Transcript Photosynthesis

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
Aerobic respiration of glucose is the most
basic means for cells to acquire energy
C6H12O6(s) + 6O2(g)  6CO2(g)+ 6H2O(l) + energy
Energy in presence of oxygen: ~38 ATP
Photosynthesis is essentially the
respiration reaction in reverse
6CO2(g)+ 6H2O(l) + hν  C6H12O6(s) + 6O2(g)
This is still a redox reaction
LE 10-3
Leaf cross section
Vein
Mesophyll
Stomata
CO2 O2
Mesophyll cell
Chloroplast
5 µm
Outer
membrane
Thylakoid
Thylakoid
Stroma Granum
space
Intermembrane
space
Inner
membrane
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
Light
LIGHT
REACTIONS
Chloroplast
LE 10-5_2
H2O
Light’s Energy generates
ATP and electrons
Light
LIGHT
REACTIONS
ATP
NADPH
Chloroplast
O2
LE 10-5_3
Using the ATP for energy, the
electrons link CO2 molecules
together to form glucose
H2O
CO2
Light
NADP+
ADP
+ Pi
LIGHT
REACTIONS
CALVIN
CYCLE
ATP
NADPH
Chloroplast
O2
[CH2O]
(sugar)
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
Visible Wavelengths
(Invisible)
Ultraviolet
UV
300nm
800nm
400nm
500nm
600nm
(Invisible)
Infrared IR
700nm
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
Slit moves to
pass light
of selected
wavelength
Green
light
100
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
0
Slit moves to
pass light
of selected
wavelength
Blue
light
100
The low transmittance
(high absorption)
reading indicates that
chlorophyll absorbs
most blue light.
Absorption of light by
chloroplast pigments
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
The Fluorescence Process
Stokes shift
Absorbance
Emission
Wavelength (nm)
1.
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
A Photosystem: A Reaction
Center Associated with LightHarvesting 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
H2O
CO2
Light
NADP+
ADP
CALVIN
CYCLE
LIGHT
REACTIONS
ATP
NADPH
O2
[CH2O] (sugar)
Primary
acceptor
Energy of electrons
e–
Light
P680
Photosystem II
(PS II)
LE 10-13_2
H2O
CO2
Light
NADP+
ADP
CALVIN
LIGHT
CYCLE
REACTIONS
ATP
NADPH
Photosystem II
splits water
O2
Primary
acceptor
Energy of electrons
Water is
oxidized
2H2O  4H+
+O2
[CH2O] (sugar)
2
H+
1/ 2
+
O2
Light
H2O
e–
e–
e–
P680
Photosystem II
(PS II)
LE 10-13_3
H2O
CO2
Light
NADP+
ADP
CALVIN
CYCLE
LIGHT
REACTIONS
ATP
NADPH
O2
[CH2O] (sugar)
Primary
acceptor
Energy of electrons
Pq
2 H+
+
1/ 2 O 2
Light
H2O
e–
Cytochrome
complex
Pc
e–
e–
P680
ATP
Photosystem II
(PS II)
LE 10-13_4
H2O
CO2
Light
NADP+
ADP
CALVIN
CYCLE
LIGHT
REACTIONS
ATP
NADPH
O2
[CH2O] (sugar)
Primary
acceptor
Primary
acceptor
e–
Energy of electrons
Pq
2
H+
1/ 2
+
O2
Light
H2O
e–
Cytochrome
complex
Pc
e–
e–
P700
P680
Light
ATP
Photosystem II
(PS II)
Photosystem I
(PS I)
LE 10-13_5
H2 O
CO2
Light
NADP+
ADP
CALVIN
CYCLE
LIGHT
REACTIONS
ATP
NADPH
O2
[CH2O] (sugar)
Primary
acceptor
Primary
acceptor
e–
Pq
Energy of electrons
2
H+
e–
H2O
Cytochrome
complex
+
1/2 O2
Light
Fd
e–
e–
NADP+
reductase
Pc
e–
e–
NADPH
+ H+
P700
P680
Light
ATP
Photosystem II
(PS II)
NADP+
+ 2 H+
Photosystem I
(PS I)
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
Which of these chromatograms shows purification?
Can we get the recipe for Coke from this?
B
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 Process
Stokes shift
Absorbance
Emission
Wavelength (nm)
1.
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
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