5BBB0223

Mitochondrial Function & Dysfunction

Timothy Pullen

With thanks to: Dr Clare Thornton, RVC

A few mitochondrial functions
• Oxidation of fat, protein and carbohydrate for energy
• Steroid hormone and neurotransmitter synthesis
• Nucleotide synthesis
• Calcium buffering
• Growth and proliferation
• Apoptosis and cell death

2
Anatomy of the mitochondrion
Permeable
<5000 Da

Permeable
to O2, CO2, H2O

3
Learning Outcomes
After this lecture and the TBL session you should be able to:
• Explain the electron transport chain and describe the nature of the protein
complexes involved
• Describe the function and regulation of the F0F1ATP synthase and the
generation of ATP
• Explain how respiratory control regulates the rate of oxidative
phosphorylation
• Understand the dynamic nature of mitochondria and define mitochondrial
fission, fusion and mitophagy
• Identify pathology which is underpinned by dysfunctional mitochondria and
perturbations in energy metabolism

4
Part I – The electron transport chain

5
 Cellular Respiration: Recap
3 stages:
Stage 1
Production of acetyl-CoA

Stage 2 TCA
Oxidation of acetyl-CoA

Stage 3
Electron transport and
chemiosmosis

6
 Cellular Respiration: Overview
3 stages:
Stage 1
Production of acetyl-CoA

Stage 2
Oxidation of acetyl-CoA

Stage 3
Electron transport and
chemiosmosis

7
 Cellular Respiration: Overview
3 stages:
Stage 1
Production of acetyl-CoA

Stage 2
Oxidation of acetyl-CoA

Stage 3
Electron transport and
chemiosmosis

8
 Cellular Respiration: Overview
3 stages:
Stage 1
Production of acetyl-CoA

Stage 2
Oxidation of acetyl-CoA

Stage 3
Electron transport and
chemiosmosis

9
What stage of cellular respiration is CO2
produced?
• Glycolysis
• Kreb’s Cycle
• Electron transport chain
• ATP synthesis

10
What stage of cellular respiration is O2
consumed?
• Glycolysis
• Kreb’s Cycle
• Electron transport chain
• ATP synthesis

11
 Electron transport (Respiratory) chain
Complex I takes 2e-
from NADH. Energy
released is used to
Ubiquinone (Q)
pump 4H+ across the
takes these 2e- from
membrane
complexes I and II
and transfers to
Complex II takes 2e- complex III
from FADH2. No H+
pumped across
membrane
Cytochrome
(Cyt) C takes e-
from complex III
and transfers to Cycle repeats - Complex IV
Complex III accepts accumulates 4e-
2e- from Q. Energy complex IV
Used to reduce molecular
released pumps H+ Complex IV oxygen to water
across membrane accepts e-
from Cyt C O2 + 4H+ + 4e- → 2H2O
12
Electron carriers: NAD +

Nicotinamide adenine dinucleotide

(NAD+) accepts 2e- + 1 proton

13
Electron carriers: Flavoproteins
Flavin nucleotide can accept either 1e- (semiquinone form) or 2e-
(giving FMNH2 or FADH2)

Flavin adenine dinucleotide (FAD)

Isoalloxazine ring-ribitol-phosphate-phosphate-ribitol-adenine

Flavin mononucleotide (FMN)

Isoalloxazine ring-ribitol-phosphate

14
Electron carriers: Iron-sulphur clusters

dimeric (Fe2S2) cluster tetrameric (Fe4S4) cluster Non-haem prosthetic group

associated with Flavin enzymes

Fe2+ or Fe3+ - net charge actually

between +2 and +3 as electrons
dispersed among the Fe atoms

Fe-S clusters accept and release

one e- at a time
Molecular Sulphur Cysteine side-chains

15
Electron carriers: Ubiquinone (Coenzyme Q10)
Only electron carrier not bound to a protein complex
Freely diffusible in the non-polar interior of the IMM

Oxidised Semiquinone (free radical form) Reduced

Q QH. QH2
Can exist as the anion QH.-
depending on the side
chain 16
Electron carriers: cytochromes
• c1, c, a and a3 in that order
• Characteristic - capable of absorbing visible light
due to haem groups
• Haem prosthetic groups – oscillates between Fe3+
and Fe2+ after accepting an electron
• Cytochromes carry 1e-
• Cytochrome c is mobile
Haem
(remains in Fe2+ in haemoglobin)

17
Complex I - NADH dehydrogenase
• Oxidises NADH from TCA cycle, fatty-acid
oxidation and glycolysis
• Reduces ubiquinone for the rest of the
respiratory chain
• Transports protons across the inner
mitochondrial membrane to support ATP
synthesis.
• Major contributor to cellular reactive oxygen
species production and oxidative stress
Alternative names:
NADH:ubiquinone oxidoreductase,
NADH-CoQ reductase
18
Complex I - Structure
• Integral membrane enzyme composed of 9
redox cofactors and 44 different subunits

Crystal structure of the entire

respiratory complex I.
Baradaran et al (2013) Nature.
494 443-8 Cardol, P BBA (2011) 1807 1390 19
Complex I - Structure

Key features:
• Membrane arm
• Matrix arm
• many Iron-Sulphur centres
• FMN-containing flavoprotein

Sazanov, L.A. and Hinchliffe, P. (2006) Science

311:1430–1436
20
Complex I passes electrons from NADH to Q
Flavoprotein FMN accepts 2e- from NADH,
converting it to its reduced form FMNH2
(matrix arm)

Iron-sulphur (Fe-S) clusters transfer 2e- to Fe-S protein N2

one electron at a time (matrix arm)

Electrons transfer from N-2 to ubiquinone

reducing it to QH2(membrane arm)
Ubiquinone (Q)
This also pulls 2H+ from the matrix
Ubiquinol (QH2)
Leonid A. Sazanov et al. Biochem. Soc. Trans. (2013) 41:1265-1271
21
 Complex I - Function
4H+

Q
Catalyses two simultaneous QH2 To Complex III
N-2
and coupled processes:

Takes 2e- from NADH and Fe-S

passes them to ubiquinone,
2H+
pulling 2H+ from the matrix, FMN
to generate ubiquinol
e-
e-
Transfers 4H+ from matrix to NAD+ + H+
intermembrane space NADH

22
 Complex III – Ubiquinol:cytochrome c
oxidoreductase
Key features:
• Membrane protein
• Dimer of identical monomers, essential for
function
• Each monomer has 11 different subunits
including:
• Cytochrome b with 2 haem groups
• Rieske Fe-S protein (Fe2S2)
• Cytochrome c1 with its heme group
Alternative name:
cytochrome bc1 complex
23
Complex III – Ubiquinol:cytochrome c
oxidoreductase
Cytochrome c1 and the Rieske iron-sulfur protein
project into the intermembrane space and interact
with cytochrome c
The complex has two distinct binding sites for
ubiquinol, QN and QP
The Q cycle is used to explain the path of electrons
through complex III

24
Complex III
Cyt C

FeS
Qp
BL

BH
QN

25
 The Q cycle Round 1
Cyt C

FeS
H H QH2
Qp-e- QQ.-
BL e

BH
Q.-
QN

Round 1:
QH2 + Cyt c Fe3+ Q.- + 2H+ims + Cyt c Fe2+
26
 The Q cycle Round 2
Cyt C

FeS
e-
H H .- QH2
BL e-
QQ

BH
Q.-2
QH

H
Round 2:
H
QH2 + Q.- + Cyt c Fe3+ + 2H+i Q + QH2 + 2H+o + Cyt c Fe2+
27
The Q cycle
Complex III catalyzes the
oxidation of fully
reduced coenzyme Q by
cytochrome c
Also pumps protons
across the inner
mitochondrial
membrane.
The mechanism by
which this occurs is
known as the Q cycle.

Also:
http://www.wiley.com/college/sc/voet/guided_exploration/index.html 28
 Q cycle summary

Round 1: QH2 + Cyt c(Fe3+) Q.- + 2H+ims + Cyt c (Fe2+)

Round 2: QH2 + Q.- + Cyt c(Fe3+) + 2H+m Q + QH2 + 2H+ims + Cyt c (Fe2+)

Summary: QH2 + 2Cyt c(Fe3+) + 2H+m Q + 4H+ims + 2Cyt c (Fe2+)

Cyt c OX – Fe3+
Cyt c RED – 29Fe2+
Cytochrome c

12kDa
104 amino acids

If it leaks out of the

mitochondrial membrane, it
can trigger apoptosis

30
Complex IV – Cytochrome c oxidase

14 protein subunits
2 catalytic subunits

2 haems
Cytochrome a
Cytochrome a3

2 copper centres:
CuA and CuB

31
Complex IV - reduce oxygen WITHOUT generating superoxide
• Two molecules of reduced cytochrome c each donate
an electron to the Cu centre (CuA)
• First e- passes through the Haem group of subunit 1 to
the Fe-CuB centre, reducing copper
• Second e- stops at Haem a3 reducing Fe3+ to Fe2+
• Oxygen now recruited, forming a Haem a3-oxygen
complex
• Proximity of reduced copper to the
haem complex reduces it to peroxide
O22- which bridges haem and Cu
centres

32
Complex IV - reduce oxygen WITHOUT generating superoxide

• Cytochrome c delivers third electron which

cleaves the O – O peroxide bond with the help
of two matrix protons, generating Fe4+ within
heme a3
• Cytochrome c delivers a final electron which
reduces Fe4+ to Fe3+
• Two more protons release two molecules of
water and resets the system

4 Cyt c RED + 4H+ + O2 4 Cyt c OX + 2H2O

Not the end of the story ..
33
Complex IV - reduce oxygen WITHOUT generating superoxide

Cytochrome oxidase
not only removes 4
protons from the
matrix for chemical
reactions, it also
pumps four H+ into the
intermembrane space

4 Cyt c RED + 8H+ + O2 4 Cyt c OX + 2H2O + 4H+

34
Complex II – Succinate dehydrogenase
succinate + Q fumarate + QH2

Couples TCA to ETC

SDH comprised of 4 subunits SDHA-D
SDHA: FAD as proton acceptor
SDHB: 3 Fe-S clusters
SDHC/D: b-type cytochrome,
Q binding site

Located on the matrix site of the IMM

Does not pump protons
(energetically unfavourable)

Alternative name:
succinate-CoQ reductase 35
The electron transport chain generates a proton gradient to drive ATP synthase

36
 Redox reactions power the ETC
- calculate the free energy cost and direction of reactions leading to electron transfer

redox potential
redox potential is a measure of the ease with which a molecule
will accept electrons, which means that the more positive the
Oxidation Is Loss (of electrons)
redox potential, the more readily a molecule is reduced.
Reduction Is Gain (of electrons)

Simultaneous oxidation and reduction

resulting in the transfer of electrons
37
 Changes in redox potential are linked to Gibbs
free energy

ΔG ΔE

Spontaneous reactions always proceed

“downhill” to create products containing
less free energy than the reactants

Therefore, for a spontaneous reaction, ΔG0’ is always negative and ΔE0’ is always positive
38
 Common Reduction Potentials
REDUCTION POTENTIALS
Half Reaction E°' (Volts)
O2 + 2 H+ + 2 e– H2O +0.816 V
SO42– + 2 H+ + 2 e– SO32– + H2O +0.480 V
fumarate + 2 H+ + 2 e– succinate +0.030 V
acetaldehyde + 2 H+ + 2 e– ethanol – 0.163 V
oxaloacetate + 2 H+ + 2 e– malate – 0.175 V
FAD + 2 H+ + 2 e– FADH2 – 0.180 V
NAD+ + 2H+ + 2 e– NADH + H+ – 0.320 V
pyruvate + CO2 + 2 H+ + 2 e– malate – 0.330 V
The half reaction with the higher E°' will
act as the reduction reaction, and the
Oxidation half reactions are the reverse half reaction with the lower E°' will act
as the oxidation reaction
39
Part II – ATP synthesis

40
“Chemiosmotic coupling” generates a proton-
motive force (PMF)
Total electrochemical
gradient of H+ across the
inner mitochondrial
membrane consists of a
large force due to the
membrane potential (ΔV)
and a smaller force due
to the H+ concentration
gradient (ΔpH)

41
F1F0 ATP synthase

500kDa complex
F0 hydrophobic unit in membrane
with 10 identical c-subunits

F1 hydrophilic catalytic unit

with 3 identical a/b subunits

Spins at 150Hz
42
F1F0 ATP synthase

Flow of protons down their electrochemical

gradient drives the FO rotor that lies in the
membrane
Protons bind to empty FO subunits
Once protonated FO subunits have completed a
full circle, protons exit to the matrix
In doing this, the energy stored in the proton
gradient is converted to rotational energy

43
Complex V – F1F0 ATP synthase

Rotary Catalysis Model

In the "open" state, ADP and phosphate enter the active

site.
The protein then closes up around the molecules and binds
them loosely
The enzyme then undergoes another change in shape and
forces these molecules together, with the active site in the
resulting "tight" state binding the newly produced ATP
molecule with very high affinity.
Finally, the active site cycles back to the open state,
releasing ATP and binding more ADP and phosphate, ready
for the next cycle of ATP production.
44
Complex V – F1F0 ATP synthase

45
 ATP Synthesis – the numbers
Total H+ # ATP made
e- carrier I II III IV
pumped 4H+/ATP

Glycolysis
(1 mol Glucose → 2
mol Pyruvate)
2 NADH
? 8H+ - 8H+ 4H+ 20H+ 5

Pyruvate
decarboxylation 2 NADH 8H+ - 8H+ 4H+ 20H+ 5
(2 mol pyruvate → 2
mol acetyl CoA)

TCA/Krebs Cycle 6 NADH 24H+ - 24H+ 12H+ 60H+ 15

2 FADH2 - - 8H+ 4H+ 12H+ 3

46
 ATP Synthesis – the numbers
Total H+ # ATP made
e- carrier I II III IV
pumped 4H+/ATP

Glycolysis
(1 mol Glucose → 2 2 NADH 8H+ - 8H+ 4H+ 20H+ 5
mol Pyruvate)
NADH needs to be shuttled from cytosol into the
Pyruvate mitochondrial matrix:
decarboxylation 2 NADH
1. Glycerol
8H+
phosphate
-
shuttle4H+
8H+ 20H+ 5
(2 mol pyruvate → 2 Delivers electrons via FADH2
mol acetyl CoA)
2. Malate-aspartate shuttle
TCA/Krebs Cycle 6 NADH 24H+Delivers
- electrons
24H+ via NADH
12H+ 60H+ 15

2 FADH2 - - 8H+ 4H+ 12H+ 3

47
 ATP Synthesis – the numbers
Total H+ # ATP made
e- carrier I II III IV
pumped 4H+/ATP

Glycolysis
(1 mol Glucose → 2 2 NADH 0-8H+ - 8H+ 4H+ 12 - 20H+ 3-5
mol Pyruvate)

Pyruvate
decarboxylation 2 NADH 8H+ - 8H+ 4H+ 20H+ 5
(2 mol pyruvate → 2
mol acetyl CoA)

TCA/Krebs Cycle 6 NADH 24H+ - 24H+ 12H+ 60H+ 15

2 FADH2 - - 8H+ 4H+ 12H+ 3

48
 ATP Synthesis – the numbers
Total H+ # ATP made
e- carrier I II III IV
pumped 4H+/ATP

Glycolysis
(1 mol Glucose → 2 2 NADH 0-8H+ - 8H+ 4H+ 12 - 20H+ 3-5
mol Pyruvate)
+ 2 ATP
Pyruvate OXPHOS
decarboxylation 2 NADH 8H+ - 8H+ 4H+ 20H+ 5
(2 mol pyruvate → 2
mol acetyl CoA) 26-28
ATP
TCA/Krebs Cycle 6 NADH 24H+ - 24H+ 12H+ 60H+ 15
+ 2 ATP/GTP 2 FADH2 - - 8H+ 4H+ 12H+ 3
49
Part III – Mitochondrial Dysfunction

50
Mitochondrial Dysfunction
1. Impairment of electron transport and ATP-synthesis machinery
Single enzyme disorders
2. Inadequate number of mitochondria
Impaired mitochondrial dynamics / biogenesis
3. Accumulation of damaged mitochondria
Impaired mitophagy

51
Complex I disorders
• Most common single enzyme mitochondrial disorder
• Mutations discovered in 26/44 genes: 7 mtDNA & 21 nDNA
• Range of symptoms and severity:
• 50% fatal under 2 years
• 75% fatal under 10 years
Lactic acidosis Mitochondrial encephalomyopathy, lactic
acidosis and stroke-like episodes (MELAS)

Leigh syndrome

52
Heteroplasmy
• Heteroplasmy – presence of >1 type of mitochondrial genome
• Multiple copies of mtDNA per cell and not segregated like nuclear genome
(estimated 100 – 10 000 copies per cell)
• Proportion of mutant DNA vary
between individuals &
even tissues
• Bottleneck at myosis –
few mitochondria transmitted
so variable mutation burden
in offspring

53
Diabetes causes marked inhibition of
mitochondrial metabolism in
pancreatic β-cells

• Lower insulin
Mitochondrially-generated Increased
ATP
secretion insulin secretion
stimulates hyperglycaemia

• Mouse model of type 2 diabetes had

reduced mitochondrial metabolism
and ATP production
Lower
mitochondrial
function

Haythorne et al (2019) Nature Communications 10:2474 54

 Mitochondrial Dynamics: Fission and Fusion

Fusion allows mito contents to mix e.g antioxidants, mtDNA, and may increase ATP production
Fission required for cell division, increased mitochondrial number and segregation of damaged
mito Volume 22, Issue 2, 4 August 2015,
60
Pages 207–21
 Fission and fusion can be a rapid response

Photoactivated green fluorescent

protein-labeled mitochondrion
divides (fission at 0.07),

and then fuses with a red

fluorescent protein--labeled
mitochondrion to create a fused
yellow mitochondrion.

Archer SL (2013) N Engl J Med 369;23 https://www.youtube.com/watch?v=CIXY-Ns5vks 61

 Mitochondrial fission
ER, Others?
• Cytosolic Dynamin-related protein
(Drp)1 is phosphorylated (or
sumoylated)
• Drp1 recruited to mitochondria and Drp1, Mff,
binds receptors MiD49/51

• Forms cuff around mitochondrion

which constricts the organelle
• Constriction severs both membranes
• Generates new mitochondria

62
Mitochondrial fusion
• Mitofusin (Mfn)1 and 2 localise to the outer
membranes and dock the two mitochondria
together, fusing the outer membrane
• Optic Atrophy (OPA)1 localised on the inner
membrane, responsible for fusing the two
inner membranes together
• Shares contents of mitochondria – “dilutes”
any effect of damage OPA1

63
Fusion protein OPA1 also critical for ETC
function and apoptosis

• OPA1 bridges the entrance to the

cristae within the mitochondrion
• Disruption of OPA1 bridge disrupts
the ETC and releases cytochrome c
• Cytochrome c escapes the
mitochondrion and promotes
apoptosis

64
Mitochondrial Quality Control - Mitophagy

65
Mitochondrial Quality Control - Mitophagy
• Selective mitochondrial degradation/recycling
• Usually upregulated in response to stress

66
 Mitochondrial Pathologies

PINK1/Parkin ROS

Complex IV

Complex II OXPHOS

OXPHOS ALS: fission/fusion

67
Further reading

Free pdf download

Ch 13, 14 Ch 4, 12, 13

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