Mitochondrial matrix
Intermembrane space
Aerobic respiration
Occurs in the mitochondrion in eukaryotes
Two major steps
Tricarboxylic acid (TCA) cycle
• a.k.a. Krebs cycle, citric acid cycle
• Role #1: generate NADH
• Role #2: entry point for amino acids into metabolism
Oxidative phosphorylation (OXPHOS)
• a.k.a. electron transport chain
• Role #1: create a proton gradient by oxidation of coenzymes
• Role #2: use the proton gradient to drive synthesis of ATP
TCA cycle
Generates NADH and
another reduced
coenzyme, FADH2
Consumes water and
yields CO2 as a
byproduct.
Coenzyme A (CoA)
CoA is a shuttle for
acetyl groups
Functions
• Entry point into
aerobic respiration
• Product of fat
oxidation
• Substrate for fat
synthesis
TCA cycle: entry
Pyruvate is oxidized and two of its three carbons are
linked to Coenzyme A, yielding 1 NADH, 1 CO2 and
acetyl CoA.
Acetyl CoA donates these two carbons to oxaloacetate
with the
hydrolysis of
water to
yield citrate.
TCA cycle: NADH and ATP generation
Subsequent steps oxidize the molecule to yield NADH
and, at one point, GTP.
The GTP may or may not be used to generate ATP.
More CO2 is
generated.
TCA cycle: FAD and completing the cycle
FAD is used as an electron acceptor at one point in the
cycle, yielding FADH2 (next slide)
With the reduction of one
last NAD+ in the next step,
we once again have
oxaloacetate, ready to
accept a new acetyl group
from acetyl CoA
Flavin adenine dinucleotide
TCA cycle: the complete reaction
acetyl CoA + 3H2O + 3NAD+ + 5[H] + FAD + ADP + Pi 
CoA + 2CO2 + 3 NADH + 3H+ + FADH2 + ATP + H2O
Remember, this happens twice for every glucose
molecule (1 glucose  2 pyruvate  2 acetyl CoA)
Net yield per glucose so far:
• 4 ATP
• 10 NADH
• 2 FADH2
The NADH and FADH2 are next utilized to make more
ATP via oxidative phosphorylation
Electron transport system: respiratory complexes
Uses the transfer of electrons from NADH/FADH2 to drive the
transmembrane transport of protons up their concentration gradient
Pumping protons by
electron transport
Flavin
mononucleotide
Coenzyme Q
Nature 494:443-8 (2013)
Electron transport system: respiratory complexes
Uses the transfer of electrons from NADH/FADH2 to drive the
transmembrane transport of protons up their concentration gradient
Cytochrome c
FADH2 oxidation moves fewer protons
A result of
passing electrons
to respiratory
complex II instead
of complex I
4 fewer protons
moved
F1F0 ATP synthase
ATP can power rotation of
the stalk within F1
F1 + stalk
http://nature.berkeley.edu/~hongwang/Project/ATP_synthase/
In the cell, 3
protons flowing
through F0
rotate the stalk
120° and
drive synthesis
of 1 ATP from
ADP
Oxidative phosphorylation overview
Each NADH drives the transport of 10 H+ across the
inner membrane
Each FADH2 drives the transport of 6 H+ across the
inner membrane
Synthesis of 1 ATP by F1 requires the flow of 3 H+
back through F0
Per glucose:
• 10 NADH + 10 H+ + 5O2 + 30ADP + 30Pi 
10 NAD+ + 10H2O + ~30ATP
• 2 FADH2 + O2 + 4ADP + 4Pi 
2FAD + 2H2O + 4ATP
Complete overview
Yield from 1 glucose
• 2 ATP from the glycolytic pathway
• 2 ATP from the TCA cycle
• 30 ATP from oxidative phosphorylation via 10 NADH
• 4 ATP from oxidative phosphorylation via 2 FADH2
• = 38 ATP/glucose
Varies depending on a number of things, but 38 is the
maximum theoretical yield.
Complete reaction
C6H12O6 + 6O2 + 38ADP + 38 Pi  6CO2 + 38 ATP + 6 H2O
Transport
The inner membrane
is impermeable to
large or polar species,
just like any lipid
bilayer.
The mitochondrion
provides transporters
for species such as
ATP, ADP, pyruvate,
water (as OH-) and Pi
Amino acids
Reactions permit
conversion of some
amino acids into
pyruvate or
elements of the
TCA cycle
Permits the
catabolism
(breakdown) or
anabolism
(synthesis) of amino
acids
β-oxidation of fats
A series of oxidation reactions allows CoA to be linked to the
carboxyl group on fatty acids.
Cleavage at the β carbon yields acetyl-CoA, which feeds
directly into TCA
The process also yields NADH and FADH2
b-oxidation
Where synthesis and breakdown of fats fit within
the metabolic pathways
Figure from http://www.np.edu.sg/~dept-bio/biochemistry/aab/topics/aab_lipid.htm
Bringing it all together – metabolism,
alcohol, obesity, and oxidative stress
The low fat diet: the old standard
A low fat diet does not protect one from fat
accumulation, since fats can still be synthesized via
• glycerol (from glycolysis) and
• acetyl CoA (from the first step of aerobic respiration)
Inevitably, fat is where any excess
calories will end up, whether from
ingestion of fat or carbohydrates.
Starvation diet
A person on a low carbohydrate/low protein diet will use
oxaloacetate for gluconeogenesis.
Oxaloacetate can be produced
from certain amino acids, which are
in turn derived from protein storage
tissue – muscle – leading to muscle
wasting.
Acetyl-CoA from fatty acid breakdown
will need to be disposed of by formation
of ketones (a.k.a. ketone bodies) because
of the oxaloacetate shortage.
High protein, low carb diet (Atkins)
Effectively a protein-supplemented starvation diet
Amino acids from the diet rather than muscle provide
oxaloacetate.
Acetyl CoA and glycerol
are provided from breakdown of fats.
Requires careful monitoring,
especially if combined with
a vigorous exercise program.
Fails to keep weight
off upon return to a
normal diet.
The best and in some ways only effective diet…
Lifestyle change
Balanced calorie diet combined with…
Exercise
…both of which contribute to lipolysis via
• Lessened fat burden from dietary sources
• Increased oxidative capacity (more mitochondria) which
metabolize fat
Low alcohol intake
My Vitamin Water experiment
183
Stopped
Vit H2O
182
Weight (lbs)
181
180
179
178
177
176
Wedding #1
Wedding #2
175
0
10
20
30
Days
40
50
60
Why alcohol can make you fat
ADH
alcohol dehydrogenase
ALDH
acetaldehyde dehydrogenase
NAD+
NAD+
NADH
H
O
H C OH
C
CH3
CH3
ethanol
acetaldehyde
H
NADH
acetyl CoA
CoA
Alcohol is oxidized to acetaldehyde by “reverse”
action of alcohol dehydrogenase using NAD+
Acetaldehyde may be metabolized to acetyl CoA by
acetaldehyde dehydrogenase using another NAD+
Acetyl CoA is raw material for synthesis of fat!
…and sick
ADH
alcohol dehydrogenase
ALDH
acetaldehyde dehydrogenase
NAD+
NAD+
NADH
H
O
H C OH
C
CH3
CH3
ethanol
acetaldehyde
H
NADH
acetyl CoA
CoA
Acetaldehyde is not a normal product in animal cells, and is
toxic!
•
•
•
•
Causes mitochondrial failure, increasing generation of free radicals
Depletion of anti-oxidants, like glutathione
Formation of acetaldehyde adducts yielding neoantigens
Triggers collagen secretion
Accumulation of fat in hepatocytes, death of cells, immune
attack and synthesis of collagen are the hallmarks of cirrhosis
Intermittent fasting diet (“5–2”)
Glycogen depletion in the liver occurs ~12–16 hours, triggering
gluconeogenesis (first) and then fat/protein catabolism after
oxaloacetate depletion
Fasting beyond the 12–16 hour window
triggers a mild state of ketosis and an
elevated sensitivity to insulin
Eating carbohydrates, proteins, fats
immediately thereafter is largely
dedicated to restoring the depleted
metabolic intermediates and regaining
glycogen stores in the liver
My 5–2 experiment
Vitamin
Water range
600 cal
day
180
178
176
174
Weight
172
170
168
166
164
162
160
-10
10
30
50
Days
70
90
110
Metabolism and oxidative stress
Oxidative stress is an imbalance between the production
of oxidizing compounds, and a biological system's
ability to detoxify the reactive intermediates or repair
the resulting damage.
The oxidizing compounds usually include:
• Reactive oxygen species (ROS)
• Superoxide (·O2-) and peroxide (O2-2)
• Reactive nitrogen species (RNS)
• Nitric oxide (·NO) and peroxynitrite (ONOO-)
Aerobic metabolism constantly generates ROS
Electron transport system: respiratory complexes
Uses the transfer of electrons from NADH/FADH2 to drive the
transmembrane transport of protons up their concentration gradient
Superoxide production in Complex I
Free electrons “leak” from reduced FMN
e–
O2  O2–
Similar electron leakage can happen with Complex III
When good molecules go bad…
Superoxide
• Among other things, carbonylates proteins
N
N
N
O O
O
+
arginine
• Can react with other reactive compounds to make
something worse (especially lipids)
DNA is also damaged by reactive oxygen species
How you mitigate their effects
Scavenger enzymes
Glutathione (GSH), an
“antioxidant”
2H2O + O2
Catalase
2 H 2O 2
2GSH
GSH Peroxidase
GSSG + 2H2O
O2
· O2Superoxide
dismutase
SOD
Why oxidative stress is a pain to understand
· O2affects
• proteins
• lipids
• nucleic acids
becomes
• :O2
• · OH
• ONOO• HOCl
• …etc…
superoxide (for example)
is mitigated by
• Superoxide dismutase
affects
becomes
mitigated by
affects
becomes
mitigated by
affects
becomes
mitigated by
An awful lot depends upon the exact conditions:
Light, pH, ions, metals, gasses and other compounds.
Interested in metabolism?
Thurl Harris, PhD
Associate Professor of Pharmacology
Research Interests: Molecular mechanisms controlling
insulin signaling and fat synthesis
Zhen Yan, PhD
Associate Professor of Medicine
Research Interests: Molecular and signaling mechanisms
of skeletal muscle plasticity and mitochondrial function
Alyssa Hasty, PhD
Professor of Molecular Physiology & Biophysics at Vanderbilt Univ.
Research Interests: Immunometabolism and obesity
Evangelia Bellas, PhD
Assistant Professor of BME @ Temple Univ.
Research Interests: Engineering models to identify variables that
regulate adipocyte behavior and study that in metabolic disease
Closing Remarks