Virtual Lab Manual

Electron Transport Chain

Synopsis
In the Electron Transport Chain simulation, you’ll meet Roxy, the lead engineer for a project
in north Alaska. Here, environmental protection has been made a priority. With VR, you will
fly over the ocean on a journey to discover that the most problematic facilities are the coal
power plant and the fish farm, which are causing glacier melting and eutrophication. But
fear not, Roxy has come up with a brilliant idea to produce biofuel from algae using the
excess of nutrients from the fish farm and the heat and CO2 from the coal plant. When
scuba diving, she found some mysterious dark algae and now she needs your help to figure
out if the algae can use green light to perform photosynthesis.

Use the Hill reaction and spectrophotometry

Most plants are green, so they reflect this colour and absorb other wavelengths, which they
use for photosynthesis. However, green is a major component of the visible light spectrum,
so combining algae that use green light with algae that use other wavelengths maximises the
use of sunlight. In this simulation, you will use the Hill reaction and spectrophotometry to
measure electron flow and find out if the pigments can use green light for photosynthesis.

Look inside a chloroplast

To understand how photosynthesis works, you will shrink to a tiny size and see what
happens during the electron transport chain. You will be able to click on the different
molecules to see electrons and protons moving, water molecules splitting and the ATP
synthase spinning around to pump protons and produce ATP.

Don’t forget your controls!

Dr. One has already extracted pigments for you. It can be very handy to have a virtual
assistant! Now, to check out if the pigments can use green light, you will set up an
experiment. In order to properly test your hypothesis, you will need to prepare and
measure your sample, as well as positive and negative controls.

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Measure photosynthesis
The Hill reaction uses DCPIP, a redox dye, which helps measure the electron flow from
photosystem II. By comparing the absorption spectra of your pigment sample exposed to
green light only in the spectrophotometer to those of the controls, you will be able to
figure out if the dark algae are able to perform photosynthesis using green light or not.

Learning Objectives
At the end of this simulation, you will be able to…
● Understand the importance and uses of photosynthesis
● Understand the photolysis of water and electron transport
● Understand properties of light and why pigments are colorful
● Develop a hypothesis and set up an experiment to test it
● Understand how to measure the redox potential of the electron transport chain

Techniques in Lab
● Centrifugation
● Hill reaction
● Spectrophotometry

Theory
Photosynthesis
Photoautotrophs (Greek for "self-feeding" organisms) can use the sun's energy to build
complex organic molecules from carbon dioxide and water. The overall chemical reaction
can be written as:

6 H₂O + 6 CO₂ -> C₆H₁₂O₆ + 6 O₂

This reaction is only superficially correct and the underlying mechanisms are extremely
complex. The many reactions involved in photosynthesis can be divided into two separate
processes; the light reaction and the Calvin cycle. In this simulation, we will focus on the
light reaction — the conversion of sunlight into chemical energy in the form of ATP and the
reducing agent NADPH.

Location
In eukaryotic organisms, photosynthesis takes place in the chloroplast. These cell
organelles consist of three membranes. The innermost membrane forms stack-like
structures called thylakoids. More specifically, photosynthesis takes place in the thylakoid
membrane.

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There is an interesting story to the two surrounding membranes: Chloroplasts used to be
free-living photosynthetic cells/bacteria that were ingested by early eukaryotic cells.
Instead of being digested, they turned out to be useful in providing energy for the cell. The
two symbiotic cells became dependent on each other, and the photosynthetic one became
what we now call an organelle. The two membranes are remnants from the vacuole of the
eukaryote (outer membrane) and the plasma membrane of the photosynthetic cell (inner
membrane).

Pigments
Membrane-surrounded compartments are essential for many biological processes. The
thylakoid membrane, for example, is where photopigments are located, within protein
complexes.

Pigments can absorb different wavelengths, often contain conjugated systems and their
different chemical properties determine their solubility in organic or inorganic solvents.

Light reaction
The light reaction is how sunlight is converted into chemical energy. It involves several
protein complexes.

Plants and algae contain two separate photosynthesizing complexes: Photosystem I and II.
The two photosystems contain different pigment molecules that absorb specific
wavelengths of light. These pigment molecules act like a funnel; the pigments absorb
sunlight and transfer the excitation energy to the neighboring pigment molecule until it
reaches the bottom of the funnel, the reaction center.

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The reaction center consists of several molecules that enable the conversion of the
excitation energy into an electron flow. Electrons are propelled out of one of the reaction
center chlorophyll molecules and are quickly absorbed by nearby molecules.

In photosystem II, this electron is used to reduce a carrier molecule called plastoquinone.
The plastoquinone takes up two electrons and two protons and transfers the electrons to
the next complex in the electron transport chain.

The oxygen-evolving complex is located right next to the two reaction center chlorophylls
of photosystem II. This structure catalyzes the water-splitting reaction. The water-splitting
reaction is essential for replacing the electron of the chlorophyll and keeps the electron
transport chain running. The oxygen-evolving complex breaks water into molecular oxygen,
protons and electrons. The protons diffuse into the thylakoid lumen, where they
accumulate. For every two water molecules that are split, one molecule of molecular
oxygen is produced. Oxygen is basically the waste product of photosynthesis.

This is the chemical equation for the light reaction:

2 H₂O + 2 NADP⁺ + nADP + nPi → O₂ + 2 NADPH + 2 H⁺ + nATP

Electron transport chain

From photosystem II the electrons are first transferred to a plastoquinone which shuttles
the electrons on the electron transport chain. The energy of the electrons is used to
actively pump protons into the thylakoid lumen. The low energy electrons are then
transferred to photosystem I, which uses light energy to re-energize the electrons to
reduce NADP+ to NADPH.

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Each electron carrier in the electron transport chain is both an electron acceptor and an
electron donor. Plastoquinone, for example, is reduced by the reaction center, because it
has a higher reduction potential than the previous electron donor in the reaction center.
The next complex in line, the cytochrome proton pump, has once again a higher reduction
potential. This drives the electrons to move along the electron transport chain and the
energy of the electrons is used to pump protons into the thylakoid lumen.

This is the chemical equation for the light reaction:

2 H₂O + 2 NADP⁺ + nADP + nPi → O₂ + 2 NADPH + 2 H⁺ + nATP

Hill reaction
In 1937, Robert Hill discovered that isolated chloroplasts can produce oxygen even if no
carbon dioxide is present. The only requirements to convert water to molecular oxygen are
an electron acceptor and light.

We can use a special electron acceptor called a redox dye to measure the electron flow in
the electron transport chain. DCPIP is a redox dye. In its oxidized state it absorbs light in
the red spectrum and it appears dark blue. In its reduced state however it does not absorb
light of the visible spectrum and is, therefore, colorless.

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These properties in combination with its ability to diffuse into biological membranes make
it the ideal indicator to measure the redox potential of the electron transport chain. So, we
can measure the electron flow from photosystem II.

To measure the activity of the photosystems, the cells should be kept in the dark for a day
before starting with the experiment. Dark incubation ensures that all the components of
the electron transport chain are in their lowest energy state.

DCPIP inside the thylakoid membrane gets reduced by the plastoquinone that would
naturally transfer the electrons onto the electron transport chain. Because of its color
change, the redox potential of the electron transport chain can easily be visualized with
DCPIP.

DCPIP reduction can be halted by DCMU, which is a very effective herbicide. It blocks the
plastoquinone binding site of photosystem II. Hence, it disables the whole electron
transport chain.

Redox
The term "redox" is a combination of the words reduction and oxidation. It refers to all
reactions that involve the transport of electrons from an electron donor to an electron
acceptor.

The most common electron acceptor (oxidizing agent) is molecular oxygen. Metal corrosion
is the result of its oxidation by oxygen. In turn the oxygen gets reduced. The result is rust
on the surface of the metal object.

Not intuitively, a molecule loses electrons when it is oxidized, and gains electrons when it
is reduced. With the two words OIL RIG, it is easy to remember the processes: Oxidation Is
Loss and Reduction Is Gain.

Each electron transfer in the electron transport chain is a redox reaction. The electron
donor (i.e. Plastoquinone) gets oxidized and the electron acceptor (i.e. DCPIP) is reduced.

Each redox reaction of the electron transport chain is exothermic. This means the potential
energy of the electrons decreases. The energy released through these reactions is
converted into a different form of energy.

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In photosynthesis, in photosystem II for example, the plastoquinone is reduced with two
high-energy electrons, which leads to the uptake of two protons from the stroma. In close
proximity to the cytochrome complex, the plastoquinone is oxidized because the electrons
are able to jump to a lower energy state. Imagine this like rolling balls on stairs. The
plastoquinone is the top stair and the cytochrome is one stair further down. If the balls
have the chance to drop down to the lower stair, they won’t come back up anymore. The
reaction site of the cytochrome is towards the thylakoid side of the membrane. That’s why
the protons will be released into the thylakoid lumen; hence, the energy is converted into a
proton gradient.

Experimental controls
A control is a comparison.

In an experiment, you test a hypothesis by adjusting something - an independent variable.

A control is something you put through the same procedure, without adjusting that
independent variable. You can then compare the results of the control to the other results
of the experiment.

We will use an analogy to explain the concept of experimental controls

Imagine you are standing on a hill, which is surrounded by several other similar hills and
you start wondering if it would be possible to climb one of those nearby hills with your
shoelaces tied together – an obvious thought when one looks at a beautiful panorama!

Luckily, you don’t have to wonder for long because you are accompanied by three boy
scouts.

You choose the following experimental setup.

Each boy scout will walk up a hill and light a fire as soon as they reach the top. This way
you will be able to see and record the result from a distance.

You are a skilled scientist and know that you have to introduce controls to support your
results. Using experimental controls is the way of knowing if your results are due to the
variable you are testing, or caused by the experimental procedure itself.

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Following the example, you have no idea what awaits your little boy scouts down in the
forest. There could be a river or a hungry bear in that valley. You would never know if it
were the tied shoelaces that stopped them from making the fire or something else. To
avoid such uncertainties, you decide to send off one of the boy scouts as a positive control.
You won’t tie his laces together. This should guarantee that he would be able to make it up
to one of the hills and light a fire. If there is no fire on that hill, you know there is
something wrong with your experimental setup. This is an excellent positive control.

For creating your negative control, you become very creative and find an interdisciplinary
approach directly from the Mafioso Repertoire. You fill a bucket with concrete and use it to
immobilize one boy scout. You know with such a weight on his feet he might be able to
hop down the valley, but he will never make it up the other hill.

Again, you have no idea what will happen down in that valley. You ensured that the shoes
of all your boy scouts are well tied and can’t be easily taken off. But in the case they do
find a way to take off the shoes and walk up barefoot, you know the boy with his cement
shoes would do the same and there would be a fire on his hill as well.

Perfect, this is all you need to run the experiment. With such an excellent setup, you can
lean back and wait for the fire signals.

Whatever experiment you are performing, you should always come up with good controls
that are truly comparable to the experimental sample. Sending the positive control off with
a helicopter would be a useless positive control, just like tying the third boy scout to a tree
would be a bad negative control.

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