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

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

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

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

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

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

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)

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

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?

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

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

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

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

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

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

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.

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

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

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

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?

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

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?

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 (@550 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!

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

Time to work!