• What is its function?
• How does it work?

Photosynthesis is the manufacture of food using energy from the sun
• Leaves are solar panels for plants
• CO
2 is taken in from the air
• Evaporation of water from leaves brings up water from roots
• All earth’s O
2 is a waste product from plants

Aerobic respiration of glucose is the most basic means for cells to acquire energy
C
6
H
12
O
6(s)
+ 6O
2(g)
 6CO
2(g)
+ 6H
2
O
(l)
+ energy
Energy in presence of oxygen: ~38 ATP

Photosynthesis is essentially the respiration reaction in reverse
6CO
2(g)
+ 6H
2
O
(l)
+ h ν  C
6
H
12
O
6(s)
+ 6O
2(g)
This is still a redox reaction

LE 10-3
Mesophyll
Leaf cross section
Vein
Chloroplast
Stomata CO
2
O
2
Mesophyll cell
Stroma
Thylakoid
Granum
Thylakoid space
Outer membrane
Intermembrane space
Inner membrane
5 µm
1 µm

• Chloroplasts have their own DNA, and a double bilayer system as do mitochondria
• They were once independent living creatures…

• Double bilayer
• Grana made of
Thylakoid membranes
• Stroma is the liquid in which the grana sit
• Photosynthesis occurs in chloroplasts in two stages- light reactions and dark

2
6CO
2(g)
+ 6H
2
O
(l)
+ h ν  C
6
H
12
O
6(s)
+ 6O
2(g)

Photosynthesis is actually 2 reactions:
Light and Dark reactions
• Light-dependent reactions: Generate ATP
– Water is split
– ATP is formed
– O2 is evolved
• Light-independent reactions-:CO2  Glucose
– Carbon is fixed

Light
H
2
O
Water is split using the sun’s energy
LIGHT
REACTIONS
Chloroplast

LE 10-5_2
H
2
O
Light’s Energy generates
ATP and electrons
Light
Chloroplast
LIGHT
REACTIONS
ATP
NADPH
O
2

LE 10-5_3
Light
Using the ATP for energy, the electrons link CO2 molecules together to form glucose
H
2
O
CO
2
NADP +
ADP
+ P i
LIGHT
REACTIONS
ATP
NADPH
CALVIN
CYCLE
Chloroplast
O
2
[CH
2
O]
(sugar)

• Visible light is only a small subset of the electro-magnetic spectrum
• 400-700nm
• Short wavelengths~ higher energy

Light can excite electrons in atoms

Chlorophyll is a light-absorbing pigment
• Electrons in double bonds absorb light energy easily
• 2 kinds: Chlorophyll a and b
• There are other light absorbing pigments
• Its absorption spectrum can be measured in vitro

(Invisible)
Ultraviolet
UV
Visible Wavelengths (Invisible)
Infrared IR
300nm 400nm 500nm 600nm 700nm
800nm Spectrum of “White” Light
• Which wavelengths are the shortest, and which are the longest?
• Which wavelengths have the highest energy, which have the lowest?
• Which do you think are ABSORBED by Chlorophyll?
• Which do you think are TRANSMITTED by Chlorophyll?

Chlorophyll’s ability to absorb light can be measured using a spectrophotometer
White light
Refracting prism
Chlorophyll solution
Photoelectric tube
Galvanometer
0 100
Slit moves to pass light of selected wavelength
Green light
The high transmittance
(low absorption) reading indicates that chlorophyll absorbs very little green light.

Chlorophyll does not absorb all light wavelengths equally
White light
Refracting prism
Chlorophyll solution
Photoelectric tube
Slit moves to pass light of selected wavelength
Blue light
0 100
The low transmittance
(high absorption) reading indicates that chlorophyll absorbs most blue light.

LE 10-9a
Chlorophyll a
Chlorophyll b
Carotenoids
400 500 600
Wavelength of light (nm)
700
Absorption spectra- will these be the same in vivo?

Other pigments absorb different wavelengths
Different pigments can cooperate to transfer energy

1.
excitation - energy is provided by an external source (mercury lamp) and used to raise the energy state of a fluorochrome
Stokes shift
2.
excited state lifetime - fluorochrome undergoes conformational change that helps dissipate its energy
Absorbance
Emission
3.
emission - the fluorochrome emits a photon of energy and generates fluorescence, at the same time returning to its ground state while emitting this energy as a photon of visible light; this shift is called the
Stokes shift
Wavelength (nm)

• A photosystem consists of a reaction center surrounded by light-harvesting complexes
• The light-harvesting complexes (pigment molecules bound to proteins) funnel the energy of photons to the reaction center

LE 10-13_1
Light
Light
H
2
O CO
2
NADP +
ADP
LIGHT
REACTIONS
ATP
NADPH
CALVIN
CYCLE
O
2
[CH
2
O] (sugar)
Primary acceptor e
–
P680
Photosystem II
(PS II)

LE 10-13_2
Photosystem II splits water
Water is oxidized
2H
2
O  4H +
+O
2
Light
H
2
O CO
2
NADP +
ADP
CALVIN
LIGHT
ATP
NADPH
O
2
[CH
2
O] (sugar)
Light
2 H +
1 /
2
+
O
2
H
2
O
Primary acceptor e
– e
– e
–
P680
Photosystem II
(PS II)

LE 10-13_3
Light
H
2
O CO
2
NADP +
ADP
LIGHT
REACTIONS
ATP
NADPH
CALVIN
CYCLE
O
2
[CH
2
O] (sugar)
Light
2 H +
1 /
2
+
O
2
H
2
O
Primary acceptor e
– e
– e
–
P680
Pq
Cytochrome complex
Pc
ATP
Photosystem II
(PS II)

LE 10-13_4
Light
H
2
O CO
2
NADP +
ADP
LIGHT
REACTIONS
ATP
NADPH
CALVIN
CYCLE
O
2
[CH
2
O] (sugar)
Light
2 H +
1 /
2
+
O
2
H
2
O
Primary acceptor e
– e
– e
–
P680
Pq
Cytochrome complex
Pc
Primary acceptor e
–
P700
ATP
Photosystem I
(PS I)
Photosystem II
(PS II)
Light

LE 10-13_5
Light
H
2
O CO
2
NADP +
ADP
LIGHT
REACTIONS
ATP
NADPH
CALVIN
CYCLE
O
2
[CH
2
O] (sugar)
1 /
2 H +
2
+
O
2
H
2
O
Primary acceptor e
– e
– e
–
Light
P680
Pq
Cytochrome complex
Pc
ATP
Primary acceptor e
–
P700
Fd e
– e
–
NADP + reductase
NADP +
+ 2 H +
NADPH
+ H +
Light
Photosystem I
(PS I)
Photosystem II
(PS II)

We will investigate photosynthetic pigment mixtures found in spinach leaves: a. Purify and isolate their constituents using
Chromatography b. Investigate their fluorescent properties using a spectroscope ( aka spectrometer )

• Chromatography: The separation of substances in a mixture by the different properties of the substances
• Always involves a “Stationary phase” (a solid) and a “mobile phase” (usually a liquid)
• Substances separated based on affinity for the respective phases
• A means of purification or analysis

• The winner has the shoes that don’t stick to the track.

A Column containing a solid phase
• Some constituents bind to the stationary phase better than others
• All substances are gradually washed through
• Which has most solid-phase affinity?
Most liquid-phase affinity?

Shows the results of a chromatographic separation
A B A B
Which of these chromatograms shows purification?
Can we get the recipe for Coke from this?

Large-scale purification using chromatography
Affinity chromatography column Biotech
• Drugs manufactured by bacteria can be purified from bacterial ingredients
• In affinity chromatography, the solid phase can be antibodies….
• …or the drugs can be antibodies…
• …or both!

• Spectrometer- Separates out light for analysis at different wavelenths
• Place photopigment sample in the light pathway- measure absorption of each wavelength

1.
excitation - energy is provided by an external source (mercury lamp) and used to raise the energy state of a fluorochrome
Stokes shift
2.
excited state lifetime - fluorochrome undergoes conformational change that helps dissipate its energy
Absorbance
Emission
3.
emission - the fluorochrome emits a photon of energy and generates fluorescence, at the same time returning to its ground state while emitting this energy as a photon of visible light; this shift is called the
Stokes shift
Wavelength (nm)

• discovered in 1960s by Dr. Frank
Johnson and colleagues
• closely related to jellyfish aequorin
• absorption max = 470nm
• emission max = 508nm
• 238 amino acids, 27kDa
• “ beta can ” conformation: 11 antiparallel beta sheets, 4 alpha helices, and a centered chromophore
• amino acid substitutions result in several variants, including YFP, BFP, and CFP
30 Å
40 Å