photosynthesis. what is this molecule? what is its function? how does it work?
TRANSCRIPT
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Photosynthesis
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What is this molecule?
• What is its function?• How does it work?
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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
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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
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6CO2(g)+ 6H2O(l) + hν C6H12O6(s) + 6O2(g)
This is still a redox reaction
Photosynthesis is essentially the respiration reaction in reverse
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LE 10-3
Leaf cross sectionVein
Mesophyll
Stomata CO2O2
Mesophyll cellChloroplast
5 µm
Outermembrane
Intermembranespace
Innermembrane
Thylakoidspace
Thylakoid
GranumStroma
1 µm
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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…
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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
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Where does the oxygen come from, water or CO2?
6CO2(g)+ 6H2O(l) + hν C6H12O6(s) + 6O2(g)
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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
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Water is split using the sun’s energy
H2O
LIGHTREACTIONS
Chloroplast
Light
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LE 10-5_2
H2O
LIGHTREACTIONS
Chloroplast
Light
ATP
NADPH
O2
Light’s Energy generates ATP and electrons
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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
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Light energy: E = h ν = hc/λ
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The electromagnetic spectrum
• Visible light is only a small subset of the electro-magnetic spectrum
• 400-700nm• Short wavelengths~
higher energy
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Light can excite electrons in atoms
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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
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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
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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
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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
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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
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Other pigments absorb different wavelengths
Different pigments can cooperate to transfer energy
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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Chromatography is like a race…
• The winner has the shoes that don’t stick to the track.
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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?
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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?
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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
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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
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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
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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