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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