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Oxidative Phosphorylation: Critical Thinking
Approach to Mitochondrial Function
Background Material: NADH and FADH2 made during the previous steps of aerobic respiration
of glucose, fatty acids, or amino acids are ultimately transformed into ATP during Oxidative
Phosphorylation in the mitochondria!
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Dept. Biology Penn State ©2004
https://www.bioscience.org/2009/v14/af/3509/fig5.jpg
© Kristin Rosler, Johnson & Wales University 2014
Passive Transport is the flow of particles from a region of high concentration to a region of low concentration
& uses NO energy to do so (spontaneous).
Active Transport is the flow of particles from a region of low concentration to a region of high concentration &
USES energy to do so (not spontaneous).
The intermembrane space (aka outer matrix) is acidic (or high [H+])
The inner matrix is basic (or low [H+])
NADH & FADH2 are in the inner matrix and now are utilized for the production of ATP via oxidative
phosphorylation.
A series of proteins in the inner membrane will be used to do so.
Active transport utilized to move H+ against concentration gradient in the Electron Transport Chain!
A series of redox reactions are employed as H+ ions are moved against a concentration gradient via active
transport. Protein Complexes I, III, & IV are H+ protein complex ““pump”s” which use the energy harnessed
from the oxidation of NADH and FADH2 (electron movement = energy!) in the Electron Transport Chain (ETC)
to ““pump”” H+ against a concentration gradient.
Passive transport is coupled to the synthesis of ATP during chemiosmosis!
Protein Complex V (ATP Synthase) couples the passive flow of H+ back into the inner matrix to the formation
of ATP. This process can be likened to a water wheel at an old time mill. H+ pour from the high concentration
of H+ on one side of the mitochondria membrane to the low concentration of H+ on the other. As the H+ pour
passively down the concentration gradient through Protein Complex V, the inner domain of the protein
complex “rotates.” This energy that is released from the passive movement of H+ (exergonic reaction) is now
coupled to the endergonic synthesis of ATP, ADP + Pi  ATP.
Oxidative Phosphorylation (occurs in the inner membrane of mitochondria)
I. Electron Transport Chain in inner mitochondrial membrane
A. NADH oxidation:
NADH → Complex I → Q → Complex III → Cytochrome c → Complex IV → O2 where Complexes I, III
and IV are active proton ““pump”s,” while Q and Cytochrome c are mobile electron carriers. The final
electron acceptor is molecular oxygen
 total of 3 H+ ““pump”s” move 12 H+ against concentration gradient
B. FADH2 oxidation:
FADH2  Complex II  Q  Complex III  Cytochrome c  Complex IV  O2 where Complexes III
and IV are active proton ““pump”s,” while Q and Cytochrome C are mobile electron cariers. The final
electron acceptor is molecular oxygen
 total of 2 H+ ““pump”s” move 8 H+ against concentration gradient
© Kristin Rosler, Johnson & Wales University 2014
B. Protein Complexes Involved
a. Complex I: NADH dehydrogenase, oxidizes NADH and reduces FMN (Flavin mononucleotide)
FMNH2, & passes its 2 e- to Q  QH2; energy released upon electron oxidation used to
actively moves 4 H+ across membrane, against gradient
b. Q: Ubiquinone Q, mobile electron carrier that passes the 2 e- from FMNH2 via Q  QH2
from Complex I to Complex III for NADH and from Complexes II to III for FADH2
c. Complex II: Succinate-CoQ reductase, oxidizes FADH2 and reduces Q  QH2
d. Complex III: CoQH2-cytochrome c reductase, oxidizes QH2 and reduces 2 Cytochrome C/Fe+3
 Cytochrome C/Fe+2; active transport of 4 H+ across membrane, against gradient
e. Cyt C: Cytochrome C, water-soluble e- carrier that passes its 2 e- from Complex III to
Complex IV
f. Complex IV: Cytochrome C oxidase, passes 2 e- from Cyt C to oxygen  1/2 water; it is
important to note that the final electron acceptor in aerobic organisms is oxygen and the endproduct is water. Each NADH or FADH2 1/2 H2O
* Interestingly, many animals (camels, hibernating bears) rely upon the water being produced
at the end of the ETC as its source of water while metabolizing fatty acids that lead to the
formation of many NADH and FADH2.
* Anaerobes, on the other hand, can use alternative terminal oxidases in an inducible manner
depending upon cellular environments. Final electron acceptors can be nitrites, nitrates,
DMSO, and fumarates to mention a few.
II. Chemiosmosis in the inner mitochondrial membrane:
V: ATP synthase couples the passive flow of H+ from the outer matrix back into the inner matrix to the
production of ATP. So, for each H+ ““pump”” that was active during the electron transport chain, the
H+ flow back, 1 ATP is made.
For each NADH, 3H+ ““pump”s” are actively utilized, therefore 3 ATP are made.
For each FADH2, 2H+ ““pump”s” are actively utilized, therefore 2 ATP are made.
Sources Used:
Nelson DL; Cox MM (April 2005). Lehninger Principles of Biochemistry (4th ed.). W. H. Freeman. ISBN 978-0-7167-4339-2.
White D. (September 1999). The Physiology and Biochemistry of Prokaryotes (2nd ed.). Oxford University Press.
ISBN 978-0-19-512579-5.
© Kristin Rosler, Johnson & Wales University 2014
Tally Sheet for Glucose Aerobic Respiration
Overall Equation:
C6H12O6 + 6 O2  6 CO2 + 6 H2O + 38 ATP
ATP PRODUCTION
I. GLYCOLYSIS:
- 2 ATP in energy investment phase
+ 4 ATP in energy pay-off phase *substrate-level phosphorylation
+2 NADH
X
3 ATP/NADH
+ 2 ATP
=
+ 6 ATP
=
+6 ATP
II. PREP PHASE, PYRUVATE GROOMING
+ 2 NADH
X
3 ATP/NADH
III. CITRIC ACID CYCLE
+ 3 NADH
X 2 rotations per glucose
X 3 ATP/NADH
=
18 ATP
+ 1 FADH2
X 2 rotations per glucose
X 2 ATP/FADH2
=
4 ATP
+ 1 ATP
X 2 rotations per glucose
=
2 ATP
WATER PRODUCTION
Each NADH and FADH2  ½ H20 at the end of the ETC
Therefore, for the 10 NADH + 2 FADH2 made = 6 H2O
© Kristin Rosler, Johnson & Wales University 2014
+ 38 ATP
Critical Thinking Time: Based upon your understanding of the pathways, determine the values in the
blanks below.
Under NORMAL + O2 Circumstances:
For each ACETYL-co A 
________ NADH X _____ turn X _______ ATP/NADH = _______ ATP
________ FADH2 X______ turn X _______ ATP/FADH2 = _______ ATP
________ ATP
X _____ turn
+
= ______ ATP
Total yield
_______ ATP
A. Imagine a drug blocks H+ passive diffusion back through protein V, ATP synthase.
How many ATP would each Acetyl-coA make in this scenario? ________________
1 Acetyl-co A turns CAC  _____ NADH X _____ turn X _____ ATP/NADH = ________ ATP
_____ FADH2 X _____ turn X _____ ATP/FADH2 = ________ATP
_____ ATP X
_____ turn =
+ ________ATP
Total yield = ___________ATP
B. Imagine a drug blocks FADH2 binding to succinate dehydrogenase, protein II.
How many ATP would each Acetyl-coA make in this scenario? _______________
1 Acetyl-co A turns CAC  _____ NADH X _____ turn X _____ ATP/NADH = ________ ATP
_____ FADH2 X _____ turn X _____ ATP/FADH2 = ________ATP
_____ ATP X
_____ turn =
+ ________ATP
Total yield = ___________ATP
C. Imagine a drug damages the inner cell membrane of the mitochondria and causes “pores” to
form between these two domains.
How many ATP would each Acetyl-coA make in this scenario? _________________
1 Acetyl-co A turns CAC  _____ NADH X _____ turn X _____ ATP/NADH = ________ ATP
_____ FADH2 X _____ turn X _____ ATP/FADH2 = ________ATP
_____ ATP X _____ turn =
+ ________ATP
Total yield = ___________ATP
© Kristin Rosler, Johnson & Wales University 2014
Fatty Acid Oxidation: Fatty acids yield an immense amount of ATP! See how below!
A. Step 1: Fatty Acid Primed (-1 ATP per fatty acid) in cytoplasm
- 1 ATP
Fatty acid + Coenzyme A
Fatty acyl-co A (now imported into mitochondria)
B. Step 2: Fatty Acyl-co A “cleavage”  2-C Acetyl-co As (# Carbons / 2)
* Cleaves = (# carbons/ 2) -1
* Each “cleave”  1 FADH2 + 1 NADH
C. Step 3: Acetyl-coAs enter CAC
Acetyl-co A
(2-C)
Turns 1 X per Acetyl-coA
CAC
Each turn of the CAC yields:
3 NADH
1 FADH2
1 ATP
© Kristin Rosler, Johnson & Wales University 2014
Calculate the number of ATP made from the following molecules.
Stearic Acid: 18-C fatty acid (use the tally sheet below to determine the number of total ATP
I. Fatty acid priming? (how many ATP needed?)
_______ ATP
II. 18-C chain produces _______ cleaves.
Each cleave produces:
_____ NADH X _____ cleaves X _____ ATP/NADH = ________ ATP
_____ FADH2 X _____ cleaves X _____ ATP/FADH2 = ________ ATP
III. 18-C chain forms ______ Acetyl-co As (2-C)
Each Acetyl-co A turns CAC: _____ NADH X _____ turns X _____ ATP/NADH = ________ ATP
_____ FADH2 X _____ turns X _____ ATP/FADH2 = ________ATP
_____ ATP X _____ turns =
________ATP
Total yield = I. ________ATP
II. ________ ATP
+
III. ________ ATP
NET: ____________ ATP
IV. A triglyceride is made up of ____ fatty acids. Just from the fatty acid components, how
many ATP are made? _________
© Kristin Rosler, Johnson & Wales University 2014
ALIEN MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION
Imagine additional proteins in the electron transport chain evolved in an organism’s mitochondria as
illustrated below. Assume protein I is the site of NADH oxidation, and protein III is the site of FADH 2
oxidation. Also assume that proteins I, II, IV, V and VI “pump” 1 Hydrogen against the concentration
gradient.
A. How many ATP would be generated for the oxidation of 1 Acetyl-co A for this organism?
1 Acetyl-coA turns CAC 
_____ NADH X _____ turn X _____ ATP/NADH = ________ ATP
_____ FADH2 X _____ turn X _____ ATP/FADH2 = ________ATP
_____ ATP X _____ turn =
+ ________ATP
Total yield = ___________ATP
B. How many ATP would be generated for the oxidation of 1 Acetyl-co A if treated with a
toxin that blocked NADH binding to protein I, NADH dehydrogenase?
1 Acetyl-co A turns CAC 
_____ NADH X _____ turn X _____ ATP/NADH =
________ ATP
_____ FADH2 X _____ turn X _____ ATP/FADH2 =
________ATP
_____ ATP X
_____ turn =
+
________ATP
Total yield = ___________ATP
C. How many ATP would be generated upon the oxidation of 1 Acetyl-co A if treated with a
toxin that blocked protein VII function?
1 Acetyl-coA turns CAC 
_____ NADH X _____ turn X _____ ATP/NADH =
________ ATP
_____ FADH2 X _____ turn X _____ ATP/FADH2 =
_____ ATP X
_____ turn =
________ATP
+
________ATP
Total yield = ___________ATP
© Kristin Rosler, Johnson & Wales University 2014
Answers:
Under NORMAL + O2 Circumstances:
For each ACETYL-co A  __3_NADH X _1___ turn X ___3____ ATP/NADH = ___9____ ATP
___1_FADH2 X_1__ turn X ____2___ ATP/FADH2 = ____2___ ATP
___1_ ATP X _1__ turn
+
Total yield
= ____1__ ATP
__12__ ATP
A. Imagine a drug blocks H+ passive diffusion back through protein V, ATP synthase.
How many ATP would each Acetyl-coA make in this scenario? 1 ATP (all ATP made from oxidative phosphorylation negated, just
from substrate-level phosphorylation during the CAC)
1 Acetyl-co A turns CAC  __3___ NADH X ___1__ turn X ___0__ ATP/NADH = ____0____ ATP
__1___ FADH2 X __1___ turn X __0___ ATP/FADH2 = ____0____ATP
___1__ ATP X __1___ turn =
+ _____1___ATP
Total yield = _____1______ATP
B. Imagine a drug blocks FADH2 binding to succinate dehydrogenase, protein II.
How many ATP would each Acetyl-coA make in this scenario? ____10 ATP (all the ATP made from FADH2 oxidation negated!)
1 Acetyl-co A turns CAC  ___3__ NADH X __1___ turn X __3___ ATP/NADH = _____9___ ATP
__1___ FADH2 X __1___ turn X __0___ ATP/FADH2 = ___0_____ATP
__1___ ATP X __1___ turn =
+ _____1___ATP
Total yield = ___10____ATP
C. Imagine a drug damages the inner cell membrane of the mitochondria and causes “pores” to form between these two
domains.
How many ATP would each Acetyl-coA make in this scenario? 1 ATP, again if there is no difference in H+ concentration between
the two sides of the mitochondrial matrices, there will be no chemiosmosis specifically
1 Acetyl-co A turns CAC   __3___ NADH X ___1__ turn X ___0__ ATP/NADH = ____0____ ATP
__1___ FADH2 X __1___ turn X __0___ ATP/FADH2 = ____0____ATP
___1__ ATP X __1___ turn =
+ _____1___ATP
Total yield = _____1______ATP
Stearic Acid: 18-C fatty acid (use the tally sheet below to determine the number of total ATP
I. Fatty acid priming? (how many ATP needed?)
___- 1 ATP____ ATP
II. 18-C chain produces ___8____ cleaves.
Each cleave produces:
__1___ NADH X __8___ cleaves X ___3__ ATP/NADH = ___24_____ ATP
__1___ FADH2 X __8___ cleaves X ___2__ ATP/FADH2 = ___16_____ ATP
© Kristin Rosler, Johnson & Wales University 2014
III. 18-C chain forms __9____ Acetyl-co As (2-C)
Each Acetyl-co A turns CAC: ___3__ NADH X __9___ turns X __3___ ATP/NADH = ___27_____ ATP
___1__ FADH2 X __9___ turns X __2___ ATP/FADH2 = __18______ATP
___1__ ATP X __9___ turns =
____9____ATP
Total yield =
I. __-1______ATP
II. ___40_____ ATP
+
III. ___54_____ ATP
NET:
______93______ ATP
IV. A triglyceride is made up of __3__ fatty acids. Just from the fatty acid components, how
many ATP are made? _279________
CRITICAL THINKING TIME!
ALIEN MITOCHONDRIAL OXIDATIVE PHOSPHORYLATION
A. How many ATP would be generated for the oxidation of 1 Acetyl-co A for this organism? 19 ATP, If 5 H are “pump”ed per
NADH  5 ATP/NADH; if 3 H+ are “pump”ed per FADH2, then 3 ATP per FADH2
1 Acetyl-coA turns CAC 
__3___ NADH X __1___ turn X __5___ ATP/NADH = ___15_____ ATP
__1___ FADH2 X __1___ turn X __3___ ATP/FADH2 = __3______ATP
__1___ ATP X
___1__ turn =
+ ___1_____ATP
B. How many ATP would be generated for the oxidation of 1 Acetyl-co A if treated with a toxin that blocked NADH binding to
protein I, NADH dehydrogenase? 4 ATP
1 Acetyl-coA turns CAC 
__3___ NADH X __1___ turn X __0___ ATP/NADH = ___0_____ ATP
__1___ FADH2 X __1___ turn X __3___ ATP/FADH2 = __3______ATP
__1___ ATP X
___1__ turn =
+ ___1_____ATP
C. How many ATP would be generated upon the oxidation of 1 Acetyl-co A if treated with a toxin that blocked protein VII
function? 1 ATP
1 Acetyl-coA turns CAC 
__3___ NADH X __1___ turn X __0___ ATP/NADH = ___0_____ ATP
__1___ FADH2 X __1___ turn X __0___ ATP/FADH2 = _0_____ATP
__1___ ATP X ___1__ turn =
© Kristin Rosler, Johnson & Wales University 2014
+ ___1_____ATP