Photosynthesis

Schedule

• 9-910: Collect lab reports

• 910-940- prepare chromatography of pigments

• 940-1000- Run chromatography

• 1000-1015- evaluate chromatography

• 1015- end- review

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, NADPH are 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

Oxygen is a waste product

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 via flourescence

The Fluorescence Process1. excitation - energy is provided by an

external source ( sun, 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

The Calvin Cycle

C6H12O6(s) + 6O2(g) 6CO2(g)+ 6H2O(l) + energy

ΔG= +685kcal/mole

18ATP + 18 H2O 18ADP + 18Pi

12NADPH12NAD+ + 12H+ + 24e-

What is the purpose of cyclic electron flow?

RubisCO

• AKA Ribulose Bisphosphate Carboxylase Oxidase

• The carbon fixing enzyme

• The most common enzyme on the planet

• Adds 3CO2’s to 3 RuBP’s at a time

LE 10-18_1

[CH2O] (sugar)O2

NADPH

ATP

ADP

NADP+

CO2H2O

LIGHTREACTIONS

CALVINCYCLE

LightInput

3

CO2

(Entering oneat a time)

Rubisco

3 P P

Short-livedintermediate

Phase 1: Carbon fixation

6 P

3-Phosphoglycerate6 ATP

6 ADP

CALVINCYCLE

3 P P

Ribulose bisphosphate(RuBP)

Step 1: Carbon Fixation

LE 10-18_2

[CH2O] (sugar)O2

NADPH

ATP

ADP

NADP+

CO2H2O

LIGHTREACTIONS

CALVINCYCLE

Light Input

CO2

(Entering oneat a time)

Rubisco

3 P P

Short-livedintermediate

Phase 1: Carbon fixation

6 P

3-Phosphoglycerate6 ATP

6 ADP

CALVINCYCLE

3

P P

Ribulose bisphosphate(RuBP)

3

6 NADP+

6

6 NADPH

P i

6 P

1,3-BisphosphoglycerateP

6 P

Glyceraldehyde-3-phosphate(G3P)

P1

G3P(a sugar)Output

Phase 2:Reduction

Glucose andother organiccompounds

Each lap generates 1 G3P

Radioactive CO2 allows tracking of these molecules (with liquid N2)

LE 10-16

MITOCHONDRIONSTRUCTURE

Intermembranespace

MembraneElectrontransport

chain

Mitochondrion Chloroplast

CHLOROPLASTSTRUCTURE

Thylakoidspace

Stroma

ATP

Matrix

ATPsynthase

Key

H+ Diffusion

ADP + P

H+

i

Higher [H+]

Lower [H+]

• The current model for the thylakoid membrane is based on studies in several laboratories

• Water is split by photosystem II on the side of the membrane facing the thylakoid space

• The diffusion of H+ from the thylakoid space back to the stroma powers ATP synthase

• ATP and NADPH are produced on the side facing the stroma, where the Calvin cycle takes place

Animation: Calvin Cycle

LE 10-17

STROMA(Low H+ concentration)

Light

Photosystem IICytochrome

complex

2 H+

Light

Photosystem I

NADP+

reductase

Fd

PcPq

H2O O2

+2 H+

1/2

2 H+

NADP+ + 2H+

+ H+NADPH

ToCalvincycle

THYLAKOID SPACE(High H+ concentration)

STROMA(Low H+ concentration)

Thylakoidmembrane ATP

synthase

ATP

ADP+P

H+i

[CH2O] (sugar)O2

NADPH

ATP

ADP

NADP+

CO2H2O

LIGHTREACTIONS

CALVINCYCLE

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

Spectral analysis of pigments- what do we expect?

• What colors should we see passing through the bottle of chlorophyll?

• What colors should the chlorophyll absorb?• Do we expect fluorescence? Where would we

see it?

Bottle of chlorophyll