or expelled;
    4. the transitions occur at the surfaces of the membrane;
    5. PQ is the major source of the proton gradient;
    6. reduction of PQ occurs at the stroma side of the thylakoid and removes 2 protons from the stroma;
    7. PQ shuttles electrons to the cyt b/f complex on the lumen side of the thylakoid. Only electrons are passed to the cyt b/f complex, the protons are expelled into the lumen. Thus, PQ is oxidized on the lumen side of the membrane;
    8. the Q cycle shuttles additional protons across the membrane;
    9. the thylakoid is impermeable to protons;
    10. a pH gradient is generated across the membrane, the stoma pH is ca. 8 while the lumen is ca. 5;
    11. the pH gradient provides the energy for the synthesis of ATP;
    12. ATP is synthesized by an ATPase associated with the CF complex; and
    13. protons escaping through the channel drives ATP synthesis something like water turning a mill.

Evidence: lots!

    1. a pH gradient exists in the chloroplast. Discharging the gradient with buffers prevents ATP synthesis;
    2. artificially creating a gradient allows for ATP synthesis;
    3. uncouplers like DNP "poke holes" in the thylakoid making it "leaky" and discharging the gradient preventing ATP synthesis. Note that this doesn’t stop electron flow - in fact, it usually increases the rate.

VII. How many photosystems? Two
    Emerson observed that the rate of photosynthesis was greater than the sum of the rates when red light (660 nm) and far red light (710 nm) were given separately. This synergistic effect, called the Emerson enhancement Effect, suggested two cooperating systems which has been the conventional wisdom for a long time.

VIII.  Reference:

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Last updated:  01/07/2009     � Copyright  by SG Saupe