Introductory

Biochemistry
Oxidative Phosphorylation
Catabolism
 Catabolic pathways serve two purposes:
 Breakdown of larger molecules into smaller building
units
 Release and (temporary) storage of energy in high-
energy molecules
 ATP/NTPs
 Reduced cofactors (NADH/FADH 2)
 Catabolic pathways are oxidative:
 Metabolites are oxidized as cofactors are reduced.
 Re-oxidation of cofactors is used to generate ATP.
Oxidative Phosphorylation
 Two separate but connected processes
 Oxidation of reduced cofactors (NADH, FADH2)
and reduction of molecular oxygen
 NADH  NAD+ + H+ + 2e-
 FADH2  FAD + 2H+ + 2e-
 4H+ + 4e- + O2  2H2O (or 2H+ + 2e- + ½O2  H2O)
 Phosphorylation of ADP to ATP
 ADP + Pi + H+  ATP + H2O
 Processes are linked through a proton
gradient across the mitochondrial membrane
Overview
Reduced cofactors (NADH, FADH2) generated from glycolysis and CAC
(oxidative catabolism)

Electron transport chain (reoxidation of NADH/FADH2)

(reduction of O2 to H2O)

Proton gradient (inner mitochondrial membrane)

(Potential energy gradient)

ATP synthesis
(Chemical energy)
Where does all this occur?

Proteins associated with oxidative

phosphorylation in eukaryotes are
found associated with the inner
mitochondrial membrane.
Components of the Electron
Transport Chain
Coenzyme Q Cytochrome c
(lipid soluble coenzyme) (peripheral membrane protein)

inter-membrane space

matrix

Complexes I - IV
(integral membrane proteins)
Cofactors in Oxidative
Phosphorylation
 Multiple cofactors are reversibly
oxidized/reduced during electron transport.
 Flavin mononucleotide
 Iron-sulfur clusters
Prosthetic groups
 Copper (Cu2+)
 Cytochrome heme groups
 Coenzyme Q Lipid-soluble cofactor

 Each cofactor has a characteristic reduction

potential or affinity for electrons.
 Electrons move from cofactors with lower reduction potential to
those with higher reduction potentials
Flavin Mononucleotide (FMN)

FMN + 2H+ + 2e- FMNH2

Similar to FAD/FADH2 but no Adenosine

Iron-sulfur clusters

Fe3+ + e- Fe2+
Cytochromes are hemoproteins that carry
out electron transport.

Fe3+ + e- Fe2+

Unlike hemoglobin and myoglobin,

these heme groups switch between
oxidized and reduced states.
 Coenzyme Q (Q)
Lipid-soluble molecule
(hydrophobic)
Transports electrons to Complex III
from Complexes I and II in the inner
mitochondrial membrane
(Cosubstrate for all three
complexes)

Q + 2H+ + 2e- QH2

(ubiquinone) (ubiquinol)
The Electron Transport Chain
 Redox reactions have a free energy change
related to reduction potential.
 Reduction potential is “affinity for electrons”
 Higher reduction potential change  more negative DG
 Electrons move from compounds with lower reduction
potentials to those with higher reduction potentials
 DG°′ = -nRDE°′

 Free energy changes from redox reactions can

be used to transport protons across the
membrane. (1° active transport)
The Path of Electrons from NADH
Through the Electron Transport Chain
4 H+ 4 H+ 2 H+

inter-membrane
space

2e-
matrix 2e-
½ O2 + 2H
+

H2O
NADH NAD+ Oxygen has a very high reduction
potential – terminal electron acceptor

Every NADH reoxidized results in 10 protons being moved out of the matrix
Reduction Potentials of Components
in the Electron Transport Chain

Increasing reduction
potential
Electron transport causes a conformational change
which allows these complexes to pump H + ions

Primary active transport (redox reaction provides energy)

 Complex II is Succinate Dehydrogenase
(part of the citric acid cycle)

C-C bond oxidation

 An integral membrane protein, Complex II contains FAD as a prosthetic group.
 Catalyzes oxidation of succinate to fumarate as part of the citric acid cycle.
 Electrons from succinate are ultimately transferred to coenzyme Q in the
membrane.
 No protons are moved across the membrane at Complex II
 The Path of Electrons from FADH2

4 H+ 2 H+

inter-membrane
space

2e-
2e-
matrix
FADH2 FAD ½ O2 + 2H+
(part of Complex II) H2O

Every FADH2 reoxidized results in 6 protons being moved out of the matrix
The Proton Electrochemical
Gradient

High pH
Negative side

Low pH
Positive side

Proton Motive Force

Proton Gradient (Potential Energy)
What determines the rate of
oxygen consumption?
A. The high affinity of oxygen for electrons.
B. Speed of electron transport.
C. The fact that the electron affinity of the
components increases as electrons move down
the electron transport chain.
D. A and B
E. All of the above
Overall, the potential energy of the H+
gradient is converted to the
chemical energy in the
phosphoanhydride bonds of ATP
 Oxidative Phosphorylation
The Action of ATP Synthase

H+ H+
H
+
H+
H+ H+
H+ H+
inter-membrane space
H+

matrix ATP
Approximately 3H+ are needed per ATP synthesized by
ATP synthase. ATP synthase
Based on this alone, approximately how many ATP are ADP + Pi
generated per NADH molecule?
ATP Synthase
 Two portions
 FO
 Transmembrane portion
 Protons pass through
 Triggers conformational change in F 1
 F1
 Catalytic portion
 Synthesis of ATP from ADP and Pi

 It is the rate of ATP synthesis that determines proton

movement and (ultimately) oxygen consumption
The ATP Synthase
H+
Intermembrane space

H+ conducting
component

Matrix
H+

ADP + Pi + H+

ATP

Catalytic
component
Links
 https://www.youtube.com/watch?v=3y1dO4nNaKY

 https://www.youtube.com/watch?v=PjdPTY1wHdQ

 https://www.youtube.com/watch?v=AigRzfNHCZA
ATP Synthase

Every complete turn of the central shaft is

associated with the generation of 3 ATP
(3 active sites make ATP)
The Adenine Nucleotide
Translocase & the Pi-H+ Symport.
lower pH

higher pH

Newly-synthesized ATP is exported from the mitochondrial matrix

into the cytosol where it can be used to “drive” the many energy-
requiring processes in the cell. The ADP and Pi produced in the
cytosol are then transported back into the mitochondrial matrix.
 Oxidation and Phosphorylation are
Coupled
The rate of oxygen consumption is connected
(coupled) to the rate of ATP synthesis
H+ H+ H+
H +
H+ H+
H+
inter-membrane
H+
space

2e
2e-
matrix -

H2O
NADH ½ O2 + 2H+ H+
NAD+

The rates of re-oxidation of NADH, of electron ADP + Pi

transport, and of oxygen consumption are coupled to
the rate of consumption (and synthesis) of ATP through ATP
the magnitude of the H+ electrochemical gradient.
The P:O Ratio
 P:O Ratio is the amount of ATP made (P) per
oxygen atom reduced to water (O)
 1 water made for each NADH or FADH2
reoxidized (each 2e-)
 Non-stoichiometric
 P:O ratio is ~2.5 for NADH reoxidized
 P:O ratio is ~1.5 for FADH2 reoxidized
 P:O ratio may vary with uncoupling
The rate of oxidative phosphorylation is
determined largely by the relative
concentration of ADP

O2 consumption (via electron transport) is connected to ATP

production at the ATP Synthase.

Oxygen consumption increases when ADP concentration rises.

ADP concentration reflects the energy-consumption of the cell

Coupling in Oxidative Phosphorylation
Low energy use

Low [ADP], [Pi]

Low ATP Synthase activity

Increase in H+ gradient

Decreased e- transport
O2 consumption drops
[NADH] and [FADH2] increase
Inhibition of CAC, PDH
Coupling in Oxidative Phosphorylation
High energy use

High [ADP], [Pi]

Increased ATP Synthase activity

Decrease in H+ gradient

Increased e- transport
O2 consumption increases
[NADH] and [FADH2] decrease
Activation of CAC, PDH
Coupling of ATP synthesis to
electron transport

Oxygen consumption increases in

isolated mitochondria when ATP
synthesis is stimulated (addition of ADP)
 Uncoupled systems allow protons to enter
the matrix without ATP synthesis
Uncoupling protein
H+ H+ H
+
H+ H+ H+
H+
inter-membrane
H+
space

2e
2e-
matrix -

H+
H2O
NADH ½ O2 + 2H+ H+
NAD+

ADP + Pi
Protons may enter matrix through a separate
ATP
process, generating heat instead of ATP
Brown Adipose Tissue

Mammals sometimes uncouple

oxidative phosphorylation
deliberately to generate heat
Oxygen consumption increases in
the presence of an uncoupler
• This refers to situations when electron transport
occurs without ATP synthesis (and thus, also, when
catabolism of fuel molecules occurs without ATP
synthesis).

• The proton gradient is then dissipated faster, and the

rate of electron transport increases (O2 consumption
goes up). The rate of re-oxidation of reduced
electron carriers increases, and the rate of reactions
in the citric acid cycle increases.
The P:O ratio refers to the amount of ATP
synthesized per oxygen converted to water. What
effect will the presence of an uncoupling protein
have on this ratio?

A. It will increase.
B. It will decrease.
C. It depends on if NADH or FADH2 is being
oxidized.
D. It will not change.
2,4-dintrophenol: An effective “diet
pill” that can prove to be fatal

Certain poisons work by dissipating the proton electrochemical gradient.

Uncoupling of ATP synthesis
to electron transport

Oxygen consumption continues with

uncouplers, even in the absence of
ATP synthesis
What is a potential consequence of
uncoupling mitochondrial oxidative
phosphorylation?

A. It allows continued mitochondrial ATP formation, but

halts O2 consumption.
B. It halts all mitochondrial metabolism.
C. It slows mitochondrial ATP formation but allows
continued O2 consumption.
D. It will cause the pH of the intermembrane space to
drop.
E. It reduces the rate at which NADH and FADH2 are re-
oxidized.
 Isolated mitochondria are suspended in a medium
containing succinate. The graph below shows O 2
consumed and ATP synthesized over time in the
mitochondria following the addition of various
substances.
What is most likely added at time 1?

A. ADP + Pi
B. An uncoupler of oxidative 2 3
phosphorylation

O2 consumed

ATP Synthesized
C. NADH
1
D. An inhibitor of ATP synthase
E. None of the above

Time