1. Light Reactions of Photosynthesis
Lab 7: The Light Reactions of Photosynthesis
Rationale:
Today you will investigate some of the fundamental principles underlying photosynthesis, one of the most important physical/chemical processes responsible for life on earth.
Biology 171L, Fall 2000

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

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

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

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
How are they replaced?
Splitting of water
Each water molecule: provides 2 electrons
An atom of oxygen
Two atoms of oxygen form O2
What happens to electrons at the primary electron acceptor?

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

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

12. Study of Hill Reaction: 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. Light Reactions of Photosynthesis
Study of Hill Reaction: DCMU
DCMU
DCPIP
DCMU inhibitor 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
Biology 171L, Fall 2000

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

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 lower energy
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”
What about green light nm) on isolated chlorophyll, the intensity of fluorescence will be low, moderate, or high?
Little energy from green light is absorbed
Most is reflected or transmitted
Few electrons boosted
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!