Pigments of Life

Chlorophill
Dr. Samir Kumar Patra
Professor of Biochemistry and Molecular Biology
National Institute of Technology Rourkela

LS-401 & LS 4103, Biochemistry

OCTOBER 23, 2018
Heme & Chlorophyll
4
Energy conservation in
photosynthesis: Harvesting Sunlight

1 2 3 4 5 6 7 8
Fig.
9 10 11 12 13 14 15 16 17
18 19 20
The primary function of leaves is
photosynthesis.
 Focus of this chapter (1)
• The structure of higher plant leaves with respect to
the interception of light
• Photosynthesis as the reduction of carbon dioxide
to carbohydrate
• The photosynthetic electron transport chain, its
organization in the thylakoid membrane, and its
role in generating reducing potential and ATP
• Problems encounters by chloroplasts when they
are subjected to varying amount of light
 Focus of this chapter (2)
• 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 as accessory pigments and
in photoprotection of chlorophyll and
• The use of herbicides that specifically interact
with photosynthetic electron transport
 The structure of the leaf
• The architecture of a typical higher plant
leaf is particularly well suited to absorb
light.
• The photosynthetic tissues (mesophyll) are
located between the two epidermal layers.
• Dicotyledonous leaf is structurally different
from monocotyledonous leaf.
The structure of dicotyledonous
leaf
• One-to-three layers of
palisade mesophyll
cells forms the upper
photosynthetic tissue.
• Below is the spongy
mesophyll cells.
The structure of dicotyledonous
leaf
• Palisade mesophyll
cells are elongated,
cylindrical cells with
the long axis
perpendicular to the
surface of the leaf.
• Spongy mesophyll
cells are irregular with
lots of air spaces
between the cells.
 The structure of
monocotyledonous leaf
• Monocotyledonous
leaf lack the
distinction between
palisade and spongy
mesophyll cells.
Comparison between mesophyll
cells
• Palisade mesophyll
generally have larger
numbers of
chloroplasts than
spongy mesophyll.
Sieve effect
• When light passes through
the first layer of cells
(palisade mesophyll cells)
without being absorbed,
we call this sieve effect.
• The sieve effect is due to
the fact that chlorophyll is
not uniformly distributed
throughout cells but
instead is confined to the
chloroplasts.
Sieve effect
• To reduce sieve effect,
plant develops
multiple layers of
photosynthetic cells.
• The reflection,
refraction, and
scattering of light
inside leaf may also
reduce sieve effect.
 Photosynthesis
• Photosynthesis can be viewed as a photochemical
reduction of CO2.
• In the 1920s, C.B. van Niel discovered the O2
produced from photosynthesis is from water.
• In 1939 Robert Hill found light reaction still can
happen in isolated chloroplast when no CO2 is
consumed and no carbohydrate was produced.
• In the early 1940s S. Ruben and M. Kamen
showed O2 produced from photosynthesis is from
water by using O18 labeled water.
Photosynthetic electron transport

The principle function of the light-

dependent reactions of photosynthesis
is therefore to generate the NADPH
and ATP required for carbon
reduction.
Photosynthetic electron transport
• The effect of photosynthetic electron
transport chain is to extract low-energy
electrons from water and raise the energy
level of those electrons to produce a strong
reductant NADPH.
• The energy plant used to raise the energy
level of those electrons is the light energy
trapped by chlorophyll.
Photosynthetic electron-transport
chain

• Two large, multimolecular complexes, photosystem I (PSI)

and photosystem II (PSII), linked with a third multiprotein
aggregate called the cytochrome complex, form the
photosynthetic electron-transport chain.
Photosystems
• Photosystems contain
several different proteins
together with a collection
of chlorophyll and
carotenoid molecules that
absorb photons.
• Most of the chlorophyll in
the photosystem functions
as antenna chlorophyll.
Photosystems
• The antenna chlorophyll
absorb light but do not
participate in
photochemical reactions.
It pass its energy to the
next chlorophyll by either
inductive resonance or
radiationless energy
transfer.
Reaction center of photosystem
• For PSII, each reaction
center consisted of two
chlorophyll a called
reaction center chlorophyll.
• Reaction center
chlorophyll is the lowest-
energy absorbing
chlorophyll in the PSII
complex (energy sink).
Energy transfer efficiency of
Photosystem
• The design of
photosystems ensure
efficient energy
transfer. Only about
10% of the energy is
lost during the whole
transfer process (from
antenna to reaction
center chlorophyll).
Why photosystems?
• The principle advantage of
associating a single
reaction center with a
large number of antenna
chlorophyll molecules is
to increase efficiency in
the collection and
utilization of light energy.
Why photosystems?
• Even in bright sunlight, an
individual chlorophyll will
only be struck not more
than a few times per
second. However, energy
transfer only takes ms. So
it is more economical not
to make every chlorophyll
into reaction center.
 Light-harvesting complexes
(LHC) are closely associated
with photosystems
 Light-harvesting complexes
(LHC)
• Light harvesting complex (also consisted of
chlorophyll and proteins) serves as extended
antenna systems for harvesting additional
light energy.
• In chloroplast, there are two LHCs. The one
associated with PSI is named LHCI and the
one associated with PSII is named LHCII,
accordingly.
 Light-harvesting complexes
(LHC)
• All the chlorophyll b are contained in LHCs. Most
of the chloroplast pigments (70%) are in LHCs.
• LHCI has a chlorophyll a/b ratio about 4 and it is
tightly bound to PSI.
• LHCII has a chlorophyll a/b ratio about 1.2.
Besides owning most of the chloroplast
chlorophyll (50~60%), LHCII also contains most
of the chlorophyll b and xanthophyll.
Photosynthetic electron transport
chain
PSII  pheophytin
• P680 is located at the
lumenal side of
reaction center.
• When excited, the
excited P680 (P680*)
is rapidly (10-12s)
photooxidized as it
passes an electron to
pheophytin (primary
electron acceptor).
pheophytin
• Pheophytin is a form
of chlorophyll a with
the Mg2+ replaced by
two hydrogens.
• The photo-oxidation
of P680 is then
followed by charge
separation
(P680+Pheo-).
 phytyl

Pheophytin

Pheophytin a R1 =-CH3; R2 = phytyl

Pheophytin b R1 = -CHO; R2 = phytyl
P680  pheophytin
• Noted the direction of
electron movement in PSII.
P680 is located at the
lumen side of PSII, then
the electron is transferred
to pheophytin, which is
more towards the stromal
side, so electron will not
recombine with P680+.
Pheophytin  QA  PQ
• Reaction proteins D1
and D2 orient specific
redox carriers of the
PSII reaction center so
the probability of
charge recombination
is further reduced.
Pheophytin  QA  PQ
• D2 contains QA (quinone
electron acceptor) which
will accept electrons from
pheophytin within
picoseconds.
• Then electron from QA
will be passed to
plastoquinone (PQ), a
quinone bound transiently
to the binding site on D1
protein (QB).
Plastoquinone (PQ)
• The reduction of
plastoquinone (PQ) to
plastoquinol (PQH2)
lowering the affinity of
this molecule for the
binding site.
• After plastoquinol is
released from the reaction
center, another molecule
of PQ will occupy the
empty space.
Oxygen-evolving complex (OEC)
• P680+ got its electron
directly from a cluster of
four Mn2+ associated with
a small complex of
proteins.
• OEC is located on the
lumen side of the
thylakoid membrane and
bound to the D1 and D2
proteins of PSII reaction
center.
Oxygen-evolving complex (OEC)
• The OEC proteins
functions to stabilize
the Mn2+ cluster.
• Chloride ion (Cl-) is
also required for the
water splitting
function.
Oxygen-evolving complex (OEC)
• To generate one
molecule of O2, four
electrons must be
withdrawn from two
molecule of H2O. This
suggest that OEC
should be able to store
charges (and
experiment results
agree with this).
PQ  cyt b6f complex
• After plastoquinol is
released from PSII, it
diffuses through the
membrane until reaches
cytochrome b6f complex.
• Because plastoquinol has
to reach cyt b6f by
diffusion, this is the
slowest step in
photosynthetic electron
transport (milliseconds).
Cytb6f complex
• Electron is then
transferred from
plastoquinol to Rieske
iron-sulfur (FeS) protein
 cytochrome f (all on
the lumenal side).
• Then electrons are
picked up by
plastocyanin (PC).
Plastocyanin (PC)
• Plastocyanin is a small
peripheral protein that
is able to diffuse freely
along the lumenal
surface of the
thylakoid membrane.
PC  PSI
• PC is then transfer
electron to PSI.
• The reaction center
chlorophyll (P700)
first become P700*,
then photooxidized to
P700+ and give its
electron to a molecule
of chlorophyll a.
Photosystem I
• The electron is then
passed to a quinone
(phylloquinone).
• Electron transfer then
proceeds through a
series of Fe-S centers
and ultimately to the
soluble iron-sulfur
protein, ferredoxin
(Fdx).
Ferredoxin  NADP+
• Ferredoxin-NADP+
reductase (Fd-NADP+
reductase) then uses
ferredoxin to reduce
NADP+.
Although PSI do accept electrons
from plastocyanin, PSI …
…can also be activated by light.
• When PSI is directly
activated by light, the
electron it lost is
satisfied by
withdrawing an
electron from reduced
PC.
 Photosynthetic efficiency
• The efficiency of photosynthesis can be
expressed as quantum yield ().
• Quantum yield is number of photochemical
product produced per photon absorbed.
 Noncyclic electron transport

• When electron transport is operating according to the

figure above, electrons are continuously supplied from
water and withdrawn as NADPH. This flow-through form
of electron transport is known as noncyclic or linear
electron transport.
Cyclic electron transport
• Cyclic electron
transport is referring
to a condition when
electrons from PSI is
transported not to Fd-
NADP+-reductase but
to a Fd-PQ reductase.
 Photophosphorylation

The light-driven production of ATP

by chloroplasts is known as
photophosphorylation.
How is ATP generated?

The light-driven accumulation of protons

in the lumen by oxidation of water and
PQ-cytochrome proton pump is the
energy source of ATP production.
How cytb6f complex moves
+
protons (H ) across the
membrane
• The most widely
accepted model for
this question is known
as the Q-cycle.
Q-cycle (1)
Q-cycle (2)
 ATP
synthase
complex
Thylakoid ATP synthase
complex : 400kDa, 9
subunit.
CF1 (hydrophilic stromal
part): 33
CF0 (transmembrane
segment): I II III12IV
 Binding change mechanism of
ATP synthesis by the CF0-CF1
complex

O-site (open): available to bind ADP and Pi

L-site (loose): ADP and Pi are loosely bound
T-site (tight): nucleotide-binding site
Proton translocation  conformation change  rotation of  
interconversion of these sites
 Lateral heterogeneity
• Lateral heterogeneity is referring to the condition
that two photosystems (PSI and PSII) are
distributed unevenly.
• PSI is mainly located in the stromal membranes
and PSII is in the granal membranes.
• ATP synthase is found mostly in stromal
membrane.
• Cytochrome b6f complex is distributed evenly.
Lateral heterogeneity
Lateral heterogeneity
• Lateral heterogeneity
is referring to the
uneven distribution of
PSI, PSII, and ATP
synthase complexes on
thylakoid membranes.
 Lateral heterogeneity
PSI/LHCI and
ATP synthase

Cytb6f is
uniformly
distributed
PSII/LHCII
 Consequences of lateral
heterogeneity
• The ratio between PSI and PSII is
adjustable.
• Output of NADPH and ATP can be adjusted
to meet cellular demands because non-
cyclic and cyclic photophosphorylations can
happen more or less simultaneously.
Role of LHCII in photosynthesis
• LHCII contains more than half of the chlorophyll
a and almost all of the chlorophyll b, however it is
not directly involved in photochemical reduction.
• Functions of LHCII:
(1) increase the activity of PSII under conditions
of low irradiance (shade plants)
(2) regulate PSII activity when light condition
fluctuates for a short period of time
(phosphorylation/dephosphorylation)
 Shade plants
• Plants grown under shade have more
thylakoids with large grana, therefore they
have higher proportion of apressed
thylakoids.
• Sun plants have less LHCII but with more
cytochrome b6f complex, plastoquinone,
plastocyanin, ferredoxin, and ATP
synthease (CF0-CF1 complex).
 Phosphorylation/
dephosphorylation of LHCII
• LHCII can be
phosphorylated by a
protein kinase. The
phosphorylation causes
LHCII becoming more
negatively charged.
• Phosphorylated LHCII can
be dephosphorylated by a
protein phosphatase.
 Phosphorylation/
dephosphorylation of LHCII
• Under high irradiance
of light, PSII will be
preferentially excited
(state 2). The
activation of PSII will
result in accumulation
of PQH2, which will
activate (LHCII)
protein kinase.
 Phosphorylation/
dephosphorylation of LHCII
• The protein kinase is
then phosphorylate
LHCII.
• The phosphorylation
makes LHCII
becoming more
negatively charged.
 Phosphorylation/
dephosphorylation of LHCII
• LHCII moves to the
stromal thylakoid
because charge
repulsion, making
PSII antenna size
smaller.
• Granal thylakoid also
loosens due to lack of
LHCII.
 Phosphorylation/
dephosphorylation of LHCII
• Now PSI is
preferentially excited
(state 1).
• [PQH2], [PQ]
• (LHCII) phosphatase
is activated.
 Phosphorylation/
dephosphorylation of LHCII
• Phosphatase
dephosphyrylates
LHCII and LHCII
moves back to the
granal side, which
increase the antenna
size of PSII.
• Granal membrane is
stacked again.
Figure 4.12
 LHCII and photoprotection
• PSII is the component of the thylakoid membrane
that is most sensitive to excess light.
• Phosphorylation/dephosphorylation of LHCII will
protect PSII from thermal damage due to excess
light energy.
• Photodamage happens when excess light causes
the oxidation of the D1 protein of PSII, which is
slowly reversible to some extent.
 Carotenoid and photoprotection

• The principle carotene in most higher plants is -

carotene.
• Carotenoids serve two functions in photosynthesis:
light harvesting and photoprotection.
Carotenoid and photoprotection
• Carotenoid-deficient
mutant and norflurazon-
treated plants
(Norflurazon is an
inhibitor of phytoene
desaturase and subsequent
blocking of carotenoid
biosynthesis) are bleached
in spite of their ability of
chlorophyll biosynthesis is
still functional.
 Carotenoid and photoprotection
• Carotenoids will trap and dissipate excess
excitation energy before it reaches reaction center.
• If excess excitation energy (happens during
periods of peak irradiance) reaches reaction center
chlorophyll, the chance of 1O2 production
(reactive oxygen species, ROS) will increase, and
ROS will result in cell damage, even death.
Xanthophylls
 Zeaxanthin and photoprotection

• Zeaxanthin can dissipate excess excitation energy

as heat.
Zeaxanthin is formed by
xanthophyll cycle
 Xanthophyll cycle

• Under conditions of excess light, violaxanthin is

enzymatically converted to zeaxanthin through de-
epoxidation.
• De-epoxidation can also be induced by a low pH in the
lumen, which also happens under high light conditions.
• Violaxanthin can also act as a light-harvesting carotenoid.
 Xanthophyll cycle

• Violaxanthin is a diepoxide. The de-epoxidation of it is

progressing one by one, first producing antheraxanthin
(monoepoxide), then zeaxanthin.
• Antheraxanthin and zeaxanthin will be converted back to
violaxanthin in the dark by enzymatic actions.
 Xanthophyll cycle

• Both antheraxanthin and zeaxanthin can lose excess energy

in the form of heat.
• However, neither of they can transfer their energy to
chlorophyll because even when they are in excited states,
their energy levels are still lower than antenna chlorophylls.
 Xanthophyll cycle

• Although they cannot pass their energy to antenna

chlorophyll, antenna chlorophyll can transfer
excess energy to them and dissipate it as heat.
Xanthophyll cycle
• So xanthophyll cycle acts
as a switch, generating
antheraxanthin and
zeaxanthin whenever
dissipation of excess
energy is required but
removing the zeaxanthin
under conditions of low
irradiance.
 Potential value of xanthophyll
cycle
Shade leaves Sun leaves

Xanthophyll 13% 32%

content

Absorbed light 91% 12%

used in
photosynthesis
Light allocated to 6% 79%
dissipation as heat
 Mehler reaction and Asada-
Halliwell pathway

• Sometimes (about 5~10%) O2 can react with electrons

generated by PSI, producing superoxide radical (O2-). This
is called Mehler reaction.
• Superoxide dismutase (SOD) will remove the O2-,
producing H2O2 (peroxide). H2O2 is then reduced to water
by ascorbate.
 Mehler reaction and Asada-
Halliwell pathway

• Plant chloroplasts normally exhibit relatively high

concentrations of ascorbate (0.5~1.0 mmol/mg of
chlorophyll).
• This pathway is to prevent H2O2 react with O2-, producing
OH·(hydroxyl radical).
Chlororespiratory pathway –
reducing O2 in the dark
O2 H2O
 Chlororespiratory pathway
• Chlororespiartory pathway is probably have
a role in photoprotection because it is not
only operating in the dark.
• This pathway also operate in the light when
organisms are exposed to excess irradiance.
The D1 repair cycle
 The D1 repair cycle
• PSII reaction center exhibit an inherent
lifetime because D1 polypeptide of PSII
will be irreversibly damaged due to photo-
oxidative damage after absorption of 105 to
107 photons.
• The life span for each D1 polypeptide of
PSII reaction center is about 30 minutes.
The D1 repair cycle

psbA marked for

degradation
 D1 polypeptide
• In addition to prone to photooxidation damage, D1
polypeptide is also the binding site of many
herbicides. Therefore it is also called herbicide
binding protein).
• Herbicides belong to urea derivative and triazine
groups inhibit photosynthesis by binding to QB
site of D1 polypeptide, interrupting photochemical
electron transport.
Urea derivatives and triazines
 Triazines

• Some plants are resistant to triazines so it can be used as a

selective herbicides.
• Corn roots contain an enzyme that degrade the herbicide.
Cotton sequesters the herbicide in special glands.
• Some weeds also develop resistance toward this herbicides.
 Bipyridylium viologen dye
herbicides

• This class of herbicides act by intercepting electrons on the

reducing side of PSI, thus interrupting electron transport.
• After accepting electrons from PSI, they auto-oxidize and
reduce oxygen to superoxide, which cause oxidative
damage to plants.
• Herbicides in this class is also toxic to animal, therefore
the usage is highly regulated.