Oxidative Phosphorylation

 Conventional view of
mitochondrial
matrix
structure is at right.
 Respiratory chain is inter-
in cristae of the inner cristae membrane
space
membrane.
 Spontaneous electron inner outer
membrane mitochondrion membrane
transfer through
respiratory chain complexes I, III & IV is coupled to
H+ ejection from the matrix to the intermembrane space.
Because the outer membrane contains large channels,
these protons may equilibrate with the cytosol.
 Respiration-linked H+ pumping out of the matrix conserves some of the
free energy of spontaneous e transfers as potential energy of an
electrochemical H+ gradient.
 matrix

inter-
cristae membrane
space

inner outer
membrane mitochondrion membrane

3-D reconstructions based on electron micrographs of

isolated mitochondria taken with a large depth of field, at
different tilt angles have indicated that the infoldings of
the inner mitochondrial membrane are variable in shape
and are connected to the periphery and to each other by
narrow tubular regions.
Electron micrograph by Dr. C.
Mannella of a Neurospora
mitochondrion in a frozen sample
in the absence of fixatives or
stains that might alter appearance
of internal structures.
Wadsworth Center website.
Tubular cristae connect to the
inner membrane via narrow
passageways that may limit
the rate of H+ equilibration
between the lumen of cristae & the intermembrane space.
There is evidence also that protons pumped out of the matrix
spread along the anionic membrane surface and only slowly
equilibrate with the surrounding bulk phase, maximizing the
effective H+ gradient.
 Matrix
Spontaneous H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O
electron flow 
through each 2 e ––
I Q III IV
of complexes I,
III, & IV is ++
coupled to H +
+ + cyt c +
ejection from 4H 4H 2H
the matrix.  Intermembrane Space

A total of 10 H+ are ejected from the mitochondrial matrix

per 2 e transferred from NADH to oxygen via the
respiratory chain.
The H+/e ratio for each respiratory chain complex will be
discussed separately.
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

++
cyt c
4H+ 4H+ 2H+
Intermembrane Space

Complex I (NADH Dehydrogenase) transports

4H+ out of the mitochondrial matrix per 2e
transferred from NADH to CoQ.
 Peripheral domain of a bacterial Complex I

NAD+ A B

NADH FMN peripheral  FMN  FMN

domain
matrix
inner mitochondrial membrane domain
membrane
membrane  N2
domain
Complex I 
PDB 2FUG

Lack of high-resolution structural information for the

membrane domain of complex I has hindered elucidation
of the mechanism of H+ transport.
Direct coupling of transmembrane H+ flux & e transfer is
unlikely, because the electron-tranferring prosthetic groups,
FMN & Fe-S, are all in the peripheral domain of complex I.
Thus is assumed that protein conformational changes are
involved in H+ transport, as with an ion pump.
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

++
+ + cyt c
4H 4H 2H+
Intermembrane Space

Complex III (bc1 complex):

H+ transport in complex III involves coenzyme Q (CoQ).
 O O
C H 3O CH 3 C H 3O CH 3
e
CH 3 CH 3
C H 3O (CH 2 CH C CH 2 ) n H C H 3O (CH 2 CH C CH 2 ) n H
O O
c o e n zy m e Q c o e n zy m e Q • 
e + 2 H +
OH
C H 3O CH 3

CH 3
C H 3O (CH 2 CH C CH 2 ) n H
OH c o e n zy m e Q H 2

The “Q cycle” depends on mobility of coenzyme Q within

the lipid bilayer.
There is evidence for one-electron transfers, with an
intermediate semiquinone radical.
 matrix 2 H+
Q Q. QH2 QH2

cyt bH
Complex III

cyt bL e e
 
Q Q· Fe-S cyt c1
One version 2 H+
of Q Cycle: cyt c
intermembrane space

Electrons enter complex III via coenzyme QH2,

which binds at a site on the positive side of the inner
mitochondrial membrane, adjacent to the intermembrane
space.
 matrix 2 H+
Q.
QH2 gives up 1e Q QH2 QH2
to the Rieske
iron-sulfur center, cyt bH
Fe-S. Complex III

Fe-S is reoxidized cyt bL e e

by transfer of the  
Q Q· Fe-S cyt c1
e to cyt c1, which

2 H+
passes it out of the intermembrane space cyt c
complex to cyt c.

The loss of one electron from QH2 would generate a

semiquinone radical, shown here as Q·, though the
semiquinone might initially retain a proton as QH·.
 matrix 2 H+
A 2nd e is


transferred from Q Q. QH2 QH2

the semiquinone to
cyt bL (heme bL) cyt bH
Complex III
which passes it via
cyt bH across the
cyt bL e e
membrane to  
another CoQ Q Q· Fe-S cyt c1
+
bound at a site on 2 H
cyt c
the matrix side. intermembrane space
The fully oxidized CoQ, generated as the 2nd e is passed to the
b cytochromes, may then dissociate from its binding site
adjacent to the intermembrane space.
Accompanying the two-electron oxidation of bound QH2, 2H+
are released to the intermembrane space.
 matrix 2 H+
Q Q. QH2 QH2

cyt bH
Complex III

cyt bL e e
 
Q Q· Fe-S cyt c1
2 H+
cyt c
intermembrane space

In an alternative mechanism that has been proposed, the

2 e transfers, from QH2 to Fe-S & cyt bL, may be
essentially simultaneous, eliminating the semiquinone
intermediate.
 matrix 2 H+
Q Q. QH2 QH2

cyt bH
Complex III

cyt bL e e
 
Q Q· Fe-S cyt c1
2 H+
cyt c
intermembrane space

It takes 2 cycles for CoQ bound at the site hear the matrix
to be reduced to QH2, as it accepts 2e from the b hemes,
and 2H+ are extracted from the matrix compartment.
In 2 cycles, 2 QH2 enter the pathway & one is regenerated.
 matrix 2 H+
Q Q. QH2 QH2
Animation
cyt bH
Overall reaction Complex III
catalyzed by
complex III, cyt bL e e
 
including net Q Q· Fe-S cyt c1
inputs & outputs 2 H+
cyt c
of the Q cycle : intermembrane space

QH2 + 2H+(matrix) + 2 cyt c (Fe3+) 

Q + 4H+(outside) + 2 cyt c (Fe2+)
Per 2e transferred through the complex to cyt c, 4H+ are
released to the intermembrane space.
 matrix 2 H+
Q Q. QH2 QH2

cyt bH
Complex III

cyt bL e e
 
Q Q· Fe-S cyt c1
2 H+
cyt c
intermembrane space

While 4H+ appear outside per net 2e transferred in 2

cycles, only 2H+ are taken up on the matrix side.
In complex IV, there is a similarly uncompensated proton
uptake from the matrix side (4H+ per O2 or 2 per 2e).
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

++
cyt c
4H+ 4H+ 2H+
Intermembrane Space

Thus there are 2H+ per 2e that are effectively transported
by a combination of complexes III & IV.
They are listed with complex III in diagrams depicting
H+/e stoichiometry.
Complex III: PDB Complex III
1BE3
Half of the homodimeric (bc1 Complex)
structure is shown.
Approximate location of
the membrane bilayer is
indicated.
Not shown are the CoQ
binding sites near heme

membrane
heme bH
bH and near heme bL.
The b hemes are heme bL
positioned to provide a Fe-S
pathway for electrons heme c1
across the membrane.
 PDB Complex III
1BE3
The domain with (bc1 Complex)
attached Rieske Fe-S has
a flexible link to the rest
of the complex.
(Fe-S protein in green.)
Fe-S changes position
during e transfer.

membrane
heme bH
After Fe-S extracts an
heme bL
efrom QH2, it moves
closer to heme c1, to Fe-S
which it transfers the e. heme c1
View an animation.
 PDB Complex III
1BE3
After the 1st e transfer (bc1 Complex)
from QH2 to Fe-S, the CoQ
semiquinone is postulated
to shift position within the
Q-binding site, moving
closer to its e acceptor,
heme bL.

membrane
heme bH
This would help to
prevent transfer of the heme bL
2nd electron from the Fe-S
semiquinone to Fe-S. heme c1
Complex III is an PDB-1BGY Complex III
obligate homo-dimer. homo-dimer
Fe-S in one half of the
dimer may interact with
bound CoQ & heme c1
in the other half of the
dimer.
Arrows point at:
• Fe-S in the half of
complex colored
white/grey Fe-S
• heme c1 in the half of heme c1
complex with proteins
colored blue or green.
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

Complex IV ++
(Cytochrome 4H+ 4H+
cyt c
2H+
Oxidase): Intermembrane Space
Electrons are donated to complex IV, one at a time, by
cytochrome c, which binds from the intermembrane space.
Each e passes via CuA & heme a to the binuclear center,
buried within the complex, that catalyzes O2 reduction:
4e + 4H+ + O2 → 2H2O.
Protons utilized in this reaction are taken up from the matrix
compartment.
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

++
cyt c
4H+ 4H+ 2H+
Intermembrane Space

H+ pumping by complex IV:

In addition to protons utilized in reduction of O2, there
is electron transfer-linked transport of 2H+ per 2e
(4H+ per 4e) from the matrix to the intermembrane
space.
Structural & mutational studies indicate that protons pass
through complex IV via chains of groups subject to
protonation/deprotonation, called "proton wires."
These consist mainly of chains of buried water molecules,
along with amino acid side-chains, & propionate side-
chains of hemes.
Separate H+-conducting pathways link each side of the
membrane to the buried binuclear center where O2
reduction takes place.
These include 2 proton pathways, designated "D" & "K"
(named after constituent Asp & Lys residues) extending
from the mitochondrial matrix to near the binuclear center
deep within complex IV.
Images in web pages of: IBI, & Crofts.
A switch mechanism controlled by the reaction cycle is
proposed to effect transfer of a proton from one half-
wire (half-channel) to the other.
There cannot be an open pathway for H+ completely
through the membrane, or oxidative phosphorylation
would be uncoupled. (Pumped protons would leak back.)
Switching may involve conformational changes, and
oxidation/reduction-linked changes in pKa of groups
associated with the catalytic metal centers.
Detailed mechanisms have been proposed.
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

++
cyt c
4H+ 4H+ 2H+
Intermembrane Space

Ejection of a total of 20 H+ from the matrix per 4 e

transferred from 2 NADH to O2 (10 H+ per ½O2).
Not shown is OH that would accumulate in the matrix as
protons, generated by dissociation of water (H2O
 H+ + OH), are pumped out.
Also not depicted is the effect of buffering.
 ADP + Pi ATP

F1 3 H+
matrix
Fo
intermembrane
space

ATP synthase, embedded in cristae of the inner

mitochondrial membrane, includes:
 F1 catalytic subunit, made of 5 polypeptides
with stoichiometry .
 Fo complex of integral membrane proteins that
mediates proton transport.
 ADP + Pi ATP

F1 3 H+
matrix
Fo
intermembrane
space

F1Fo couples ATP synthesis to H+ transport into the

mitochondrial matrix. Transport of least 3 H+ per ATP is
required, as estimated from comparison of:
 G for ATP synthesis under cellular conditions (free
energy required)
 G for transfer of each H+ into the matrix, given the
electrochemical H+ gradient (energy available per H+).
 Matrix ADP + Pi ATP

H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

F1
2 e ––
I Q III IV
Fo
++
cyt c 3H+
4H+ 4H+ 2H+
Intermembrane Space

The Chemiosmotic Theory of oxidative phosphorylation,

for which Peter Mitchell received the Nobel prize:
Coupling of ATP synthesis to respiration is indirect,
via a H+ electrochemical gradient.
Matrix ADP + Pi ATP

H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

F1
2 e ––
I Q III IV
Fo
++
cyt c 3H+
4H+ 4H+ 2H+
Intermembrane Space

Chemiosmotic theory - respiration:

Spontaneous e transfer through complexes I, III, & IV is
coupled to non-spontaneous H+ ejection from the matrix.
H+ ejection creates a membrane potential (, negative
in matrix) and a pH gradient (pH, alkaline in matrix).
 Matrix ADP + Pi ATP

H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

F1
2 e ––
I Q III IV
Fo
++
cyt c 3H+
4H+ 4H+ 2H+
Intermembrane Space
Chemiosmotic theory - F1Fo ATP synthase:
Non-spontaneous ATP synthesis is coupled to spontaneous
H+ transport into the matrix. The pH & electrical gradients
created by respiration are the driving force for H+ uptake.
H+ return to the matrix via Fo "uses up" pH & electrical
gradients.
 Transport of ATP, ADP, & Pi

 ATP produced in the mitochondrial matrix must exit to

the cytosol to be used by transport pumps, kinases, etc.
 ADP & Pi arising from ATP hydrolysis in the cytosol
must reenter the matrix to be converted again to ATP.
 Two carrier proteins in the inner mitochondrial
membrane are required.
 The outer membrane is considered not a permeability
barrier. Large outer membrane VDAC channels are
assumed to allow passage of adenine nucleotides and Pi.
 ADP + Pi ATP matrix
lower [H+]
ATP4
__

++
3 H+ ATP4 ADP3 H2PO4 H+
energy
requiring higher [H+]
reactions ADP + Pi cytosol

Adenine nucleotide translocase (ADP/ATP carrier) is an

antiporter that catalyzes exchange of ADP for ATP
across the inner mitochondrial membrane.
At cell pH, ATP has 4 () charges, ADP 3 () charges.
ADP3/ATP4 exchange is driven by, and uses up,
membrane potential (one charge per ATP).
 ADP + Pi ATP matrix
lower [H+]
ATP4
__

++
3 H+ ATP4 ADP3 H2PO4 H+
energy
requiring higher [H+]
reactions ADP + Pi cytosol

Phosphate re-enters the matrix with H+ by an electroneutral

symport mechanism. Pi entry is driven by, & uses up, the
pH gradient (equivalent to one mol H+ per mol ATP).
Thus the equivalent of one mol H+ enters the matrix with
ADP/ATP exchange & Pi uptake. Assuming 3H+ transported
by F1Fo, 4H+ total enter the matrix per ATP synthesized.
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

++
cyt c
4H+ 4H+ 2H+
Intermembrane Space

Questions: Based on the assumed number of H+ pumped

out per site shown above, and assuming 4 H+ are
transferred back to the matrix per ATP synthesized:
 What would be the predicted P/O ratio, the # of ATP
synthesized per 2e transferred from NADH to ½ O2?
 What would be the predicted P/O ratio, if the e source is
succinate rather than NADH?
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
For, summing up I Q III IV
synthesis of ~P ++
bonds via ox cyt c
4H+ 4H+ 2H+
phos, assume: Intermembrane Space

 2.5 ~P bonds synthesized during oxidation of NADH

produced via Pyruvate Dehydrogenase & Krebs Cycle
(10 H+ pumped; 4 H+ used up per ATP).
 1.5 ~P bonds synthesized per NADH produced in the
cytosol in Glycolysis (electron transfer via FAD to CoQ).
 1.5 ~P bonds synthesized during oxidation of QH2
produced in Krebs Cycle (Succinate Dehydrogenase –
electrons transferred via FAD & Fe-S to coenzyme Q).
 All Quantities Per Glucose
QH2 ~P bonds ~P bonds
~P bonds
NADH produced 1.5 or 2.5 1.5 per Total ~P
Pathway ATP or
produced (via per NADH QH2 in bonds
GTP direct
FADH2) in oxphos oxphos
Glycolysis
Pathway
Pyruvate
Dehydrogenase

Krebs Cycle
Sum of
Pathways
 a ADP added
An oxygen electrode
may be used to record
b ADP all
[O2] in a closed vessel. [O2] c
converted
to ATP
Electron transfer, e.g.,
NADH  O2, is
monitored by the rate
time
of O2 disappearance.
Above is represented an O2 electrode recording while
mitochondria respire in the presence of Pi and an e donor
(succinate or a substrate of a reaction to generate NADH).
The dependence of respiration rate on availability of ADP,
the ATP Synthase substrate, is called respiratory control.
 a ADP added

b ADP all
[O2] c
converted
to ATP

time

Respiratory control ratio is the ratio of slopes after and

before ADP addition (b/a).
P/O ratio is the moles of ADP divided by the moles of O
consumed (based on c) while phosphorylating the ADP.
Chemiosmotic explanation of respiratory control:
Electron transfer is obligatorily coupled to H + ejection
from the matrix. Whether this coupled reaction is
spontaneous depends on pH and electrical gradients.
Reaction G
e transfer (NADH  O2) negative value*
H+ ejection from matrix positive; depends on H+
gradient**
e- transfer with H+ ejection algebraic sum of above
*Go' = nFEo' = 218 kJ/mol for 2 e NADH  O2.
**For ejection of 1 H+ from the matrix:
G = RT ln ([H+]cytosol/[H+]matrix) + F
G = 2.3 RT (pHmatrix  pHcytosol) + F
 Matrix ADP + Pi ATP

H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

F1
2 e ––
I Q III IV
Fo
++
+ + cyt c + 3H+
4H 4H 2H
Intermembrane Space

With no ADP, H+ cannot flow through Fo. pH &  are

maximal. As respiration/H+ pumping proceed, G for H+
ejection increases, approaching that for e transfer.
When the coupled reaction is non-spontaneous,
respiration stops. This is referred to as a static head.
In fact there is usually a low rate of respiration in the
absence of ADP, attributed to H+ leaks.
 Matrix ADP + Pi ATP

H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

F1
2 e ––
I Q III IV
Fo
++
cyt c 3H+
4H+ 4H+ 2H+
Intermembrane Space

When ADP is added, H+ enters the matrix via Fo, as ATP

is synthesized. This reduces pH & .
G of H+ ejection decreases.
The coupled reaction of electron transfer with H+ ejection
becomes spontaneous.
Respiration resumes or is stimulated.
 OH

NO2

NO2
2,4-dinitrophenol

Uncoupling reagents (uncouplers) are lipid-soluble

weak acids. E.g., H+ can dissociate from the OH group
of the uncoupler dinitrophenol.
Uncouplers dissolve in the membrane and function as
carriers for H+.
Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e
I Q III IV uncoupler

+ + cyt c
4H 4H 2H+ H+
Intermembrane Space

Uncouplers block oxidative phosphorylation by

dissipating the H+ electrochemical gradient.
Protons pumped out leak back into the mitochondrial
matrix, preventing development of pH or .
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e
I Q III IV uncoupler

+ + cyt c
4H 4H 2H+ H+
Intermembrane Space

With uncoupler present, there is no pH or .

G for H+ ejection is zero
G for e transfer coupled to H+ ejection is maximal
(spontaneous).
Respiration proceeds in the presence of an uncoupler,
whether or not ADP is present.
 ADP + Pi ATP

F1
3 H+
matrix
Fo
intermembrane
space

ATPase with H+ gradient dissipated

G for H+ flux is zero in the absence of a H+ gradient.

 Hydrolysis of ATP is spontaneous.
The ATP Synthase reaction runs backward in presence
of an uncoupler.
 Uncoupling Protein
An uncoupling protein (thermogenin) is produced in
brown adipose tissue of newborn mammals and
hibernating mammals.
This protein of the inner mitochondrial membrane
functions as a H+carrier.
The uncoupling protein blocks development of a H+
electrochemical gradient, thereby stimulating
respiration. G of respiration is dissipated as heat.
This "non-shivering thermogenesis" is costly in terms
of respiratory energy unavailable for ATP synthesis,
but provides valuable warming of the organism.