Lab 7: The Light Reactions of Photosynthesis

1. Lab 7: The Light Reactions of Photosynthesis

2. Purpose of the lab exercises: • Study the Hill Reaction and the effects of DCMU on electron transport • Determine absorption spectrum of chlorophyll • Observe fluorescence in chlorophyll

3. Properties of Light • Source of energy • wave and particle (photons) • Wavelength of light: Peak to Peak • Different wavelengths have different characteristics and energies  (wavelength)

4. The Electromagnetic Spectrum Long wavelengths have lower energies Short wavelengths have high energies Visible portion between 380 and 750 nm Different wavelengths = different colors.

5. Photosynthesis Today, you will examine the Hill Reaction The chemical equation for photosynthesis is: 6CO2 + 6 H2O + ENERGY C6H12O6 + 6O2

6. Photosynthesis • 2)The Dark Reactions • Calvin Cycle Combines H2O and CO2 to produce sugars in stroma • Two sets of reactions: (1) The light reactions • Light energy trapped by chlorophyll • (NADPH) and (ATP) are formed in thylakoid membranes

7. Light Reactions of Photosynthesis • Complexes embedded in thylakoid membrane Organized cluster of chlorophyll and proteins • Harvest light energy, resonance transfer • Reaction centers = chlorophyll a + primary electron acceptor • Two Photosystems: PSII and PSI • Contain chlorophyll a in reaction center • PSII chlorophyll a is P680 (Absorbs 680) • PSI chlorophyll a is P700 (Absorbs 700)

8. Light Reactions of Photosynthesis • Primary electron acceptors • associated w/ chlorophyll a of reaction center • traps high-energy electrons (excited) • prevent return to ground state.

9. Light Reactions of Photosynthesis Photosystem II (P680) Electrons lost to primary electron acceptor • Splitting of water • Each water molecule: provides 2 electrons • An atom of oxygen • Two atoms of oxygen form O2 How are they replaced? What happens to electrons at the primary electron acceptor?

10. Light Reactions of Photosynthesis Electron Transport Chain Plastoquinone (Pq) Complex of two cytochromes Plastocyanin (Pc) Lose energy Lower energy level Produce ATP Electrons move from PS II to PS I As electrons move through electron transport chain

11. Light Reactions of Photosynthesis • Hill Reaction • Named after Robin Hill • Chloroplast preparations can split water

12. Study of Hill Reaction: DCPIP DCPIP • Colorimetric indicator (DCPIP) • Intercepts electrons in electron transport chain • Between Pq and cytochrome complex • Reduced (gains electrons)

13. Study of Hill Reaction: DCPIP • As DCPIP becomes reduced, gradually turns from blue to colorless • Over the 30s intervals, drop in absorbance and readings in spectrophotometer

14. Study of Hill Reaction: DCMU DCMU DCPIP • DCMUinhibitor of electron transport • Blocks passage of electrons from primary acceptor of PS II - plastoquinone • Prevents DCPIP from being reduced • Degree of inhibition depends on concentration

15. Study of Hill Reaction: DCMU High concentrations of DCMU, electrons are almost completely blocked from passing to Pq very little reduction of DCPIP, little change in spec readings • Lower concentrations of DCMU, electrons are only moderately inhibited from passing to Pq, • DCPIP continues to be reduced

16. Interaction of Light with Matter Light can be reflected transmitted absorbed Color of objects due to reflected or transmittedlightChlorophylls absorb red and blue light Reflects and transmits green light.

17. Pigments: Chlorophyll • Pigments Absorb visible light • Chlorophyll a and b: • Two primary pigments in photosynthesis • Differ slightly in chemical structure Chlorophyll molecule

18. Absorption Spectrum Graph of light absorbence vs. wavelength Today you will create your own absorbance spectrum using isolated chlorophyll.

19. Fluorescence! Isolated chlorophyll molecules Fluorescence in Isolated Chlorophyll But what happens if chlorophyll is isolated from the intact structure of chloroplast, and then illuminated with light?

20. Fluorescence in Isolated Chlorophyll • Electrons still boosted to higher energy levels • No electron acceptor • They quickly drop back down to ground state • Energy released as light and heat. Why does it fluoresce red?

21. Fluorescence in Isolated Chlorophyll • Looking at the spectrum, red is associated with lowerenergy • In returning back to ground state, some energy is lost as heat • Energy of fluorescing light is less than that which illuminated it • Longer wavelengths have lower energy

22. Fluorescence in Isolated Chlorophyll • Today you will illuminate isolated chlorophyll with different wavelengths (colors) from the visible portions of the spectrum • Observe the INTENSITY and red fluorescence. • Must use absorption spectrum. • Would you expect the intensity of fluorescence to be high, moderate, or low if chlorophyll was exposed to blue light?

23. Fluorescence in Isolated Chlorophyll Chlorophyll absorbs most of the blue light Electrons boosted to higher orbitals fall back to ground state: “ High Fluorescence” • Little energy from green light is absorbed • Most is reflected or transmitted • Few electrons boosted What about green light (@550 nm) on isolated chlorophyll, the intensity of fluorescence will be low, moderate, or high? Low!

24. Experiment 1: The Hill Reaction • DCPIP (e- acceptor; blue to non-blue) • DCMU (e- inhibitor) • Experiment 2: Determining the Absorption Spectrum of Chlorophyll • Spinach leaf pigment extract • Experiment 3: Fluorescence of Chlorophyll Extract in Acetone

25. Time to work!