B Med Sci: Fundamentals of Life Science

Tricarboxylic Acid Cycle, Electron

Transport Chain & ATP Synthesis
Dr Scott A. White
School of Biosciences
s.a.white@bham.ac.uk

Alternatve texts: "Biochemistry" 8th Edn, Berg et al. (Barnes). "Lehninger's Principles of
Biochemistry", Cox & Nelson (Barnes). "Fundamentals of Biochemistry" 5th Edn, Horton et al.,
(Barnes). "Molecular Biology of the Cell" 5th Edn, Alberts et al.
Aerobic Respiration in Mitochondria
acetyl CoA
pyruvate

Tricarboxylic
Acid NADH & CO2
NADH Cycle

NADH & CO2

QH2
ATP
The process by which the acetyl group is oxidatively
decarboxylated to produce CO2, NADH and ubiquinol.

NADH Oxidative Phosphorylation

H2O
NAD+ ½ O2

ADP
Pi ATP

The process by which the respiratory electron transport

chain (ETC) is coupled to ATP synthesis.

S.A.White TCA, ETC & ATP

The Mitochondrion Outer membrane
Intermembrane Space
Inner membrane

Matrix
pyruvate/ pyruvate/ Cristae
fatty acids fatty acids acetyl CoA

H+
H+ Tricarboxylic
Acid
CO2 CO2
+
H Cycle
H+
H+ 3NADH ADP Pi ADP
3NAD+ 2H2O Pi
ATP ATP H+
H+ H+ O2
I III H+
+ IV H+
H
H+
+ H+ H + + H+ H+
H H
H+ H+ H+
O2
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Irreversible maximum energy release
Glycolysis
• only a fraction of the potential energy of glucose released in glycolysis
Aerobic conditions
• full energy potential released using the Tricarboxylic Acid cycle (TCA) & Electron
Transport Chain (ETC)
Pyruvate dehydrogenase
• pyruvate has to be converted to Acetyl CoA in the mitochondrial matrix first

CO2– S-CoA
C=O + HS-CoA + NAD+ C=O + CO2 + NADH
pyruvate
deH2ase
CH3 CH3 ΔG°´ = -31 kJ.mol-1
Pyruvate AcetylCoA

AcetylCoA Pyruvate TCA aka Citric Acid Cycle or Krebb’s

Cycle
irreversible ETC aka Respiratory Chain
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Coenzyme A
2-Mercaptothylamine Pantothenate ADP with 3’ phosphate

Abbreviated HS-CoA

Coenzyme A
• used to activate acetyl groups so they can be readily transferred to other
metabolites

CO2– S-CoA
+
C=O + HS-CoA + NAD C=O + CO2 + NADH
pyruvate
deH2ase
CH3 CH3
Pyruvate AcetylCoA

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Pyruvate dehydrogenase complex E1

An enormous protein complex

• 106 Da to 107 Da, depending on organism E3
Cofactors
• thiamine pyrophosphate (TPP) [vitamin B1]
E2
• lipamide
• flavin adenine dinucleotide (FAD) [from
vitamin B2 riboflavin]
Berri Berri disease (B1 deficiency) results in
weakness, fatigue & increased pyruvate

(Azotobacter)
Really 3 different enzyme types
Prosthetic
Copies* Enzyme Function
Group
60 E1 pyruvate dehydrogenase TPP oxidative decarboxylation of pyruvate
60 E2 dihydrolipoyl transacetylase lipoamide transfer of acetyl group to CoA
12 E3 dihydrolipoyl dehydrogenase FAD regeneration of oxidised lipoamide
(* eukaryotic)
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Architecture of eukaryotic Pyruvate dehydrogenase
pentagonal dodecahedron
• 12 faces, 20 vertices, 30 edges

the inner core

E2 60 copies of the E2 dihydrolipoyl transacetylase
E3 form the core
• 20 trimers of E2, one on each of the 20 (3-fold
symmetry) vertices of a dodecahedron
• 12 copies of the E3 protein are positioned in
between the core E2 proteins

the outer shell

60 copies (2 per edge) of the E1 pyruvate
E1 (α2β2) dehydrogenase form the outer shell
• each E1 forms contacts to an underlying E2
from the inner core, as well as E1 neighbours
• each copy of the E1 protein is formed of an
α2β2 heterotetramer

S.A.White TCA, ETC & ATP

Acetyl CoA - a key intermediate

Pyruvate
amino acids fatty acids

Acetyl CoA

TCA cycle

why high carbohydrate rich diet results in fat (obesity)

reverse not true, as Acetyl CoA → pyruvate

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TCA Overview
C2 Overall
Acetyl CoA Acetyl CoA → CoA + 2 CO2
H2O
HS-CoA 3NAD+ → 3NADH
NADH
C4 C6 H2O
FAD → F ADH2 (Q → QH2)
NAD
⊕
citrate
oxalo- GDP + Pi → GTP
acetate
C4 malate cis-aconitate
C6 (substrate level
H 2O phosphorylation)
H 2O TCA
C4 fumarate cycle isocitrate
C6
FADH2 NAD⊕
α-keto
Q succinate glutarate
FAD
QH2
C4 C5 NADH
CO2
1st decarboxylation
GTP C4 NAD⊕
HS-CoA Pi NADH HS-CoA
GDP CO2
2 decarboxylation
nd

succinyl-CoA

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TCA Detail
⊖
COO
CH2
S-CoA
 citrate synthase
Acetyl CoA
 aconitase
H2O
⊖
⊖  aconitase
HS-CoA COO
NADH
COO
O=C
CH2
⊖
 isocitrate dehydrogenase
H 2O
CH2
⊖
 HO-C-COO
CH2
 α-ketoglutarate
NAD⊕ COO citrate COO⊖ ⊖
dehydrogenase complex
⊖  COO
oxalo-
COO acetate CH2
⊖  succinyl-CoA synthetase
HO-CH C-COO
CH2
malate cis-aconitate CH
⊖
 succinate dehydrogenase
⊖ H 2O
COO COO
(Complex II)
 COO⊖
H 2O ⊖ TCA  fumarase
COO CH2  malate dehydrogenase
CH
fumarate cycle isocitrate H-C-COO
⊖

CH HO-CH
⊖ ⊖
COO COO
FADH2
 COO
⊖  NAD⊕
⊖ α-keto
Q COO succinate CH2
glutarate
FAD CH2 CH2
CH2 ⊖ C=O NADH
QH2
COO
⊖  COO
CH2  COO
⊖ CO2
1st decarboxylation
GTP CH2 NAD⊕
HS-CoA Pi C=O NADH HS-CoA
GDP S-CoA CO2 nd
2 decarboxylation
succinyl-CoA

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Electron Transfer Chains
Ared Box Cred Dox
wh. A, B, C, D are
electron or redox
carriers.
Aox Bred Cox Dred

If we look at anyone of the reactions in the chain, eg

Bred + Cox Box + Cred

the reaction will tend to proceed from Left to Right if the redox potential
(Eh) of B is lower than that of C.

In “Biology” electron transfer chains are often depicted in other forms ...
Note “uphill”
– 400
A intermediate steps
may be possible

Em
B C
(mV)
A B C D
D
+ 300
Note direction
of axis
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Review of electrochemistry Ch.18 Housecroft & Constable, 4ed

Copper e–
V e– Zinc
Electrode Electrode
Why do electrons flow
from Zinc to Copper?
Zn (s) → Zn2+ (aq) + 2e
–

Cu2+ (aq) + 2 e– → Cu (s)

Porous
Plug
Copper Sulphate Zinc Sulphate
solution solution

Voltmeter measures the potential difference between the two half cells (ΔEh)
Zn (s) + Cu2+ (aq) → Zn2+ (aq) + Cu (s) ΔEh = + 1.1 V

Not possible to measure the potential of a half cell, so measure the potential difference to the
“standard hydrogen electrode” (potential = 0 V) to give standard reduction potential Eh.

Zn2+ + 2 e– → Zn Eh  =  – 0.76 V If the half cell reaction is written in reverse, the
potential changes sign, eg
Cu2+ + 2 e– → Cu Eh  =  + 0.34 V Zn → Zn2+ + 2 e– Eh  =  + 0.76 V
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Biting into Aluminium Foil

• Aluminium comes into contact with

Mercury filling or Gold crown in a saline
(saliva) solution.
• Electric battery is formed.
• Electrons flow due to redox potential
difference between Al and Hg (or Au)
• Nerves stimulated → pain

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Free energy (ΔG) of electron transfer
The free energy, ΔG, of an electron transfer reaction is proportional to the potential difference, ie

  wh n  =  No. of electrons
ΔG =  – n F ΔEh F = Faraday’s const. = 96,500 C.mol–1

For a reaction to proceed spontaneously, the free energy, ΔG, must be negative, therefore...

ΔEh must be positive

Why do electrons flow from Zinc to Copper?
As indicated by the reduction potentials, Copper has a higher potential to become reduced.

Cu2+ (aq)  +  2 e–  →  Cu (s)  Eh = + 0.34 V  (the higher Eh value)

ie positive!
Zn2+ (aq)  +  2 e–  →  Zn (s)  Eh = – 0.76 V
Zn (s) + Cu2+ (aq)  →  Zn2+ (aq)  +  Cu (s)  ΔEh = + 0.34 V – (– 0.76) V  =  + 1.10 V

Assuming all solution concentrations are 1 mol.dm–3...

–1 –1
ΔG  =  – n F ΔEh  =  (– 2)  x  96,500 C.mol   x  (+ 1.10) V  =  – 212,300 J.mol

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ie negative! TCA, ETC & ATP
Eh varies with [Red] : [Ox]
100
Nernst Equation
80
RT ln [red]
n=1 Eh = Em – x
nF [ox]
60

40 when [red] = [ox]

Eh = Em when [Red] = [Ox]
Eh - Em (mV)

20
[red] / [ox] = 1
n=2 ln (1) = 0
0
10 20 30 40 50 60 70 80 90 100 Eh = Em
-20 hence “mid-point potential”

-40

Eh slope = –RT/nF
-60
Em

-80
ln([Red]/[Ox])

-100

% Reduction

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pH adjustment of Em
In general Couple m H+ n e– Em,7 ΔEm/pH unit
(mV) (mV)
+ –
Ox +  m H  + n e   → Red
Ferredoxin ox / red 0 1 – 430 0

m RT x H+ / ½H2 1 1 – 420 – 60
Em, pH = Em, pH=0 – 2.303 pH
n F
NAD+ / NADH 1 2 – 320 – 30
Fum. / Succ. 2 2 + 30 – 60
At T = 298 K, 2.303RT/F ≈ 0.06 V
ie 60 mV UQ / UQH2 2 2 + 40 – 60
Cyt c  ox / red 0 1 + 220 0
mx
Em, pH = Em, pH=0 – 0.06 x pH O2 / 2H2O 4 4 + 820 – 60
n

In “Biology” mid-point potentials usually reported at pH 7, but should always check!

Also, mid-point potentials frequently reported in mV – must use V in calculations or suffer the
consequences.
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Electron-transfer chain within proteins

FAD-
binding
domain

FeS- 2Fe-2S
binding 4Fe-4S
domain 3Fe-4S

haem- Q Fe
binding
domain membrane
Fe

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Oxidative Phosphorylation from NADH to O2
Matrix: low proton potential
ADP xH+ ATP
+ Pi
H
+
NAD+
NADH
2H+ + + 2H+
+ 2H 2H +
4H ½ O2
H2O

Complex I Complex III Complex IV Complex V

NADH UQ Cyt.bc1 Cyt.aa3 ATP
deH2ase (Cyt. reductase) (Cyt. oxidase) synthase
UQH2

Cyt.c

4H+ 4H+ 2H+

Outside: high proton potential “Downhill redox reaction”

Protonation/Deprotonation
(per 2 electrons)

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Oxidative phosphorylation versus potential
-0.4 + +
NAD + H
-320 mV
NADH
-0.2
Complex I
NADH
deH2ase
0
UQ Complex III
UQH2 Cyt.bc1
+220 mV
0.2 (Cyt. reductase)
Em,7 (V)

Cyt.c
Complex IV
Cyt.aa3
0.4 (Cyt. oxidase)

Elec
tron
0.6 flow
thro
ugh +
2H +
ETC ½ O2
0.8 +820 mV
H2O

1.0

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The Chemistry of Biological Electron Carriers
o –
N of e­ (n) Examples
Oxidised / Reduced
substrates & pyruvate / lactate
2
metabolites fumarate / succinate

NAD+ / NADH
dinucleotides 2
NADP+ / NADPH

2 ubiquinone / ubiquinol
quinones
(1) (ubisemiquinone)
flavins 2 FMN / FMNH2
(1) (flavin semiquinone)

Transition Cu2+ / Cu1+

1
metals Fe3+ / Fe2+

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 –
Substrates & Metabolites 2e
O O
O + 2 H+ + 2 e– O Em,7 = + 0.031 V
O O
O O
Fumarate Succinate

Dinucleotides, e.g. NADH

Em,7 = – 0.320 V
H H

+ H–

NAD+ + Hydride NADH

2’ Position equiv. to
H plus 2 e–
+
R = H NAD+
R R = PO32- NADP+
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 – –
Quinones e.g. Ubiquinone 2e (1e )
O
O
Ubiquinone (UQ) x = 6 - 10
Lipid soluble redox
O H x = 10, a.k.a. Coenzyme Q10
carrier
O x
isoprenyl
two n = 1 steps
O OH OH

1 e– + 1 H+ 1 e– + 1 H+

ubiquinone O semiquinone O ubiquinol OH

O OH

one n = 2 step – +
2e +2H

O OH
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 – –
Flavins e.g. FMN & FAD 2e (1e )
isoalloxazine R
N N O

NH
N
fully oxidised 1 e– +
O
1 H+
ribityl tail 1 H– +
R
1 H+
N N O

+ NH
N
pyrophosphate semiquinone H – 1 e– +
O
link 1 H+
R
H
N N O
adenine

NH
N
Flavins normally remain bound to proteins in fully reduced H
Electron Transport Chains. O

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Haem = Fe-Protoporphyrin 1e– Iron-Sulphur (Fe2 2Cys4) 1e–
OH
H 2C CH3
Cys-S S-Cys
O
H3C
Fe Fe
N N
FeII/III Cys-S S-Cys
H 2C N N

OH Cys
Iron sulphur clusters remain bound to Cys
CH3 CH3 S S
O proteins in Electron Transport Chains
II III Fe
2 x tetrahedral Fe / and 2 x inorganic S
sulphurs ( , each charged –2). S
His18 octahedral FeII/FeIII Fe
Cluster is bound to protein via 4 cysteine S Cys
Horse heart sidechains. Cys S
cytochrome c Fe Despite 2 Fe atoms, only 1 e– transfer C-terminal fragment of E. coli
(n = 1) is possible. oxidative stress sensor SoxR
The transferring electron is delocalised
Met80
1hrc across the Fe2 2 cluster. 2zhh

Iron-Sulphur (Fe1 0Cys4) 1e– Iron-Sulphur (Fe4 4Cys4) 1e–

Cys-S S-Cys
Iron atom remains bound to protein Cys-S S-Cys
in Electron Transport Chains
Fe Fe Fe
FeII/III is chelated to protein via 4 S
Cys-S S-Cys
cystine sidechains. Fe

Fe
Cys Cys S-Cys
Cβ Cβ Cys-S
Sγ
Cys
Sγ
Tetrahedral FeII/FeIII
Fe Cys
S
Sγ
Cys Cys Fe
Fe S
Sγ Cβ Fe
Cβ S
Fe
Cys S

Rhodocyclus Tenuis
Clostridium pasteurianum High-Potential IronSulphur Protein 1isu
rubredoxin
5rxn Cys
(HiPIP)
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Oxidative Phosphorylation from NADH to O2
Matrix: low proton potential
ADP xH+ ATP
+ Pi
H
+
NAD+
NADH
2H+ + + 2H+
+ 2H 2H +
4H ½ O2
H2O

Complex I Complex III Complex IV Complex V

NADH UQ Cyt.bc1 Cyt.aa3 ATP
deH2ase (Cyt. reductase) (Cyt. oxidase) synthase
UQH2

Cyt.c

4H+ 4H+ 2H+

Outside: high proton potential “Downhill redox reaction”

Protonation/Deprotonation
(per 2 electrons)

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Complex II links Citric Acid Cycle with Ox.Phos.
Mitochondrial succinate dehydrogenase
(Complex II) is the only membrane-bound
enzyme in the Citric Acid Cycle.

Citric Acid
O OH O OH

+ 2H+ + 2e-
Cycle HO O HO O

Succinate Fumarate

Complex IV

UQ
UQH2

Complex V
Complex I Complex II Complex III ATP-synthase

Cytochrome c
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Respiratory Control

additions
port for
ler
up
o u ndria
d
uff rate

nco
bu ocho

ate

P ount
Pi b satu

O2 electrode
er

ruv
tN
t
-

of all am
mi
air

py

AD
250 μM

sm
[O2] sealed

P
beaker with

AD
mag. stirrer

cin
my
go
Oligomycin is a specific inhibitor

oli

P
AD
of the ATP synthase.

Why is the respiratory rate

accelerated by ADP?
0 μM
time
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Uncoupling Agents

ia
d
Can show that uncouplers inhibit the

ffe ate

dr
on
conversion of added ADP and Pi into ATP
bu ur

te
ch
Pi -sat

m r

tra
ito

r
air

bs

ple
Uncouplers are lipid souble weak acids

su

u
e.g. DNP

co
un
NO2
[O2]

NO2
OH
time
Low [H+]
Uncouplers work by increasing the permeability
of the membrane to protons.
UH U–
• Hence relief from respiratory control: bilipid
accelleration of respiratory rate, and membrane

inhibition of ATP synthesis. UH U–

• Potential slimming agents?
High [H+]
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The energy balance of Ox.Phos.
Digression: to calculate energy from the electric potential difference and the amount of
charge moved
ie the voltage drop and the number of Coulomb moved

an electric potential charge moved, say 0.1 C (e.g. 0.01

across a light bulb Amp flowing for 10 sec)
energy released = Q x ΔV
= 0.1 C x 1.5 V
ΔV e.g. 1.5 V =  0.15 J

Similarly: for ions (e.g. protons) moving across a membrane

ΔG = – m F Δp
energy the electrochemical potential
across the membrane (Volts)

Convention amount of charge

when positive ions are translocated ie number of ions pumped x F
from in to out, m is positive (Faraday const = the charge on one mole of ions)
F = 96,500 C.mol-1
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For electrons transferred in a redox reaction

ΔG  =  – n F ΔEh
energy the redox potential
Acceptor - Donor (Volts)

amount of charge
ie number of electrons transferred x F
(the charge on one mole of ions)

Energy available from the respiratory chain?

the NAD+/NADH couple (Eh) is measured at ≈ – 0.3 V pretty good
the O2/H2O couple
“ “ “ “ “ “ ≈ + 0.8 V agreement !!
ΔEh = + 0.8 V – (– 0.3 V) = + 1.1 V
ΔG = – 2 x 96,500 C.mol-1 x 1.1 V = – 212,300 J.mol-1  (per 2 e–)

Energy needed to pump 10 H+ (per 2e–) to give a Δp  =  – 0.23 V ?

ΔG = – 10 x 96,500 C.mol-1 x – 0.23 V = + 221,950 J.mol-1  (per 10 H+)

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Energy needed to make ATP?
In respiring mitochondria supplied with substrate, can reach [ATP]  =  1 mM, [ADP]  =  1 μM
Pi = 10 mM

ADP + Pi ATP ΔG0’  =  + 30 kJ.mol-1

0 10–3
ΔG = ΔG ’ + R T ln
10–6 x 10–2

=  + 30 kJ.mol-1 + 27 kJ.mol-1  =  + 57 kJ.mol-1

Energy available from Δp to make ATP in the mitochondrion?

ΔG = – m F Δp m  =  – 3.3 H+ Δp = – 0.23 V

ΔG = – (– 3.3) x 96,500 C.mol-1 x – 0.23 V = – 73.2 kJ.mol-1

ie plenty energy
3.3 H+ available !!
Δp = – 0.23 V
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Cryo-EM structure of intact mammalian
FoF1 ATPsynthase (2012)
Stator

F1
Rotor

Fo
50 Å

Three orthogonal views of bovine Schematic to highlight nomenclature:

FoF1 ATP synthase at 18 Å resolution
The F1 is the site of ATP synthesis (forward)
or ATP hydrolysis (reverse) reactions.
F1 (soluble fragment), Fo (oligomycin-
sensitive component), Stator (not
rotating) and Rotor

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Yeast Fo rotor formed from 10 helical hairpins
(subunit c)

conserved
acidic AA
on outside
helix

Yeast
C10 ring

outer ring

The crystal structure of yeast C10(Fo):F1

fragment complex inner ring

DOI
2xok 1 helical hairpin

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Proposed two channel model of Fo rotor
Estimated to rotate at 100 s–1, or 6,000 rpm

Evidence?
Conserved Asp or Glu in C
subunits
Location of Na+ ions in Na+ATP
Synthase
Covalent modification of C-ring
carboxylic sidechain by DCCD
inactivates Fo (reacts with –
CO2H, not –CO2–)
Direct observation by attachment
of fluorescent actin to C-ring
Figure 1 from Stock et al DOI
H+/ATP ratios
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Crystal Structure of the F1 Fragment
Three conformations of β subunit observed in the first crystal structure: 1) ATP-bound; 2) ADP + Pi;
3) empty
“side view”

α β

Nucleotide-binding sites are at the

interface of the α (red) and β (yellow)
1bmf subunits, but most nucleotide:protein
interactions are to the β subunit.

The γ subunit (green) goes through

the middle and attaches to the rotor
(would be at the bottom of this
image). “top view”

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Binding Change Mechanism
Developed initially (Boyer and others) from analysing binding affinities and reaction rates of the reverse reaction, ATP
hydrolysis, with the F1 fragment, then modified by analysis of crystal structures (Walker). The F1 ATP synthase head group
(α3β3) has three active sites (labelled 1-3, all in the β subunits) in which ATP synthesis is catalysed. At any moment the three
active sites have one of the three states: ATP-bound, ADP/Pi-bound, or empty.

ATP synthesis
3 3
βADP ADP βATP βADP
2 Pi 2
ADP ADP ADP
βempty
Pi γ ATP*
γ
Pi ATP
γ
Pi

ATP ATP βempty

βATP
1 1 ATP
viewed from “below” possible Note: 120°
intermediate rotation of γ
subunit

The γ subunit rotates due to rotating c-ring. The head group with the 3 active sites does not rotate, due to the stator. Consider
one rotation of the γ subunit by 120º rotation. The ATP in the βATP site (1) will be “kicked out” by the rotating γ subunit. The
ADP/Pi in the βADP site (2) becomes ATP via an activated intermediate (ATP*). The βempty active site (3) will now bind ADP/Pi.
Although the three active sites have not physically rotated, the binding status of each has.
Repeat another two rotations of 120º times gives a full 360º rotation of γ subunit and 3 ATP molecules are synthesised. The
rotating γ subunit (driven by the rotating c-ring, driven by the H+ gradient, ... etc) drives the “uphill” (energy requring) reaction
of ATP synthesis.
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