Lab 7: The Light Reactions of Photosynthesis

• Study the Hill Reaction and the effects of DCMU on electron transport

• Determine absorption spectrum of chlorophyll

• Observe fluorescence in chlorophyll

Purpose of the lab exercises:

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

Short wavelengths have high energies

Long wavelengths have lower energies

Visible portion between 380 and 750 nm Different wavelengths = different colors.

Today, you will examine the Hill Reaction

The chemical equation for photosynthesis is:

Photosynthesis

6CO2 + 6 H2O + ENERGY C6H12O6 + 6O2

2)The Dark Reactions

• Calvin CycleCombines H2O and CO2 to produce sugars in stroma

PhotosynthesisTwo sets of reactions: (1) The light reactions • Light energy trapped by

chlorophyll • (NADPH) and (ATP) are formed

in thylakoid membranes

Light Reactions of PhotosynthesisComplexes 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 PSIContain 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?

Electrons move from PS II to PS I

Light Reactions of Photosynthesis

Lose energy Lower energy level Produce ATP

Plastoquinone (Pq) Complex of two cytochromes Plastocyanin (Pc)

Electron Transport Chain

As electrons move through electron transport chain

Hill Reaction

• Named after Robin Hill

• Chloroplast preparations can split water

Light Reactions of Photosynthesis

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

Study of Hill Reaction: DCPIP

Reduction of DCPIP by Isolated Chloroplast

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 50 100 150 200 250 300 350

Time (seconds) exposed to light

A62

0

Study of Hill Reaction: DCMU

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

High concentrations of DCMU, electrons are almost completely blocked from passing to Pq very little reduction of DCPIP, little change in spec readings

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 50 100 150 200 250 300 350

Time (seconds) exposed to light

A62

0

High [DCMU]

No DCMU

Study of Hill Reaction: DCMU

• Lower concentrations of DCMU, electrons are only moderately inhibited from passing to Pq,

• DCPIP continues to be reduced

Interaction of Light with MatterLight 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.

But what happens if chlorophyll is isolated from the intact structure of chloroplast, and then illuminated with light?

Isolated chlorophyll molecules

Fluorescence in Isolated Chlorophyll

Fluorescence!

• Electrons still boosted to higher energy levels

• No electron acceptor

• They quickly drop back down to ground state

• Energy released as light and heat.

Fluorescence in Isolated Chlorophyll

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

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

• Little energy from green light is absorbed• Most is reflected or transmitted • Few electrons boosted

Fluorescence in Isolated ChlorophyllChlorophyll 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?

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!