DNP Case

Dr. Sarah Robinson

 Learning objectives
1. Define the proton motive force and explain how it is used to make ATP.
2. Compare redox potentials for different members of the electron transport chain to explain why
electrons flow toward oxygen and why that process is energetically favorable.
3. Identify the sub-cellular locations of the electron transport chain (ETC) and ATP synthase in the cell.
4. Describe and visualize electron flow through the ETC complexes and protons that are pumped by
the ETC and travel through ATP synthase.
5. Explain the coupling between the electron transport chain and ATP synthesis.
6. Explain how DNP affects oxidative phosphorylation.
7. Explain the biochemical reasons for the symptoms that result from DNP poisoning.
8. Explain the first law of thermodynamics (conservation of energy) in terms of oxidative
phosphorylation.
9. Contrast the mitochondria of brown fat and white fat and state the function of brown fat in babies and
hibernating animals.
10. Calculate the number of ATP produced from glucose metabolism and assess how metabolic enzyme
deficiencies affect ATP production.
11. Visualize glucose metabolism occurring in a single liver cell to integrate the pathways of glycolysis,
gluconeogenesis, PDH complex, TCA cycle, oxidative phosphorylation.
 Electrons Electrons carried
carried via NADH and
via NADH FADH2

Oxidative
Pyruvate
Glycolysis Citric phosphorylation:
oxidation
acid electron transport
Glucose Pyruvate Acetyl CoA cycle and
chemiosmosis

CYTOSOL MITOCHONDRION

ATP ATP ATP

Substrate-level Substrate-level Oxidative

phosphorylation phosphorylation phosphorylation
 Intermembrane
H space
H 

Protein H


complex H
Cyt c
of electron
carriers
Inner
IV mitochondrial
Q membrane
III
I
ATP
II synth-
2 H + 1/2O2 H 2O ase
FADH2 FAD

NADH NAD
ADP  P i
ATP
(carrying electrons
from food) mitochondrial matrix
H

1 Electron transport chain 2 Chemiosmosis

Oxidative phosphorylation
The patient

A 27-yr-old woman was admitted to emergency services complaining of fatigue, nausea, and
excessive sweating. She reported starting a new weight loss tablet, containing 2,4-
dinitrophenol (DNP), which she bought over the Internet a week before her admission. She had
doubled the recommended dose for faster results. Past medical history was negative. She was
a non-smoker, non-drinker, and had no known allergies. Her airway was clear, respiratory rate
was elevated (60 breaths/minute), oxygen saturation 100% (“normal”). Her temperature was
39°C (102.2°F). There were no other significant clinical findings. Six hours after admission, her
situation worsened and temp was 40°C (104°F). An hour later, she became asystolic (no
heartbeat). Cardiopulmonary resuscitation was started. It was not possible to ventilate her due
to widespread sustained muscle rigidity. After 14 cycles with epinephrine and atropine, she
remained asystolic and was declared dead.
2,4-dinitrophenol (DNP)

• DNP has a pKa of 4.1

• DNP is lipid soluble

What part of DNP is ionizable?

What does that mean about its

movement in the cell? Which
membrane is involved in
oxidative phosphorylation?
Passive vs active transport
How is it that the cell
can make ATP when
a proton travels down
concentration
gradient?

What would be the

delta G of that
process?

Hint for case: recall First Law of Thermodynamics

Leigh syndrome
• Infant male patient
• Initial symptoms at three months old: vomiting, diarrhea, and dysphagia
• High levels of pyruvate and lactate (similar symptoms to pdh deficency)
• At eleven months old:
• hypotonia, dystonia, ataxia, visual loss
• abnormal results of brain MRI
• Diagnosis: Leigh Syndrome
• Treatment: riboflavin and CoQ10
• Passed away at thirteen months old
• Mutation of gene encoding ATP synthase: subunit a
 INTERMEMBRANE SPACE

ATP Synthase H
Stator
Rotor

Internal
rod

Catalytic
knob

ADP
https://www.youtube.com/watch?v
+
=PjdPTY1wHdQ Pi ATP

MITOCHONDRIAL MATRIX
Hypotonia: ”floppy baby syndrome”

https://medlineplus.gov/ency/imagepages/17229.htm
 Intermembrane
Electrons going to complex
space
H I result in protons being
H 
pumped from ETC complex
1, III and IV = 2.5 ATP per
Protein H


complex H NADH
Cyt c
of electron
carriers
Inner
IV mitochondrial
Q membrane
III
I
ATP
II synth- Electrons going to complex II
2 H + 1/2O2 H 2O ase
FADH2 FAD result in protons being
NAD pumped from ETC complex
NADH ATP
(carrying electrons
ADP  P i III and IV = 1.5 ATP per
from food) mitochondrial matrix FADH2
H

1 Electron transport chain 2 Chemiosmosis

Oxidative phosphorylation
 This reaction
hydrolysis ATP
Glycolysis

location:
cytoplasm This reaction
hydrolysis ATP

How do we make
This reaction makes 1 NADH,
ATP from NADH
happens 2 X for every 1 glucose produced here?
Need to get
This reaction makes ATP, happens electrons to ETC
2 X for every 1 glucose

Substrate level phosphorylation

This reaction makes ATP, happens

2 X for every 1 glucose
In heart and liver, electrons from cytoplasmic NADH
are used to generate mitochondrial NADH in malate-
aspartate shuttle.

The malate-aspartate shuttle consists of two

membrane transporters and four enzymes.
Malate-aspartate shuttle

1 NADH = 2.5 ATP because electrons go to Complex 1

In muscle, electrons from cytoplasmic NADH can enter the electron-
transport chain by using the glycerol phosphate shuttle.

The electrons are transferred from

NADH to FADH2 and subsequently to Q
to form QH2.
Glycerol 3-phosphate shuttle

1 FADH2 = 1.5 ATP because electrons go to Complex II

 This reaction
Glycolysis hydrolysis ATP
location:
cytoplasm
This reaction
hydrolysis ATP Malate-aspartate
shuttle takes e- to
ETC complex 1

This reaction makes 1 NADH,

happens 2 X for every 1 glucose

This reaction makes ATP, Glycerol 3

happens 2 X for every 1 glucose phosphate shuttle
takes e- to ETC
complex 2
Substrate level phosphorylation

This reaction makes ATP, happens

2 X for every 1 glucose
Pyruvate dehydrogenase (PDH) complex

location:
This reaction makes 1 NADH,
mitochondrial matrix which goes to ETC complex 1
(no shuttle needed because it
is made in the matrix)
 NADH goes to ETC complex 1

Citric acid cycle (TCA) (no shuttle needed because it

is made in the matrix)

location: 2 FADH2 goes to ETC complex 2

(no shuttle needed because it
mitochondrial matrix is made in the matrix)
1

3
8

7 4

6 5

Substrate level phosphorylation

Tallying up glucose oxidation
2x 2x

1 glucose
Glycolysis PDH complex TCA cycle
(6C)

2 pyruvates 2 acetyl CoA (2C) 4 CO2

(3C)
Outputs: 6 NADH x 2.5 = 15 ATP
2 net ATP via 2 NADH x 2.5 = 5 ATP
substrate-level
2 FADH2 x 1.5 = 3 ATP
phosphorylation
2 CO2
2 NADH 2 net ATP via
substrate-level
phosphorylation

Glycerol 3
Malate-aspartate
phosphate shuttle
shuttle takes e- to
takes e- to ETC
ETC complex 1
complex 2
2 NADH x 1.5= 3 ATP 2 NADH x 2.5 = 5 ATP
 ATP per glucose: normal cell
SLP = substrate-
• Glycolysis: level phosphorylation

• How many net ATP via SLP: 2

• How many NADH (using malate/aspartate shuttle): 2 X 2.5 = 5
• (using glycerol 3-phosphate shuttle): 2 x 1.5 = 3
• 2 pyruvate  2 acetyl CoA
• How many ATP via SLP: 0 Net total using malate-aspartate shuttle = 32
• How many NADH X 2.5: 2 X 2.5 = 5 Net total using glycerol 3-phosphate shuttle = 30

• 2 turns citric acid cycle:

• How many ATP via SLP: 2 What is the total net ATP produced per
glucose in someone with Leigh Syndrome?
• how many NADH X 2.5: 6 X 2.5 = 15
• how many FADH2 X 1.5: 2 X 1.5 = 3
Drawing

An important part of unit 3 is visualizing where the metabolic pathways occur

in the cell and how they connect. Before we talk about lipid metabolism, let’s
review what we have done so far. This drawing is something you will want to
keep and add on to. This will be an important study tool for exam 3.

Everyone needs to complete their own drawing.

Drawing

ALL in one liver cell:

Glycolysis (see my drawing for level of detail)
Gluconeogenesis (see my drawing for level of detail)
Pyruvate  acetyl CoA (inputs and outputs)
Citric acid cycle (inputs and outputs at each step)
Electron transport chain: all complexes, flow of electrons and protons
ATP synthase: flow of protons and formation of ATP
Glycolysis + gluconeogenesis
 PDH complex and TCA cycle For TCA cycle, you
don’t need to include
structures or enzyme
names in your drawing
NAD+ NADH + H+

Pyruvate acetyl CoA

CO2
PDH Complex