Photosynthesis 1

Light Reactions and Photosynthetic Phosphorylation

Lecture 31

Key Concepts

• Overview of photosynthesis and carbon fixation

• Chlorophyll molecules convert light energy to redoxenergy

• The Z scheme of photosynthetic electron transport

• Oxidative phosphorylation and photosynthesis are related processes

How do photosynthetic reaction centers convert light energy into redox energy?

In what ways are photosynthesis and oxidative phosphorylation similar? Different?

Together, the photosynthetic electron transport system and Calvin cycle (carbon fixation) convert sunlight energy into chemical energy (ATP, NADPH, triose phosphate), and in the process, oxidize H2O to form O2.

The photosynthetic electron transport system is often referred to as the light reactions of photosynthesis, whereas, the Calvin cycle has been called the dark reactions.

However, the term "dark reactions" can be misleadingbecause the Calvin cycle is most active in the light when ATP and NADPH levels are high.

Oxygenic Photosynthesis

H2O CH2OCO2O2

Buchanon, et. al., Fig.12.2

Overview of Photosynthesis

Light excitation of Photosystems I and II results in oxygen evolution from the splitting H2O, and the generation of chemical energy in the form of ATP and NADPH. Plants use this chemical energy (ATP and NADPH) to convert CO2 into sugars via the Calvin cycle which takes place in the stroma.

Net reaction of photosynthesis and carbon fixation

Although written as a balanced reaction, O2 generation is the result of H2O oxidation, whereas, the CO2 is used to synthesize carbohydrate (CH2O) It takes 2 H2O to make an O2 and six CO2 molecules are required for the synthesis of each molecule of glucose, therefore:

∆Gº' for this reaction is +2868 kJ/mol!This is overcome by the energy potential stored in the products of photosynthetic electron transport, namely, ATP and NADPH.

H2O + CO2 ─(light energy)→ (CH2O) + O2

6 H2O + 6 CO2 ─(light energy)→ C6H12O6 + 6 O2

General equation for photosynthesis:2H2A (red. e- donor) + CO2

(CH2O) + H2O + 2A (ox. e- donor)

In “oxygenic PS” A= O

Diverse photosynthetic organisms use electron donors other than H2O for photosynthesis:

Green sulfur bacteria:light

2H2S + CO2 (CH2O) + H2O + 2S

Other PS bacteria:light

2 lactate + CO2 (CH2O) + H2O + 2 pyruvate

Joseph Priestly’s Experiment (200 yrs ago!)

Live plants and respiring animals could coexist in a closed system for a limited period of time as long as H2O and light were provided

It was later shown that chloroplasts contain light gathering pigments called chlorophyll that work with proteins to convert light energy into chemical energy and in the process release O2.

A modern Priestly experiment

This experiment did not actually work very well because CO2 levels built up inside the sealed environment and periodic CO2 removal was required.

Biosphere 2 outside of Tucson, built in the 1990s by a private company called Space Biospheres Ventures.

Metabolic pathway questions related to the photosynthetic electron transport system and

the Calvin cycle

1. What do the photosynthetic electron transport systemand Calvin cycle accomplish for the cell?

• The photosynthetic electron transport system converts light energy into redox energy which is used to generate ATP by chemiosmosis and reduce NADP+ to form NADPH.

• Calvin cycle enzymes use energy available from ATP and NADPH to reduce CO2 to form glyceraldehyde-3-P, a three carbon carbohydrate used to synthesize glucose.

• Photosynthetic cells use the carbohydrate produced by the Calvin cycle as a chemical energy source for mitochondrial respiration in the dark.

Photosynthetic organisms are autotrophs because they derive energy from light rather than from organic materials (as food).

Questions: Do plants use oxidative phosphorylation? Do plants have mitochondria?

2. What are the overall net reactions of photosynthetic electron transport system and the Calvin cycle?

Photosynthetic electron transport system

(production of ATP and O2):

2 H2O + 8 photons + 2 NADP+ + ~3 ADP + ~3 Pi →

O2 + 2 NADPH + ~3 ATP

Calvin cycle (six turns of cycle yields glucose):

6 CO2 + 12 NADPH + 18 ATP + 12 H2O →

Glucose + 12 NADP+ + 18 ADP + 18 Pi

3. What are the key enzymes in the photosynthetic electron transport system and the Calvin cycle?

Protein components of the photosynthetic electron transport system– three protein complexes are required for the oxidation of H2O and reduction of NADP+; photosystem II (P680 reaction center), cytochromeb6f (proton pump) and photosystem I (P700 reaction center).

Chloroplast ATP synthase – enzyme responsible for the process of photophosphorylation which converts proton-motive force into net ATP synthesis; this enzyme is very similar to mitochondrial ATP synthase.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) - is responsible for CO2 fixation in the first step of the Calvin cycle. Rubisco activity is maximal in the light when stromal pH is ~8 and Mg2+ levels are elevated due to proton pumping.

4. What are examples of the photosynthetic electron transport system and Calvin cycle in real life?

DCMU (dichlorophenyl dimethylurea) is a broad spectrum herbicide that functions by blocking electron flow through photosystem II and is used to reduce weeds in non-crop areas. Another herbicide, paraquat, prevents reduction of NADP+ by accepting electrons from intermediate reductantsin photosystem I.

Chloroplast Structure

StromaStroma

thylakoidsGranalthylakoids

Outer envelope

Inner envelope

Chloroplast Structure

Buchanon et al., Fig.12.1

Light energy is absorbed by numerous accessory pigments which can transfer the absorbed energy to nearby reaction centerscontaining specialized chlorphyll molecules.

These accessory pigments function as light harvesting antenna.

Chlorophyll (and Other Pigments)

Grab hold of those photons!

Chlorophylls are the primary light gathering pigments.

They have a heterocyclic ring system that constitutes an extended polyene structure, which typically has strong absorption in visible light.

Buchanon et al., Fig.12.4

Chlorophyll absorption spectra

Buchanon et al., Fig.12.6

Other Photosynthesis Pigments

Buchanon et al., Fig.12.6

Expression ofcarotenoidbiosyntheticgenes in E. coli

Buchanon et at., Fig.12.8

Light Energy:In photosynthesis, light energy is converted to chemical energy, therefore it is important to understand the energy content of light.

Light energy is expressed as: Einsteins = moles of photons

Light energy is wavelength dependent:E = hνh = Planck’s constant (6.626 x 10-34 J sec)ν = light frequency = c/λ (speed of light/wavelength)

How is the energy of light related to light wavelength?

Energy in the electromagnetic

spectrum

Buchanon et al., Fig. 19-36:

1) Light energy is wavelength dependent:

E = hn = c/λ

h = Planck’s constant (6.626 x 10-34 J sec)

n = light frequency = c/λ (speed of light/wavelength)

2) Standard Free Energy change of redox reactions:

∆G°’ = - nF·∆E°’ ∆E°’= E°’ acceptor - E°’ donor

∆G = RT·ln(C2/C1) + ZF·∆Ψ

∆G = 2.3 RT·∆pH + F·∆Ψ

3) Free Energy of PMF:

Chlorophyll energy levels

Buchanon et al., Fig.12.3

Organization of Photosynthetic pigments

• Light absorbing pigments are organized in functional arrays called Photosystems.

• “Light harvesting” or “antenna” pigment molecules are specialized to absorb light and transfer the energy to neighboring pigment molecules.

• “Photochemical reaction center” pigment molecules are specialized to transduce light energy into chemical energy.

• Several hundred light harvesting pigment molecules funnel light to one reaction center molecule.

Steps in PhotochemistryOr, How Reaction Centers Work

1) Light harvesting chlorophyll (LHC) electron is excited by light.

2) Energy of LHC electron is passed to successive LHC chlorophyll molecules by resonance energy transfer.

3) LHCs near reaction center (RC) transfer energy to RC chlorophyll.

4) Excited RC chlorophyll donates electron to an electron acceptor in the RC.

5) RC regains electron from electron donor creating “charge separation” in the RC.

Chl* = excited chlorophyll

In the chloroplast PSII reaction center, the electron acceptor is a molecule called pheophytinwhich becomes negatively charge as denoted by •Pheo-.

Importantly, the oxidized chlorophyll molecule (now positively charged, Chl+) returns to the ground state by accepting an electron through a coupled redox reaction involving the oxidation of H2O.

This process of O2 evolutiontakes place in the manganese center present in the thylakoidmembrane and is ultimately the source of electrons needed for the photosynthetic electron transport system.

The Z Scheme of Photosynthetic Electron Transport

Light energy captured by chlorophyll molecules actually involves the interplay of two reaction centers called photosystem I and photosystem IIthat are linked together by redox reactions.

The reaction center complexes are functionally linked by an electron carrier protein called plastocyanin that shuttles electrons one at a time from PSII to PSI.

The oxidation of 2 H2O requires 8 photons to transport 4 e- through the system, resulting in the accumulation of 12 H+ in the thylakoid spaceand generation of 2 NADPH in the stroma.

The Z Scheme of Photosynthetic Electron Transport

Z Scheme Described

The photosynthetic electron transport system in plants consists of two linked electron circuits, each requiring an input of energy from light absorption at PSII and PSI reaction center complexes to initiateelectron flow. The Z scheme energy diagram showing how photon absorption by the PS II reaction center complex results in electron flow from H2O to plastocyanin, providing energy to translocate H+ across the thylakoid membrane. A second photon absorption event at PS I drives electron transport from plastocyanin to NADP+.

Electron flow through protein-linked redox reactions involves numerous electron carriers, including Fe-S centers, the hydrophobic molecule plastoquinone (Q) which is reduced to form plastoquinol (QH2) and analogous to ubiquinone/ubiquinol in the mitochondrial electron transport system. Plastocyanin has the same job in photosynthetic electron transport as does cytochrome c in mitochondrial electron transport.

Z Scheme of Photosynthetic Electron Transport

Buchanon et al., Fig.12.22

TIPS 7: 183-185

TIPS 7: 183-185

Photosystem II (PSII)

Photosystem II contains chlorophylls a and b and absorbs light at 680nm. This is a large protein complex that is located in the thylakoidmembrane.

Schematic drawing of electron flow through PS II

The absorption of light energy by PS II results in electron flow leading to generation of O2 from the splitting of H2O and to the reduction of plastoquinone (Q) to form plastoquinol (QH2).

Water Oxidation at PSII

A single photon of 680 nm light does not have enough energy to break the bonds in water.

Instead, the 4 e- are passed one at a time to oxidized P680 through a Tyr residue in the D1 subunit of PSII.

4 P680+ + 4 Tyr . 4 P680 + 4 Tyr

4 Mn ions store the positive charge and coordinate the oxygen until oxidation of water is complete.

4 Tyr . + [Mn complex]0 4Tyr + [Mn complex]4+

[Mn complex]4+ + 2 H2O [Mn complex]0 + 2H+ + O2

2H2O 2H+ + 4e- + O2

Manganese cluster of the PS II Reaction Center from Synechocystis elongatus Nature 409:739 (2001)

The electron flow resulting from the absorption of 4 photons leads to the reduction of two molecules of plastoquinone, and in the process, generates a net increase of 4 H+ inside the thylakoid lumen.

The net reaction from oxidation of QH2, and from proton pumping through the cytochrome b6f complex, is the addition of 8 H+ to the thylakoid lumen. This brings to 12 the total number of H+ moleculesaccumulated inside the thylakoid lumen for every O2 generated in PS II.

Photosystem I (PSI)

The final stage of photosynthesis: the absorption of light energy by PS I is at a maximum of 700 nm. Again 4 photons are absorbed, but in this case, the energy is used to generate reduced ferredoxin, which is a powerful reductant.

Structure of PS I complex showing Fe-S clusters

Electron flow diagram of PS I leading to generation of reduced ferredoxin

Ferrodoxin-NADP+ reductase converts NADP+ to NADPH through a semiquinone intermediate involving FADH˙

We can put it all together to showing that ATP synthesis results from protons flowing OUT of the thylakoid lumen back into the stroma.

Note that the chloroplast ATP synthase is structurally and functionally similar to the mitochondrial ATP synthase we have already described, with the exception that 4 H+ are required for every ATP synthesized based on experiments showing that ~3 ATP are synthesized for every O2 generated (12 H+ transported/O2

generated).

This difference (3 H+/ATP in mitochondria versus 4 H+/ATP in chloroplasts) could be due to differences in the "gear ratio" of the complex, or uncertainties in the experimental measurements.

ATP synthesis occurs from protons flowing OUT of the thylakoidlumen back into the stroma.

Understand the compartmentalization here!

Comparison of Photosynthesis and Oxidative Phosphorylation

These processes are related.

You should understand how they are related.

The light induced proton motive force generated during photosynthesis is used to generate ATP in the stroma as a

result of protons moving out of the thylakoid lumen.

In contrast, protons pumped out of the mitochondrial matrix as a result of NADH oxidation by the electron transport

system, flow back into the matrix to generate ATP inside the mitochondria.