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 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

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 Aerobic respiration of glucose is the most basic means for cells to acquire energy

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 Photosynthesis is essentially the respiration reaction in reverse

LE 10-3 Leaf cross section Vein Mesophyll Stomata CO 2 O2O2 Mesophyll cell Chloroplast 5 µm Outer membrane Intermembrane space Inner membrane Thylakoid space 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 CO 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

Water is split using the sun’s energy H2OH2O LIGHT REACTIONS Chloroplast Light

LE 10-5_2 H2OH2O LIGHT REACTIONS Chloroplast Light ATP NADPH O2O2 Light’s Energy generates ATP and electrons

LE 10-5_3 H2OH2O LIGHT REACTIONS Chloroplast Light ATP NADPH O2O2 NADP + CO 2 ADP P + i CALVIN CYCLE [CH 2 O] (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 nm 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 White light Refracting prism Chlorophyll solution Photoelectric tube Galvanometer The high transmittance (low absorption) reading indicates that chlorophyll absorbs very little green light. Green light Slit moves to pass light of selected wavelength 0 100

White light Refracting prism Chlorophyll solution Photoelectric tube The low transmittance (high absorption) reading indicates that chlorophyll absorbs most blue light. Blue light Slit moves to pass light of selected wavelength 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? Absorption of light by chloroplast pigments

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

The Fluorescence Process 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 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 Light P680 e–e– Photosystem II (PS II) Primary acceptor [CH 2 O] (sugar) NADPH ATP ADP CALVIN CYCLE LIGHT REACTIONS NADP + Light H2OH2O CO 2 Energy of electrons O2O2

LE 10-13_2 Light P680 e–e– Photosystem II (PS II) Primary acceptor [CH 2 O] (sugar) NADPH ATP ADP CALVIN CYCLE LIGHT REACTIONS NADP + Light H2OH2O CO 2 Energy of electrons O2O2 e–e– e–e– + 2 H + H2OH2O O2O2 1/21/2 Photosystem II splits water Water is oxidized 2H 2 O  4H + +O 2

LE 10-13_3 Light P680 e–e– Photosystem II (PS II) Primary acceptor [CH 2 O] (sugar) NADPH ATP ADP CALVIN CYCLE LIGHT REACTIONS NADP + Light H2OH2O CO 2 Energy of electrons O2O2 e–e– e–e– + 2 H + H2OH2O O2O2 1/21/2 Pq Cytochrome complex Electron transport chain Pc ATP

LE 10-13_4 Light P680 e–e– Photosystem II (PS II) Primary acceptor [CH 2 O] (sugar) NADPH ATP ADP CALVIN CYCLE LIGHT REACTIONS NADP + Light H2OH2O CO 2 Energy of electrons O2O2 e–e– e–e– + 2 H + H2OH2O O2O2 1/21/2 Pq Cytochrome complex Electron transport chain Pc ATP P700 e–e– Primary acceptor Photosystem I (PS I) Light

LE 10-13_5 Light P680 e–e– Photosystem II (PS II) Primary acceptor [CH 2 O] (sugar) NADPH ATP ADP CALVIN CYCLE LIGHT REACTIONS NADP + Light H2OH2O CO 2 Energy of electrons O2O2 e–e– e–e– + 2 H + H2OH2O O2O2 1/21/2 Pq Cytochrome complex Electron transport chain Pc ATP P700 e–e– Primary acceptor Photosystem I (PS I) e–e– e–e– Electron Transport chain NADP + reductase Fd NADP + NADPH + H 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 Process 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 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