1

Chapter 18

Takusagawas Note

Chapter 18: Photosynthesis

1. CHLOROPLASTS
- Photosynthesis is carried out at chloroplasts.
Structure of chloroplast

Outer membrane
Stroma lamellae

Inner membrane
Thylakoid

Chloroplast

Granum
Stroma

Dark reaction

Light reaction

Composition of innermembrane and granum membrane are unusual.

- Phospholipid (negatively charged) ~10%
- Neutral lipid (galactose) ~80%

Photosynthesis occurs in two distinct phases:

1. Light reactions --- Generates NADPH & ATP by using light energy at thylakoid membrane
(prokaryotes: inner membrane).
2. Dark reactions --- Synthesize carbohydrates from CO2 + H2O by using NADPH & ATP at
stroma.

Chapter 18
2.
-

Takusagawas Note

LIGHT REACTION
Initially believed: CO2 + H2O light
CH2O + O2
van Niel showed: CO2 + 2H2S light
CH2O + 2S + H2O
In general:
CO2 + 2H2A light
CH2O + 2A + H2O

This suggested that photosynthesis is two step reactions:

1. Light energy oxidizes H2A (light reaction):
2H2A light
2A + 4[H]
2. The reducing agent [H] reduces CO2 (dark reaction): 4[H] + CO2 CH2O + H2O

A. Absorption of light
- The principal photoreceptor is chlorophyll (Chl) derived from protoporphyrin IX.
- Major differences between chlorophyll and hemes are:
- Chl has an extra ring (V) (cyclopentanone).
- Ring IV of Chl is more reduced.
- Mg is present instead of Fe.
- Side chains are different --- some Chl have a long chain at R4 site.

Chapter 18
-

Takusagawas Note

Eukaryotes have:
- Chlorophyll a (Chl a)
- Chlorophyll b (Chl b)
Prokaryotes have:
- Bacteriochlorophyll a (BChl a)
- Bacteriochlorophyll b (BChl b)

- Note: No absorption of Chl a and b in 480 - 620 nm region. But other pigments, carotenoids
(such as -carotene), phycoerythrin and phycocyanin absorb the light between 480 and 620.

Chapter 18

Takusagawas Note

Photo-energy
-

Photo-energy E = h =

hc

, where h = Plancks constant (6.626 10-34 J/s), c = speed of

light (2.998 108 m/s), and = wavelength of light.

- One mole of red light (6.02 1023 photons = 1 einstein) with = 700 nm has 171 kJ/einstein:
Nhc (6 10 23 )(6.6 10 34 J s 1 )(3 10 8 m s 1 )
E=
=
= 171kJ / einstein .

700 10 9 m
- In general, when molecules absorb photon energy, they move from ground state to various
excited states.
- There are four pathways that an excited state returns to lower energy state.
1. Losing energy as heat (kinetic).
2. Losing energy as light (fluorescence) that normally shifts to longer wavelength.
3*. Losing energy as energy transfer to another molecule.
M*A + MB MA + M*B
MA + MB light
*
4 . Losing energy by losing electron (photooxidation)
Chl+ + eChl light
* 3 and 4 are important for photosynthesis.

Chapter 18

Takusagawas Note

Light absorbed by antenna chlorophylls and accessory pigments is transferred to

photosynthetic reaction centers
- Light is absorbed in light-harvesting complexes (LHCs) located on the membrane of
thylakoid in chloroplast.
- LHC is membrane bound proteins containing Chl molecules and other pigments.

Light-harvesting complex in pea chloroplast

Most Chl molecules and other pigment molecules absorb light and the light energy (exciton)
is randomly transferred to the neighboring Chl or other pigment until it is trapped by a
photosynthetic reaction center.
Thus, the function of most Chls is to gather light, i.e., they act as light-harvesting antennas.
Photosynthetic reaction centers contain the same Chl molecules as antennas have.
However, Chl molecules in photosynthetic reaction centers have slightly lower excited state
energies because their different environments.
Thus, excitons are trapped or funneled into reaction centers.
A photosynthetic reaction center is surrounded by ~300 photosynthetic antenna complexes.
Photosynthetic reaction center

Schemetic diagram of light harvesting complex

Chapter 18

Takusagawas Note

Chloroplast

Takusagawas Note

Chapter 18

C. Plant Photosynthesis is two-center electron transport

- Bacteria photosynthesis is one center process, i.e., one light absorbing event, whereas
- Photosynthesis in plants and algae is two center process, i.e., two light absorbing event.

light (4 photons)
1. 2H2O
O2 + 4e- + 4H+

E = -0.815 V

light (4 photons)
2NADPH
2. 2NADP+ + 4e- + 2H+

E = -0.320 V

Thus, light is used to reduce NADP+ with H2O.

2NADP+ + 2H2O 2NADPH + O2 + 2H+

E = -1.135 V

G = -nFE = 438 kJ/mol,

where n = 4 (since 4 electrons are involved) and F=96.5 kJ/V.

As explained in previously, photo-energy of 700 nm light per one mole of photon (einstein)
is:
E = 171 kJ/einstein.

In each event, four electrons are involved. Thus the total electrons are eight (Remember:
two-light absorbing events).
One electron activation requires one photon. Thus eight photons are necessary to activate
eight electrons. The input light energy is: E = 8 171 = 1368 kJ/mol, which is much larger
than G(438 kJ/mol) of 2 mol NADPH and 1 mol O2 productions.

G can be calculated from the pair of half-cell reactions listed in Table 15-4.
NADP+ + H+ + 2e- NADPH
E = -0.320 V
+
H2O O2 + 2H + 2e
E = -0.815 V
Thus, 2 mol NADPH and 1 mol O2 production require 2 (-0.320 + -0.815) = -2.27 V.
Since G = -nFE, G = -2 96.5 -2.27 = 438.11 kJ/mol

Photosynthetic O2 production requires two-sequential photosystems

- These events are occurred in two different photosynthetic reaction centers that are connected
essentially in series.
1. Photosystem II (PSII) --- generates a weak reductant and strong oxidant to oxidize H2O.
light (4 photons)
2H2O
O2 + 4e- + 4H+

eChl

light

Chl+

Chl*

e- (from H2O)

Takusagawas Note

Chapter 18

2. Photosystem I (PSI) --- generates a strong reductant to reduce NADP+ and weak oxidant.
light (4 photons)
2NADP+ + 4e- + 2H+
2NADPH
Chl

light

eChl*

Chl-

(to NADP+) e

Herbicide dichlorophenyl-dimethylurea (DCMU) blocks photosynthetic electron transport

from PSII to cytochrome b6-f.

Chapter 18

Takusagawas Note

Three complexes in membrane

- are involved in the electron transport from (2H2O O2 + 4H+ + 4e-) event to (2NADP+ +
2H+ + 4e- 2NADPH) event. Those are:
3) PSI
1) PSII
2) Cyt b6-f complex
- The electrons are carried by the mobile electron carriers between the complexes.
Plastoquinol (PQ) --- between PSII and Cyt b6-f complex.
Plastocyanin (PC) ---between Cyt b6-f complex and PSI.
- Electron flow:
PSII (PQ) Cyt b6-f complex (PC) PSI

Plastoquinol (PQ) has two oxidation states, PQ

(oxidized state) and PQH2 (reduced state).
Plastocyanin (PC) is a small Cu-containing protein.

Chapter 18

10

Takusagawas Note

Details of electron transport

1. Oxidation of H2O by O2-evolving complex (OEC) in PSII.
- OEC contains 4Mn cluster that binds 2H2O.
- Oxidation of H2O occurs 5 steps, i.e., S0, S1, S2, S3, S4. Four electrons are stripped, one at a
time in light-driven reactions (S0 S4).

- In the recovery step (S4 S0), O2 is replaced with H2O.

2H2O
O
M
M
O2
O
O
M
O
O
M
M
M
O
O
M
+
- O
O
O
M
2H+
2H + 4e
S4 (Mn4O6)
S0 (Mn4O4)
Adamantane-like
Cubane-like
- S0, S1, S2 and S3 states take the cubane-like Mn4O4 structure.
- S4 states take the adamantane-like structure, i.e., 4O + 2H2O + 4Mn.
- Then, electrons go to substance Z (Tyrosine radical).

10

O
M

O
O

M
O

S0 (Mn4O4)
Cubane-like

Chapter 18

11

Takusagawas Note

2. PSII reaction center P680 (Chl a)

- P680 = Photon-absorbing center in PSII, which gives the absorption maximum at 680 nm and
composed of Chl as.
- Light causes P680 to be a strong oxidant which accepts electrons from the substance Z via
OEC.
- Electron flow in PSII:
P680 (Chl a) Chl a Pheophytin a (Chl a without Mg) Plastoquinone-Fe (QA)
complex Plastoquinone (QB).
- The QB is exchanged with the membrane-bound plastoquinone (Qpool), i.e., the electrons flow
into a membrane from PSII.
Note: When a plastoquinone (Q) receives two electrons with two protons 2[H], the
plastoquinone becomes QH2 (see page 8). H = H+ + e3. Electrons to Cyt b6-f complex
- 2H+ per electron are transported from stroma to the thylakoid lumen (Total 8 H+).
4. Electrons to plastocyanin (a Cu protein)
5. Electrons to PSI
P700 = Photon-absorbing center in PSI, which gives the absorption maximum at 700 nm and
composed of Chl as.
- Photooxidation of P700 yields P700+ (weak oxidant), and subsequently accepts an electron
directly from PC. Electrons pass through several carriers (Chl & quinones).

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Chapter 18

12

Takusagawas Note

6. Electrons in PSI flow two possible pathways

1. to NADP+. This is noncyclic pathway.
- The electrons are transferred from PSI to the [2Fe-2S] containing soluble ferredoxin (Fd) in
stroma, and to the FAD-containing Fd-NADP+ reductase which reduces the NADP+ to
NADPH (see Fig. 22-15).
2. back to Cyt b6-f complex. This is a cyclic pathway (see Fig. 22-16).
- No NADPH formation, but ATP is produced since H+ is pumped from stroma to thylakoid at
Cyt b6-f. Bacteria use this pathway, thus do not produce NADPH.
PSI and PSII occupy different parts of the thylakoid membrane
- There are three characteristic features:
1. PSI occurs mainly in the unstacked stroma lamella.
2. PSII is located almost exclusively between the closely stacked grana.
3. Cyt b6-f is uniformly distributed.

This distribution allows chloroplasts to respond to different types of light (full and shady
sun).

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Takusagawas Note

13

Chapter 18

Full sun --- more shorter light (higher energy)

- PSII (P680) absorbs light more than PSI (P700).
- Thus PSI cannot handle electrons from PSII.
- Therefore, plastoquinones are in a reduced state (PQH2).
- The reduced PQH2 activates a protein kinase (PK) which phosphorylates the specific
threonine (Thr) of light-harvesting complex (LHC).
- The phosphorylated P-LHC migrates to unstacked region of thylakoid membrane where it
binds to PSI and funnels the incident light to PSI.
- Thus, PSI is activated in order to continue synthesis of NADPH and ATP.

PK (inactive)
PQH2
LHC
PK (active)
bind
P-LHC PSI (more active)

Shady sun --- more longer light (lower energy)

- PSI takes electrons faster than PSII produces them since lower energy light reaches less to
PSII.
- Thus, plastoquinones are mostly in oxidized forms (PQ).
- Therefore, LHCs are dephosphorylated, and migrate to PSII.
- Thus, the incident light is funneled to PSIIs, and thus PSIIs are activated.

PQH2

LHC
h

protein kinase (PK)

dephosphorylation

PSII

PQ

PSI

Full sun

Shady sun
-

LHC
h

The other reason is that PSII does not act as LHC for PSI.

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14

Chapter 18

Takusagawas Note

D. Photophosphorylation (ATP synthesis)

- In mitochondria, pH and are used to produce ATP.
- The free energy across the membrane is: G = -2.3RT pH + ZAF
- In chloroplast, only pH is used (pH =3.5 pH unit).
- The free energy across the membrane is: G = -2.3RT pH
- Stroma is basic (low [H+] or high pH) and thylakoid is acidic (high [H+] or low pH).
- No electropotential ( = 0) across the Chl membrane because the Chl membrane is
permeable to Mg2+ (out) and Cl- (in).
- When H+s are transported from stroma to thylakoid lumen, Mg2+s are oppositely
transported. Thus, maintains neutral.

H+
(stroma side)
Mg2+ (thylakoid side)
Cl
ATP productions in noncyclic and cyclic pathways are slightly different
- Noncyclic pathway:
- 12H+ (4 from 2H2O and 8 from 4PQH2 in Cyt b6-f) are transported by one O2
production, 12H+/O2 or 12H+/(8 absorbed photons).
- 3H+ produce one ATP (~ 1 ATP/ 3H+), thus, 4ATP/O2 or 0.5 ATP/ absorbed photon.
- Each NADPH produced has the free energy to produce 3ATP.
- Thus if the NADPH oxidation is taken into account, 10ATP can be produced by 8
absorbed photons (4ATP by proton gradient and 6ATP by 2NADPH oxidation) or 1.25
ATP per absorbed photon.

Cyclic pathway:
- ~4 additional protons are transported to the thylakoid lumen by the electron from PSI to
Cyt b6-f complex. The total H+ transported to thylakoid is 16 (= 12 + 4).
- Or, 16H+/8 absorbed photons.
- Thus, 16/(8 3) = 2/3 ATP per absorbed photon.

Noncyclic
Cyclic

No. H+
12
16

No. ATP
4
5 and 1/3

No. NADPH
2
0

Note: one photon (h) = one electron (e-)

14

ATP/e0.5 (1.25)
2/3

No. e8
8

15

Chapter 18

Takusagawas Note

Cartoon representation of light reactions

- Let us imagine that PSII and PSI are quite similar to a windmill.
1. Four photons hit the propeller of electron pump in PSII and rotate it several times.
2. The electron pump in PSII sucks 4 electrons from 2H2O molecules so that the water
molecules become O2 and 4H+.
3. The sucked electrons are sent to the Cyt b6-f motor which rotates the proton pump.
4. The proton pump transports 8H+ from stroma to thylakoid.
5. The electron voltage is reduced since it is used to rotate the motor of proton pump.
6. Four photons hit the propeller of electron pump in PSI and rotate it several times.
7. The electron pump in PSI sucks up electrons from the low voltage to the high voltage.
8a. The activated 4 electrons are used to reduce 2NADP+ to NADPH.
8b. In the cyclic pathway, the activated 4 electrons are sent to the Cyt b6-f motor which rotates
the proton pump and transports 4H+ from stroma to thylakoid.
9. 4H+ in (2), 8H+ in (4) and 4H+ in (8b if the cyclic pathway) are moved into the proton
reservoir of ATP synthesizer.
10. 12H+ or 16H+ (if the cyclic pathway) flow the narrow channel into the ATP synthesizer and
rotate the synthesizers propeller.
11. The ATP synthesizer phosphorylates 4ADP to produce 4ATP. For the cyclic pathway, 5 and
1/3 ATP are produced.
2NADP

2NADPH

cyclic pathway
4h

Stroma

4h
8H+

4e

4ADP

+
4ATP 12H

ATP
synthesizer

Motor

4e- Electron
pump
(PSII)

Electron
pump
(PSI)
+

12H+

8H
2H2O

O2 + 4H

Proton
pump
(Cyt b6-f)

15

Thylakoid lumen

Chapter 18
3.
-

16

Takusagawas Note

DARK REACTION
It takes place in stroma, and no light is required.
It is called Calvin cycle.
It synthesizes CH2O from CO2 and H2O using pentose phosphate enzymes and other enzymes.
The most important other enzyme is ribulose-bisphosphate carboxylase (Rubisco) which
catalyzes carboxylation to ribulose-1,5-bisphosphate (RuBP).
Overall carboxylation reaction is exergonic (G = -35.1 kJ/mol), which is driven by the
cleavage of the -keto acid intermediate to yield an additional resonance-stabilized
carboxylate group.

Evidence for the above mechanism of ribulose carboxylation.

1. H on C3 of RuBP exchanges with solvent (D2O or T2O), indicating that the hydrogen is
acidic.
2. C2 and C3 oxygen atoms remain attached to their respective C atoms, which eliminates
mechanisms involving a covalent adduct such as Schiff base between RuBP and the lysine
(Lys) residue of enzyme.
3. Carboxylated product, -keto acid
(C1-COO-) can be trapped by NaBH4.
But no -keto is detected.
4. Transition analogue, 2carboxyarabinitol-1-phosphate (CA1P)
binds tightly to the enzyme and inhibits
the enzyme.
16

Transition state analogues

17

Chapter 18

Calvin cycle

Rubisco

17

Takusagawas Note
Products of light reaction

Chapter 18

18

Takusagawas Note

Major route of Calvin cycle

- is to produce the nucleotide-glucose (XDP-G):
Ru5P ATP
RuBP CO
2 2(3PG) ATP
2(BPG) NADPH
2(GAP)
GAP
XTP
DHAP
FBP
G6P
G1P
XDP - G
GAP
where XDP = UDP, ADP, GDP or CDP.

Products from XDP-G are:

Starch XDP-G F6P sucrose-P sucrose

- Others are recovery pathways to re-produce Ru5P.

Calvin cycle can be two stage reactions
1. Stage-1: Production of GAP
3Ru5P + 9ATP + 3CO2 + 6NADPH + 6H+ 6GAP + 6NADP+ + 9ADP + 6Pi
2. Stage-2: Recovery
5GAP 3Ru5P + 2Pi
One GAP goes product
- Overall reaction:

+
3CO2 + 9ATP + 6NADPH + 6H GAP + 6NADP+ + 9ADP + 8Pi
(CO2 + 3ATP + 2NADPH + 2H+ 13 GAP + 2NADP+ + 3ADP + 8/3Pi)
B. Control of dark reaction
- Major site of control is Rubisco.
- During the day, light and dark reactions are satisfactory carried out, but at night, plants must
use their nutritional reserves to generate their required ATP and NADPH.
- Stroma contains enzymes of glycolysis, oxidative phosphorylation and pentose phosphate
pathway as well as Calvin cycle enzymes.
- Thus, it is possible to just waste ATP and NADPH by futile cycle, i.e., production of
carbohydrates using ATP and NADPH by dark reaction, and reproduction of ATP and
NADPH using the carbohydrates by glycolysis, citric acid cycle and oxidative
phosphorylation & pentose phosphate pathway.
- Therefore, the dark reaction must be control by light sensitive manner.
Calvin cycle is activated by light
1. Under light, pH in stroma increases (H+s are pumped from stroma to thylakoid).
- The pH of stroma in night and day are 7.0 and 8.0, respectively.
- The optimum pH of Rubisco is 8.0, i.e., thus Rubisco is active only daytime.
2. When H+s are transported to thylakoid, Mg2+s are transported to stroma (efflux of Mg2+ to
stroma) in order to balance the electric charges. Thus, the [Mg2+] in stroma increases.
- Rubisco is activated by Mg2+.
3. Rubisco is allosterically activated by NADPH produced by light reaction.
- At low [NADPH] (i.e., at night time), Rubisco is less active.
4. The potent inhibitor and transition analog of Rubisco, 2-carboxyarabinitol-1-phosphate
(CA1P) is only synthesized in the dark.
- Thus Rubisco is inhibited by CA1P in night.

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Chapter 18

19

Takusagawas Note

Second regulatory system (other than Rubisco regulation)---Mainly day time regulation
- In recovery stage, two phosphatases are activated by increased pH, Mg2+ and NADPH.
1. Fructose bisphosphatase, FBPase at step-7.
2. Sedoheptulose bisphosphatase, SBPase at step-10.
- Actually, FBPase and SBPase are activated by the reduced ferredoxin (Fd) via thioredoxin
reductase.
- The reduced Fd also inhibits PFK.
- Thus, glycolysis is inhibited and gluconeogenesis is stimulated in day time.

Inhibits PFK

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Chapter 18

20

Takusagawas Note

Photorespiration and the C4 cycle

- Rubisco can use O2 instead of CO2, since the KM values of [O2] and [CO2] in plants are
nearly the same.
- When Rubisco uses O2, its process is called photorespiration.
Photorespiration

Rubisco
3PG + 2-phosphoglycolate (C2)
RuBP + O2

The proposed reaction mechanism:

Carboxylation vs. oxygenation

- Carboxylation: RuBP + CO2 2(3PG)
- Oxygenation:
RuBP + O2 3PG + C2 (2-phosphoglycolate)

NADH + ATP

CO2

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Chapter 18

21

Takusagawas Note

Photorespiration is taken place in three organelles (Chloroplast, Peroxisome and

Mitochondrion)
The 2-phosphoglycolate produced by RuBP
oxygenation is dephosphorylated, and is
moved into a peroxisome (glyoxisome).
At there, glycolate is converted to glycine
(Gly) by receiving NH3.
The Gly is transported into a mitochondrion.
At there, two Gly are condensed into serine
(Ser) and CO2.
The Ser is re-transported into a peroxisome.
At there, the Ser is deaminated and becomes
glycerate.
The glycerate is phosphorylated by ATP in
cytosol, and becomes 3PG.
The 3PG is re-transported into chloroplast,
and enters the Calvin cycle.

The overall photorespiration is:

2RuBP + O2 2(3PG) + Ser + CO2 then
Ser + 3PG + NADPH + ATP
RuBP + CO2 + NADP+ + ADP

The overall processes are:

RuBP + O2 + NADPH + ATP
3PG + 2CO2 + NADP+ + ADP

Thus, photorespiration is detrimental

wasteful process.

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Chapter 18

22

Takusagawas Note

Some plants have a way to partially overcome O2 reaction

- This process is called C4 cycle.
- Photosynthesis takes place in bundle sheath cells where Calvin cycle occurs.
- In C4 plants, bundle sheath cells are covered by mesophyll cells which do not contain
Rubisco. Thus, the photorespiration does not occur in mesophyll cells.
- In mesophyll cells,
- Pyruvate is converted to PEP by consumption of two ATP.
- CO2 is fixed on PEP to produce oxaloacetate (C4 molecule).
- Reduced oxaloacetate, malate is transported to bundle sheath cells.
- In bundle sheath cells,
- Then the malate is reoxidized to pyruvate and CO2.
- The CO2 is entered into Calvin cycle.

No Rubisco

C4 plants require five ATP to fix one CO2 in their photosynthesis (C4 cycle + Calvin cycle),
where as C3 plants use three ATP to fix one CO2.
Note:
C3 plants initially fix CO2 in the form of three-carbon acids, such as 3PG.
C4 plants initially fix CO2 in the form of four-carbon acids, such as oxaloacetate.
C4 plants have advantage in warm climate, since the stomata closed much of time to avoid
H2O loss.
These plants absorb CO2 at night and store it as malate, and the stored CO2 is used at day
time.
Mesophyll cells also prevent from O2 to access to the bundle-sheath cells.

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Chapter 18

23

Takusagawas Note

CAM plants (Crassulacean acid metabolism)

- C4 plants separate CO2 fixation (Calvin cycle) by space, i.e., mesophyll cells.
- CAM plants do not have mesophyll cells, but their stomata are closed during day to prevent
loss of H2O. Thus, O2 cannot enter in day time.

At night (Rubisco activity is low), stomata is opened to absorb H2O and CO2. CO2 is stored
in PEP as malate as C4 plants.
CO2 + PEP OAA Malate (stored)

At daytime, CO2 is regenerated from the stored malate, and enters into Calvin cycle to
produce CH2O.
Calvin cycle
Malate CO2 + PEP and CO2 + RuBP 3PG

Thus, CAM plants separate CO2 fixation (Calvin cycle) in time (day and night).

23