Active Learning Module
to accompany
Principles of Life, Second Edition
Hillis • Sadava • Hill • Price
Chemiosmotic Mechanism
by Carly Jordan
This active learning module includes the following elements:
 This main document
 A pre-class video
 A PowerPoint presentation for use during the in-class exercise
 A student handout
Before beginning any planning, we recommend that the instructor read through this
document thoroughly, review the accompanying PowerPoint presentation and student
handout, and watch the pre-class video.
GETTING STARTED
General Overview
Description: This module challenges students to think about the role of individual
components of the respiratory chain complexes and predict the impact of loss of function.
Students will be engaged with this task by its framing as a forensic mystery: a man is
found dead, and the students need to figure out what caused his death. They will predict
how the major inputs and outputs of the respiratory chain would be affected if each
individual component was damaged by specific mitochondrial poisons, and then compare
their predictions to the test results of the fictional victim.
Learning objectives:
 Label the major components of the inner mitochondrial membrane
 Trace the path of electrons through the respiratory chain
 List the inputs and outputs of the respiratory chain and ATP synthase
 Explain how a loss of function of one of the inner mitochondrial membrane
complexes would impact the process of chemiosmosis
 Predict the effect of mitochondrial poisons on the inputs and outputs of respiration
Instructor prep time required: 5–10 minutes
Student prep time required: Variable, depending on options selected; 15–30 minutes of
activities, plus reading the chapter
© 2016 Sinauer Associates, Inc.
Class time required: Variable, depending on options selected; 30–50 minutes
Formats: The activity is presented as a PowerPoint presentation with accompanying
student handout, and students will prepare for class by accessing online content.
Target class size: Any
Materials needed: Computer and projector, white board and markers, notecards with
names of poisons, tablet and stylus, student handouts
Keywords: Chemiosmosis, respiratory chain (electron transport chain), metabolic poison
General Background
The student should be familiar with the following concepts before beginning this activity:
 From textbook Concept 4.3:
o Mitochondria are enclosed by an outer membrane and an inner membrane
that folds inward to form cristae. Mitochondria contain the proteins
needed for cellular respiration and the generation of ATP.
 From textbook Concept 5.2:
o Substances can diffuse across a membrane by three processes: unaided
diffusion through the phospholipid bilayer, diffusion through protein
channels, or diffusion by means of a carrier protein (facilitated diffusion).
 From textbook Concept 5.3:
o Active transport requires the use of energy to move substances across a
membrane against a concentration gradient via specialized proteins.
 From textbook Concept 6.1:
o Redox reactions transfer large amounts of energy. Much of the energy
liberated by the oxidation of the reducing agent is captured in the
reduction of the oxidizing agent.
 From textbook Concept 6.2:
o The electron transport chain oxidizes NADH + H+ and FADH2 from
glycolysis, pyruvate oxidation, and the citric acid cycle, thereby
regenerating NAD+ and FAD. Oxygen (O2) is the final acceptor of
electrons and protons, forming water (H2O).
o The chemiosmotic mechanism couples proton transport to oxidative
phosphorylation, yielding ATP.
Common Student Misconceptions
A major challenge of teaching this topic is that students feel overwhelmed with details
and miss the big picture. Students will memorize the steps of cellular respiration and of
the electron transport chain complexes, and yet they will not be able to explain the energy
transformation that leads from glucose to ATP. The main thing to emphasize for students
is that the most important function of glycolysis and the Krebs cycle is to transfer the
© 2016 Sinauer Associates, Inc.
potential energy stored in glucose to electron carriers. Then the role of the loaded
electron carriers is to release that energy to create the proton gradient that drives ATP
synthase.
If you ask students: Where do the atoms in glucose end up after respiration? they will
often answer that they are used to build ATP. Of course, it is not the atoms of glucose
that build ATP, but the energy stored in its bonds, transformed to power ATP synthase.
By working through this activity, students will see that each of the metabolic poisons in
some way disrupts the proton gradient: by limiting its formation (rotenone), neutralizing
it (DNP), or blocking its interaction with ATP synthase (oligomycin). This activity is
designed to help students understand that the proton gradient, and not glucose, is the
direct source of energy for ATP synthesis.
PREPARATION
Introduce the activity to students, describe the objectives of the lesson, and assign preclass reading, quiz, and/or video.
Textbook Content
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Chapter 4: Cells: The Working Units of Life
Chapter 5: Cell Membranes and Signaling
Chapter 6: Carbohydrate Catabolism in the Presence of Oxygen Releases a Large
Amount of Energy
The focus of the exercise is on the electron transport chain and chemiosmosis (Concepts
6.2), but knowledge of diffusion, membrane transport, redox reactions, and mitochondrial
structure is also important (Concepts 4.3, 5.2, 5.3, and 6.1).
Related Media Resources
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Animated Tutorial 5.3: Passive Transport
Animated Tutorial 6.1: Electron Transport and ATP Synthesis
Animated Tutorial 6.2: Two Experiments Demonstrate the Chemiosmotic
Mechanism
Pre-Class Video
The pre-class video for this module, entitled “Chemiosmotic Mechanism,” will provide
students a concise review of the chemiosmotic mechanism, so that they can come to class
fully prepared to participate in the exercise. The video is available to students within
LaunchPad and available for the instructor to download.
Pre-Quiz
This quiz is pre-built and available for the instructor to assign in LaunchPad.
© 2016 Sinauer Associates, Inc.
1. High-energy electrons are carried to the respiratory chain by
a. glucose.
b. NADH.
c. NAD+.
d. ATP.
Answer: b
2. As electrons are passed down the complexes of the respiratory chain, _______ are
pumped into the _______.
a. hydrogen ions; matrix
b. electrons; matrix
c. hydrogen ions; intermembrane space
d. electrons; intermembrane space
Answer: c
3. The electrons passed through the respiratory chain are ultimately accepted by
a. ATP.
b. water.
c. hydrogen.
d. oxygen.
Answer: d
4. Which enzyme(s) convert(s) the potential energy of the electrochemical gradient to
potential energy stored in a chemical bond?
a. ATP synthase
b. Pyruvate dehydrogenase
c. The respiratory chain enzymes
d. The enzyme that catalyzes the first step of glycolysis
Answer: a
5. Each complex of the respiratory chain holds electrons tighter and with greater affinity
than the one before. Are these complexes being oxidized or reduced when receiving
electrons?
a. oxidized
b. reduced
Answer: b
IN-CLASS ACTIVITY (30–50 minutes)
Activity/Exercise
In this activity, students analyze the function of the inner mitochondrial membrane
complexes by predicting the effect of their inhibition. Mitochondrial poisons are
presented, and students are challenged to determine which inputs and outputs of the
© 2016 Sinauer Associates, Inc.
respiratory chain will be affected, and whether they will accumulate or be depleted.
Through this exercise, the students learn that when the proton gradient is disrupted, ATP
production is affected.
Learning how poisons work is interesting in itself, but the problem can be further framed
as a forensic mystery: A man is found dead, and toxicology results show high levels of
NADH. What could explain this result? This question is presented to begin, and a quick
brainstorm gets the class interested. Without confirming any of the students’ hypotheses
as correct or incorrect, the instructor suggests that in order to answer this question, a
thorough understanding of the functions of the inner mitochondrial membrane is required.
Textbook Figure 6.9 shows the detailed workings of the inner mitochondrial membrane.
The unlabeled version of this figure is presented to the students for them to complete (see
the student handout; see the suggestions below for how to incorporate this task in a
variety of class settings). Once the figure is complete, the instructor asks students to
identify the inputs and outputs of the processes shown. They could be circled or
highlighted in different colors, or listed on the white board in two columns. Now that
each student has a complete and correct picture of the inner mitochondrial processes, they
are ready to explore the potential poisons. For each poison presented, the class will
predict how each of the inputs and outputs will be affected, leading to the identification
of which poison could be the cause of death.
Before presenting the full list of poisons the instructor may choose to work through one
example together as a class. The PowerPoint presentation uses cyanide as the example
poison. Cyanide inhibits complex IV (cytochrome c oxidase), preventing the transfer of
electrons from cytochrome c to oxygen. The instructor asks students to predict what will
accumulate and what will be depleted as a result. A “think-pair-share” model would work
well here to assure that each student considers the question independently before starting
group work. The instructor then guides the discussion to tease apart all of the effects that
cyanide would have.
Now the main part of the activity begins. Three poisons are presented, each with a
different target (see the suggestions below for how to adjust the activity for a variety of
class types.) In groups, the students will now analyze these poisons, predicting the impact
on the inputs and outputs of the respiratory chain and chemiosmosis. Checking in on the
progress of groups, the instructor may need to ask probing questions to get students to
think about downstream effects of a given poison. Suggestions for guiding questions:

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For rotenone: If complex I is inhibited, what does that mean about NADH? About
the movement of hydrogen ions? Which components are still working?
For DNP and oligomycin: How do hydrogen ions normally move across the inner
mitochondrial membrane (in both directions)? What is the effect of that
movement?
For all poisons: What would happen if the proton gradient was diminished?
© 2016 Sinauer Associates, Inc.
Once students are finished, collect the information about each poison in a table. The
instructor may choose to fill in the table based on student answers, or project the table
onto a white board and have students fill it in (depending on the number of
groups/students). To bring the activity back to the original prompt, the instructor reminds
the students of the elevated NADH levels and asks:
 Could any of these poisons be at fault? (Rotenone)
 What other molecule could you measure to confirm this? (NAD+ should be low.)
Potential Modifications Based on Pre-Quiz Responses
Pre-quiz questions 2 and 4 are essential to understanding this module. If these questions
are not answered correctly by most students in a class, extra time should be spent at the
beginning of class emphasizing the purpose of the respiratory chain (to convert the
chemical energy stored in the bonds of NADH into the electrochemical gradient across
the inner mitochondrial membrane) and how ATP synthase functions to create ATP (by
harnessing the energy in that gradient to add a phosphate group to ADP).
Questions to ask the students:
 What is the purpose of the respiratory chain? (To create the gradient)
 Is the movement of protons into the matrix an endergonic or exergonic reaction?
(Exergonic)
 How is the released energy used? (It is coupled to the reaction of ADP + Pi.)
Adapting for Different Class Sizes
Large classes: Ask students to bring their own hard copy of Textbook Figure 6.9 to class
(blank or annotated); assign poisons to groups by asking one member of each group to
choose a notecard with the poison and target, or if groups are numbered, assign based on
group numbers; if writing on the white board is not visible, annotate slides using a tablet
and stylus or by typing into classroom computer.
Short class period: Ask students to annotate Textbook Figure 6.9 as homework and bring
to class to use during the activity; each group makes predictions for only one poison and
then turns to a neighboring group to explain the impact of their poison.
Long class period: Each group makes predictions about all poisons.
For more challenges, additional poisons can be included: malonate (inhibits complex II),
antimycin (inhibits complex III), K+ ionophore (an open channel for potassium ions).
Wrap-Up
These questions are included in the PowerPoint presentation.
1. Which drug could most effectively neutralize the H+ gradient across the inner
mitochondrial membrane?
a. Rotenone
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b. DNP
c. Oligomycin
d. Cyanide
Answer: b
2. Which would cause the greatest decrease in ATP production?
a. Rotenone
b. DNP
c. Oligomycin
Answer: c
FOLLOW-UP (10 minutes)
Post-Quiz
This quiz is pre-built and available for the instructor to assign in LaunchPad.
1. DNP shuttles H+ ions across biological membranes in either direction. Based on what
you know about diffusion and the electrochemical gradient normally present in the
mitochondria, which direction would H+ ions initially move if DNP was introduced to the
inner mitochondrial membrane?
a. Into the intermembrane space
b. Into the mitochondrial matrix
c. Into the cytoplasm
Answer: b
2. What effect would increasing the pH of the intermembrane space have on ATP
synthesis?
a. Decrease ATP production
b. Increase ATP production
c. No effect on ATP production
Answer: a
3. Which molecule is oxidized during the processes of the respiratory chain and
chemiosmosis?
a. Glucose
b. FAD
c. ATP
d. NADH
Answer: d
4. What is the fate of the atoms in glucose at the end of cellular respiration?
a. They are used to build ATP.
b. They are rearranged to form CO2 and H2O.
c. They are transformed into NADH and FADH2.
d. They are incorporated into the sugars of DNA and RNA.
© 2016 Sinauer Associates, Inc.
Answer: b
Test Items
The following questions are provided for use in the instructor’s own quizzes and exams.
1. Which condition would most drastically increase ATP synthesis by chemiosmosis?
a. Complexes that pump twice as many H+ ions for each electron received
b. Channel proteins that allow H+ ions to pass freely through the inner mitochondrial
membrane
c. Twice as many complex IV enzymes on the inner mitochondrial membrane
d. Unlimited ADP and Pi in the mitochondrial matrix
Answer: a
2. Draw a section of the inner mitochondrial membrane showing the essential membrane
proteins. Trace the path of electrons and hydrogen ions removed from NADH, and
explain how they are used to generate ATP.
© 2016 Sinauer Associates, Inc.