Photosynthesis
What is this molecule?
• 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
• CO2 is taken in from the air
• Evaporation of water from leaves brings up water from roots
• All earth’s O2 is a waste product from plants
C6H12O6(s) + 6O2(g) 6CO2(g)+ 6H2O(l) + energy
Energy in presence of oxygen: ~38 ATP
Aerobic respiration of glucose is the most basic means for cells to acquire energy
6CO2(g)+ 6H2O(l) + hν C6H12O6(s) + 6O2(g)
This is still a redox reaction
Photosynthesis is essentially the respiration reaction in reverse
LE 10-3
Leaf cross sectionVein
Mesophyll
Stomata CO2O2
Mesophyll cellChloroplast
5 µm
Outermembrane
Intermembranespace
Innermembrane
Thylakoidspace
Thylakoid
GranumStroma
1 µm
Chloroplasts are the site of photosynthesis in plants
• Chloroplasts have their own DNA, and a double bilayer system as do mitochondria
• They were once independent living creatures…
Chloroplast structure
• 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
Where does the oxygen come from, water or CO2?
6CO2(g)+ 6H2O(l) + hν C6H12O6(s) + 6O2(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
Water is split using the sun’s energy
H2O
LIGHTREACTIONS
Chloroplast
Light
LE 10-5_2
H2O
LIGHTREACTIONS
Chloroplast
Light
ATP
NADPH
O2
Light’s Energy generates ATP and electrons
LE 10-5_3
H2O
LIGHTREACTIONS
Chloroplast
Light
ATP
NADPH
O2
NADP+
CO2
ADPP+ i
CALVINCYCLE
[CH2O](sugar)
Using the ATP for energy, the electrons link CO2 molecules together to form glucose
Light energy: E = h ν = hc/λ
The electromagnetic spectrum
• 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
The visible spectrum
• 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?
300nm 400nm 500nm 600nm 700nm 800nm
Visible Wavelengths
Spectrum of “White” Light
(Invisible) Ultraviolet UV
(Invisible) Infrared IR
Chlorophyll’s ability to absorb light can be measured using a spectrophotometer
Whitelight
Refractingprism
Chlorophyllsolution
Photoelectrictube
Galvanometer
The high transmittance (low absorption) reading indicates that chlorophyll absorbs very little green light.
Greenlight
Slit moves to pass light of selected wavelength
0 100
Whitelight
Refractingprism
Chlorophyllsolution
Photoelectrictube
The low transmittance (high absorption) reading indicates that chlorophyll absorbs most blue light.
Bluelight
Slit moves to pass light of selected wavelength
0 100
Chlorophyll does not absorb all light wavelengths equally
LE 10-9a
Chlorophyll a
Chlorophyll b
Carotenoids
Wavelength of light (nm)
Absorption spectra- will these be the same in vivo?
Ab
sorp
tio
n o
f lig
ht
by
chlo
rop
last
pig
men
ts
400 500 600 700
Other pigments absorb different wavelengths
Different pigments can cooperate to transfer energy
The Fluorescence Process1. excitation - energy is provided by an
external source (mercury lamp) and used to raise the energy state of a fluorochrome
2. excited state lifetime - fluorochrome undergoes conformational change that helps dissipate its energy
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
Stokes shift
Wavelength (nm)
Absorbance
Emission
A Photosystem: A Reaction Center Associated with Light-
Harvesting Complexes
• 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
LightP680
e–
Photosystem II(PS II)
Primaryacceptor
[CH2O] (sugar)
NADPH
ATP
ADP
CALVINCYCLE
LIGHTREACTIONS
NADP+
Light
H2O CO2
En
erg
y o
f el
ectr
on
sO2
LE 10-13_2
LightP680
e–
Photosystem II(PS II)
Primaryacceptor
[CH2O] (sugar)
NADPH
ATP
ADP
CALVINCYCLE
LIGHTREACTIONS
NADP+
Light
H2O CO2
En
erg
y o
f el
ectr
on
sO2
e–
e–
+2 H+
H2O
O21/2
Photosystem II splits water
Water is oxidized
2H2O 4H+ +O2
LE 10-13_3
LightP680
e–
Photosystem II(PS II)
Primaryacceptor
[CH2O] (sugar)
NADPH
ATP
ADP
CALVINCYCLE
LIGHTREACTIONS
NADP+
Light
H2O CO2
En
erg
y o
f el
ectr
on
sO2
e–
e–
+2 H+
H2O
O21/2
Pq
Cytochromecomplex
Electron transport chain
Pc
ATP
LE 10-13_4
LightP680
e–
Photosystem II(PS II)
Primaryacceptor
[CH2O] (sugar)
NADPH
ATP
ADP
CALVINCYCLE
LIGHTREACTIONS
NADP+
Light
H2O CO2
En
erg
y o
f el
ectr
on
s
O2
e–
e–
+2 H+
H2O
O21/2
Pq
Cytochromecomplex
Electron transport chain
Pc
ATP
P700
e–
Primaryacceptor
Photosystem I(PS I)
Light
LE 10-13_5
LightP680
e–
Photosystem II(PS II)
Primaryacceptor
[CH2O] (sugar)
NADPH
ATP
ADPCALVINCYCLE
LIGHTREACTIONS
NADP+
Light
H2O CO2E
ner
gy
of
elec
tro
ns
O2
e–
e–
+2 H+
H2O
O21/2
Pq
Cytochromecomplex
Electron transport chain
Pc
ATP
P700
e–
Primaryacceptor
Photosystem I(PS I)
e–e–
ElectronTransportchain
NADP+
reductase
Fd
NADP+
NADPH
+ H+
+ 2 H+
Light
Today’s lab
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)
Part a: Chromatography of plant leaf pigments
• 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
Chromatography is like a race…
• The winner has the shoes that don’t stick to the track.
Chromatography can purify a mixture
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?
Analysis of chemicals using a Chromatogram
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
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!
Affinity chromatography column
Part b: Spectral analysis of pigments
• Spectrometer- Separates out light for analysis at different wavelenths
• Place photopigment sample in the light pathway- measure absorption of each wavelength
The Fluorescence Process1. excitation - energy is provided by an
external source (mercury lamp) and used to raise the energy state of a fluorochrome
2. excited state lifetime - fluorochrome undergoes conformational change that helps dissipate its energy
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
Stokes shift
Wavelength (nm)
Absorbance
Emission
Green Fluorescent Protein
• 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
40 Å
30 Å