Chapter 4

ENERGY CONSERVATION IN PHOTOSYNTHESIS:

HARVESTING SUNLIGHT

4.1. Leaves are photosynthetic machines which maximize the absorption of light
4.2. Photosynthesis is an oxidation-reduction process
4.3. Photosynthetic electron transport
4.4. Photophosphorylation is the light-dependent synthesis of ATP
4.5. Lateral heterogeneity is the unequal distribution of thylakoid complexes
4.6. Light-harvesting complexes are super antenna complexes that regulate energy
distribution
4.7. Photoinhibition of photosynthesis: photoprotection versus photodamage
4.8. Inhibitors of photosynthetic electron transport are effective herbicides
Purpose
The organization of leaves with respect to the exploitation of light as the
primary source of energy and its conversion to the stable, chemical forms to
ATP and NADPH by the chloroplast

Special topics
- The structure of higher plant leaves with respect to the interception of light
- Photosynthesis as the reduction of CO2 to carbohydrate
- The photosynthetic electron transport chain, its organization in the thylakoid
membrane, and its role in generating reducing potential and ATP
- Problems encountered by chloroplasts when they are subject to varying
amounts of light, often in excess, which may decrease the efficiency of
photosynthesis or even damage components of the electron transport chain
- The dynamic nature of the thylakoid membrane, showing how changes in
the organization of light-harvesting apparatus influence the absorption and
distribution of light energy
- The role of carotenoids are accessory pigments and in photoprotection of
chlorophyll
- The use of herbicides that specifically interact with photosynthetic electron
transport
 4.1. LEAVES ARE PHOTOSYNTHETIC MACHINES
THAT MAXIMIZE THE ABSORPTION OF LIGHT
The architecture of a typical higher plant leaf is particular well suited to absorb light

The anatomy of typical dicotyledonous and monocotyledonous mesomorphic leaf is shown in

fig4.1: The leaf is sheathed with upper and lower epidermis. The photosynthetic tissues are
located between the 2 epidermal layers and identified as mesophyll. The upper photosynthetic
tissue generally consists of 1-3 layers of palisade mesophyll cells; below is the spongy
mesophyll cells.

Palisade cells generally have larger numbers of chloroplasts than spongy mesophyll cells.

Because the absorbing pigments are confined to the chloroplast, a substantial amount of
light may thus pass through the first cell layer without being absorbed. This is called sieve
effect

The impact of the sieve effect on the efficiency of light absorption is balanced by factors (1)
light may all be reflected off the many surfaces associated with leaf cells (2) light that is not
reflected but passed between the aqueous volume of mesophyll cells and the spaces that
surround them will be sent by refraction (3) light may be scattered

The longer light path increases the probability that any given photon will be absorbed by a
chlorophyll molecule before it can escape from the leaf

Some of the incident light is channeled through the intercellular spaces between the palisade
cells in much the same way that light is transmitted by an optical fiber (fig4.2)

Within the leaf mesophyll cells of plants, the chloroplast is the organelle that transforms
light energy into ATP and NADPH to convert CO2 and sugar
Fig 4.1: The structure of leaves shown in cross-section
 Fig 4.2: A simplified diagram illustrating how the optical properties of leaves help to
redistribute incoming light and maximize interception by chlorophyll
(A) Photon strikes a chloroplast and is absorbed by chlorophyll. (B) The sieve effect – a
photon passes through the first layer of mesophyll cells without being absorbed. It may
be absorbed in the next layer of cells or pass through the leaf to be absorbed by another
leaf below. (C) The plano-convex nature of epidermal cells creates a lens effects,
redirecting incoming light to. (D) The light-guide effect. Because the refractive index of
cells is greater than that of air, light reflected at the cell-air interfaces may be channeled
through the palisade layer(s) to the spongy mesophyll below
 4.2. PHOTOSYNTHESIS IS AN OXIDIZATION-REDUCTION PROCESS

Photosynthesis is fundamentally an oxidation-reduction reaction:

6CO2 + 12H2O  C6H12O6 + 6O2 + 6H2O
CO2 + 2H2O  (CH2O) + O2 + H2O

Photosynthesis can be viewed as a photochemical reduction CO2. The energy of

light is used to generate strong reducing equivalents from H2O – strong enough to
reduce CO2 to carbohydrate

Addition of energy for carbon reduction is required ATP, which is also generated at the
expense of light

The principal function of the light-dependent reactions of photosynthesis is

therefore to generate the NADPH and ATP required for carbon reduction
 4.3. PHOTOSYNTHETIC ELECTRON TRANSPORT
4.3.1. Photosystems are major components of the photosynthetic electron transport chain

The key to the photosynthetic electron transport chain is the presence of 2 large, multimolecular
complexes photosystem I (PSI) and photosystem II (PSII) (fig4.3) that operate in series linked by a
third multiprotein aggregate called the cytochrome complex

Such fraction studies have revealed that PSI and PSII, each contain several different proteins together
with a collection of chlorophyll and carotenoid molecule that absorb photons

The bulk of the chlorophyll in the photosystem functions as antenna chlorophyll (fig4.4)

The association of chlorophyll with specific proteins forms a number of different chlorophyll protein
(CP) complexes (fig4.5)

The energy of absorbed photons thus migrates through the antenna complex, passing from 1
chlorophyll molecule to another until it eventually arrives at the reaction center (fig4.4)

Each reaction center consists of a unique chlorophyll a molecule that is though to be present as a
dime.

This reaction center chlorophyll + protein + redox carriers = light-driven redox reactions

The principal adventage of associating a sinle reaction center + a large number of antenna
chlorophyll molecules is to increase efficiency in the collection and utilization of light energy

The augment antenna complexes are known as light-harvesting complexes

A schematic of the photosynthetic electron transport chain depicting the arrangement of:
PSII  PSI  cytochrome b6/f (in thylakoid membrane) (fig4.6)
 Fig 4.3: A linear representation of the photosynthetic electron-transport chain
A sequential arrangement of the 3 multimolecular membrane complexes extracts low-
energy electrons from water and, using light energy, produces a strong reductant,
NADPH + H+
 Fig 4.4: A photosystem contains atenna and a reaction center
Antenna chlorophyll molecules absorb incoming photons and transfer the excitation
energy to the reaction center where the photochemical oxidation-reduction reaction occur
Fig 4.5:
Separation of thylakoid
chlorophyll-protein
complexes by
nondenaturing
polyacrylamide gel
electrophoresis
 Fig 4.5a: Separation of thylakoid chlorophyll-protein complexes
by nondenaturing polyacrylamide gel electrophoresis
(A) In the presence of specific detergents, chlorophyll-protein complexes are removed from
thylakoid membranes structurally and functionally intact. These pigment-protein-detergent
complexes are charged and thus will migrate when an electric field is applied. The porous
matrix through which the electric field is applied is a polyacrylamide gel. Thus, the physical
separation of protein complexes through a polyacrylamide gel matrix by applying an electric
fields is called polyacrylamide gel electrophoresis. Since these proteins complexes still have
chlorophyll bound to them, you can watch the chlorophyll-protein complexes separate through
the gel matrix according to their molecular mass right in front of your eyes ! The largest
complexes remain at the top of the gel and the smallest complexes near the bottom of the
gel. The illustration shows such an electrophoretic separation with the arrow indicating the
direction of migration. Typically, 7 “green bands” can be solved. Bands 1-6 are individual
chlorophyll-protein complexes associated with PSI and PSII. The band exhibiting the greatest
migration (band 7) is free pigment
 Fig 4.5b: Separation of thylakoid chlorophyll-protein complexes
by nondenaturing polyacrylamide gel electrophoresis
(B) The relative amount represented by each green band can be qualified be scanning the
gel in a spectrophotometer. The peak areas provide an estimate of the relative content of
each chlorophyll-protein complex. Thus, green bands 2 and 6 are present at the greatest
amount in this particular thylakoid sample
 Fig 4.5c: Separation of thylakoid chlorophyll-protein complexes
by nondenaturing polyacrylamide gel electrophoresis
(C) The pigment composition of each green band can be assessed by measuring the absorption
spectrum of each band. Here, the absorption spectrum of green bands 2, 5 and 6 only are illustrated.
Note that green bands 2 and 5 exhibit a similar absorption spectrum with maxima at about 435nm and
671nm. This indicates that both of these pigment-protein complexes have chlorophyll a bound to them.
Green band 2 represents PSI plus its core antenna and green band 5 represents the PSII core complex
(CP43 + CP47 + PSII reaction center). Green band 6 exhibits absorption maxima at 435nm and 671nm
representing chlorophyll a plus maxima 470nm and 654nm, representing chlorophyll b. Thus, green
band 6 contains both chlorophyll a and chlorophyll b. This green band represents LHCII, the major
chlorophyll a/b light-harvesting complex associated with PSII. Note that LHCII and PSDI are present in
the greatest amounts in this particular thylakoid sample
Fig 4.6: The organization of the photosynthetic electron transport system
in the thylakoid membrane
4.3.2. Photosystem II (PS II) oxidizes water to produce O2

Electron (photons)  PSII (P680)  P680* (passes 1 electron to pheophytin – Pheo).

Pheophytin is the primary electron acceptor in PSII. Photo-oxidation event result P680+
and Pheo-, a charge separation

The role of reaction proteins, D1 and D2, is to bind and to orient specific redox
carriers of the PSII reaction center in such a way as to decrease the probability of
charge recombination between P680+ and Pheo-

How does this happen ?

(1) pheophytin passes 1 electron on to a quinone (QA) forming [P680+ Pheo- QA] (fig4.7)
then the electron passed from QA  plastoquinone (PQ) forming [P680+ Pheo QA]
(2) P680+ reduced P680 forming [P680 Pheo QA]

P680+ was supplied the electrons by a cluster of 4 Mg2+ ions associated with a small
complex protein called the oxygen-evolving complex (OEC). OEC is bound to the D1
and D2 proteins of PSII reaction center and functions to stabilize Mg2+ cluster
2H2O  O2 + 4H+ + 4e (4.8)
 Pheophytin (Pheo)

Fig 4.7: The light-independent,

cyclic “opening” and “closing” of photosystem II reaction centers
4.3.3. The cytochrome complex and photosystem I (PS I) oxidize plastoquinone

Following its release from PSII  plastoquinone (PQ) diffuse electron  cytochrome
b6/f complex (contain s an additional redox component called Rieske ion-sulfur (FeS)
protein)  From the Cyt f, electrons are pick up by plastocyanins (PC)

In PSI, P700  excited to P700*  photo-oxidation to P700+

The primary electron acceptor in PSI is chlorophyll a  passed through  quinone (Q)
 FeS center  ferredoxin  reduce NADP+

The over all effect of the complete electron transport scheme is to establish a
continuous flow of electron between water and NADP+, passing through PSII and PSI
and intervening cytochrome complex (fig4.6)
Water  PSII  cytochrome complex  PSI  NADP+ (Fig4.8)

From (4.8), the quantum yield of O2 evolution: : 0.125M of O2 evolved / photon

absorbed
 Fig 4.8: The Z-scheme for photosynthetic electron transport
The redox components are arranged according to their approximates mid point redox
potentials (Em, chapter2). The vertical direction indicates a change in energy level (ΔG,
chapter2). The horizontal direction indicates electron flow. The net effect of the process
is to use the energy of light to generate a strong reductant, reduced ferredoxin (fd) from
the flow-energy electrons of water. The downhill transfer of electrons between P680*
and P700 represents a negative free energy charge. Some of this energy is used to
establish a proton gradient, which in turn drives ATP synthesis. Indicated redox
potentials are only approximately
 4.4. PHOTOPHOSPHORYLATION
IS THE LIGHT-DEPENDENT SYNTHESIS OF ATP

The ATP required for carbon reduction and other metabolic activities of the chloroplast is
synthesized by photophosphorylation in accordance with Mitchell’s chemiosmotic
mechanism.

Light-driven production of ATP by chloroplast is known as photophosphorylation

Formation of ATP association with non-cyclic photophosphorylation in PSII.

PSI has a process of cyclic electron transport (fig4.9) called cyclic photophosphorylation:
4 protons are translocated from stroma  lumen:
ferredoxin  carry electron back  through Fdx-PQ  to PQ  FeS / Cyt f  PC

The key to energy conservation in photosynthetic electron transport and the

accompanying production of ATP is the light-driven accumulation of protons in the lumen:
(1) the oxidation of H2O (2 protons / each water molecule oxidized)
(2) PQ-cytochrome proton pump (fig4.10)
 Fig 4.9: Cyclic electron transport.
PSI units operating independently of PSII may return electrons from P700* to P770
through ferredoxin (fd), a ferredoxin-PQ oxireductase (Fdx-PQ), plastoquinone (PQ),
and the cytochrome b6/f complex. In cyclic electron transport, the oxidation of PQ by
the cytochrome b6/f complex generates a proton gradient that can be used for ATP
synthesis but no NADPH is produced
 Fig 4.10: The Q-cycle,
a model for coupling electron transport from plastoquinol (PQH2) to the cytochrome b6/f
complex (Cyt b6/f) with the translocation of protons across the thylakoid membrane. 4 protons
are translocated for each pair of electrons that passes through the electron transport chain. Fe,
Riesk FeS-center; Cyt f, cytochrome f of the cytochrome b6/f complex; Cyt bLP, high reduction
potential form cytochrome b6 of the cytochrome b6/f complex; Cyt bHP high reduction
potential form of cytochrome b6 of the cytochrome b6/f complex; PC= plastocyanin
 4.5. LATERAL HETEROGENEITY
IS THE UNEQUAL DISTRIBUTION OF THYLAKOID COMPLEXES

There is a distinct lateral heterogeneity with respect to their distribution of the major
protein complexes within the thylakoids (fig4.11)

Nonappressed mambrane: PSI, ATPsynthase, stroma thylakoids, grana end

membranes and grana margin exposed to the stroma

Appressed membrane: PSII, cytochrome b6/f complex, plastoquinone (PQ),

plastocyanin (PC) distributed throughout the membrane system
(A)
(B)

Fig 4.11: Lateral heterogeneity in the thylakoid membrane

(A) Nonappressed membranes of the stroma thylakoids, grana end membranes, and
grana margin are exposed to the stroma. Appressed membranes in the interior of
grana stacks are not exposed to the stroma. (B) PSII units are located almost
exclusively in the appressed regions while PSI and ATP synthase units are located
in nonappressed regions. The cytochrome b6/f complex, plastoquinone, and
plastocyanin are uniformly distributed throughout the membrane system
 4.6. LIGHT-HARVESTING COMPLEXES (LHC)
ARE SUPERANTENNA COMPLEXES
THAT REGULATE ENERGY DISTRIBUTION

PSII overexcited (relative to PSI)  electron  plastoquinone (PQ) accumultes 

protein kinase  phosphorylates LHCII

PSI  electron  oxidize PQH2  plastoquinone (PQ)  Phosphatase 

dephosphorylates LHCII (fig4.12)
 Fig 4.12: Reversible phosphorylation of LHCII (light-harvesting complex II)
When PSII is over excited relative to PSI, plastoquinone (PQH2) accumulates. A high level of
plastoquinol activates a protein kinase that phosphorylates LHCII. Addition of a phosphate
group weakens the interaction between LHCII and the PSII core antenna, causing LHCII to
dissociate from PSII. The input of light to PSII is disminished and PSII slows down, thus
allowing PSI to oxidize excess PQH2 to plastoquinone (PQ), which, in turn, deactivates the
protein kinase. LHCII is dephosphorylated by a protein phosphatase, allowing LCHII to reform
in association with PSII
 4.7. PHOTOINHIBITION OF PHOTOSYNTHESIS:
PHOTOPROTECTION VERSUS PHOTODAMAGE

4.7.1. Carotenoids serve a dual function: light-harvesting and

photoprotection (fig4.13-14)

4.7.2. O2 may protect against photoinhibition by acting as an

alternative electron acceptors (fig4.15)

4.7.3. The D1 repair cycle overcomes photodamage to PSII

(fig4.16)
 Fig 4.13: The xanthophyll cycle
De-epoxidation removes the 2 O (epoxy) groups the rings of violaxanthyl. This is a 2-step
reaction with the intermediate antheraxanthin containing only 1 epoxy group. De-epoxidation
is induced by high light, low lumenal pH and high levels of reduced ascorbate. Note that in
zeaxanthin, the number of C-C double bonds is increased by 2 relative to violaxanthin.
Zeaxanthin and antheraxanthin, but not violaxanthin, are able to accept a downhill energy
transfer from excited chlorophyll. Zeaxanthin is converted back to violaxanthin via
antheraxanthin in low light or darkness
 Fig 4.14: Energy dissipation by zeaxanthin.
Upon the absorption of low light, ground state chlorophyll (Chl) becomes excited. The energy of
excited chlorophyll (Chl*) is transferred to reaction center via other antenna chlorophyll a,
chlorophyll b, and carotenoids such as violaxanthin. Thus, the absorbed energy is preferentially
allocated to photosynthesis and the excited chlorophyll falls back to ground state (thick arrows). As
irradiance increases, violaxanthin (V) in the antenna is converted to zeaxanthin (Z) and an
increasing proportion of the absorbed excitation energy is transferred to zeaxanthin, which
becomes excited (Z*). However, Z* cannot pass its energy on to other antenna chlorophyll and the
energy is dissipated as heat (broken arrows). Thus, the reversible conversion of violaxanthin to
zeaxanthin represents a switch that regulates energy flow within the photosystem
 Fig 4.15: Oxygen as an alternative electrons acceptor in chloroplasts.
(A) The Asada-Halliwel pathway, O2 can be photoreduced by PSI directly to generate the
superoxide free radial, O2- (Mehler reaction). Superoxide dismutase (SOD) then converts this
radical to hydrogen peroxide (H2O2). Hydrogen peroxide is also toxic and reduced via the
chloroplastic enzyme, ascorbate peroxidase, to water and ascorbate is oxidized to monodehydro-
ascorbate (MDHA). Ascorbate (vitamin C) is regenerated through the action of the enzyme,
dehydro-ascorbate reductase, through the consumption of reduced glutathione (GSH). Oxidized
glutathione (GSSH) is, in turn, reduced by the enzyme, glutathione reductase, which uses NADPH
as reductant. (B) Chlororespiration pathway. NAD(P)H dehydrogenase (Ndh) present in thylakoid
membranes consumes stroma (NAD(P)H and passes the electrons (e) directly to plastoquinone
(PQ). The plastid terminal oxidase (PTOX) present in thylakoid membrane oxidizes plastoquinol
and reduce O2 to water. The stromal pool represents any metabolic pathway present in the
stroma that generates reducing power (chapter5). Ndh may also participate in cyclic electron
transport around PSI
 Fig 4.15a: Oxygen as an alternative electrons acceptor in chloroplasts.
(A) The Asada-Halliwel pathway, O2 can be photoreduced by PSI directly to generate the
superoxide free radial, O2- (Mehler reaction). Superoxide dismutase (SOD) then converts this
radical to hydrogen peroxide (H2O2). Hydrogen peroxide is also toxic and reduced via the
chloroplastic enzyme, ascorbate peroxidase, to water and ascorbate is oxidized to
monodehydro- ascorbate (MDHA). Ascorbate (vitamin C) is regenerated through the action of
the enzyme, dehydro-ascorbate reductase, through the consumption of reduced glutathione
(GSH). Oxidized glutathione (GSSH) is, in turn, reduced by the enzyme, glutathione
reductase, which uses NADPH as reductant.
 Fig 4.15b: Oxygen as an alternative electrons acceptor in chloroplasts.
(B) Chlororespiration pathway. NAD(P)H dehydrogenase (Ndh) present in thylakoid
membranes consumes stroma (NAD(P)H and passes the electrons (e) directly to
plastoquinone (PQ). The plastid terminal oxidase (PTOX) present in thylakoid membrane
oxidizes plastoquinol and reduce O2 to water. The stromal pool represents any metabolic
pathway present in the stroma that generates reducing power (chapter5). Ndh may also
participate in cyclic electron transport around PSI
 Fig 4.16: The D1 repair cycle.
Although light is the ultimate source of energy for photosynthesis, too much light can be
dangerous because it may result in damage to the photosystems, especially the D1
reaction center polypeptide present in PSII. Plants and algae have evolved and elaborate
mechanism to repair photodamage called the D1 repair cycle. Exposure to an irradiance
that exceeds the capacity of the plant either to utilize that energy in photosynthesis or to
dissipate it safely as heat results in damage to the D1 polypeptide. Functional PSII reaction
centers are converted to damaged PSII reaction centers. When this happens, PSII is
disassembled and D1 is degraded by thylakoid proteolytic enzymes. Subsequently, new D1
is synthesized de novo by the chloroplast translational machinery and inserted into the
thylakoid membrane to form functional PSII reaction centers
4.8. INHIBITORS OF PHOTOSYNTHETIC ELECTRON TRANSPORT
ARE EFFECTIVE HERBICIDES

Both urea and triazine (fig4.17) herbicides are taken up by the roots and
transported to the leaves

DCMU used to block electron transport between PSII - PSI

Triazine used to control weed in corn field

Fig 4.17: The chemical structures of some common herbicides
that act by interfering with photosynthesis