Molecular Biochemistry I

Oxidative Phosphorylation

Copyright © 1999-2004 by Joyce J.

Diwan.
All rights reserved.
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

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

Spontaneous e flow through complexes I, III, & IV is

coupled to H+ ejection from the mitochondrial matrix.
Stoichiometry: There is general agreement that
 4H+ are ejected from the mitochondrial matrix per 2e
passing through each of the complexes I and III.
 The ratio is 2H+/2e for complex IV. 
 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):

The mechanism of H+ transport linked to electron flow
through complex I is still not known. 
There is evidence for conformational changes associated
with transmembrane 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 IV (Cyt. Oxidase): Protons are thought to pass

through complex IV via a chain of groups subject to
protonation/deprotonation, called a "proton wire."
Proposed constituents of H+ transport pathways include:
amino acid side-chains, propionate side-chains of the
hemes, & water molecules within the complex.
There appears to be a separate pathway linking the
buried binuclear center to each side of the membrane.
A switch mechanism controlled by the reaction cycle is
proposed to effect transfer of a H+ 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.
The process of switching may involve conformational
changes, and oxidation/ reduction-linked changes in pKa
of groups associated with the metal centers.
 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 en 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 en zy m e Q H 2

The “Q cycle” depends on:

 mobility of CoQ in the lipid bilayer
 existence of binding sites for CoQ within the complex
that stabilize the semiquinone radical, Q·.
 matrix 2 H+
Q Q. QH2 QH2

cyt bH
Complex III
e
cyt bL 

e
Q Q·  Fe-S cyt c1
Q Cycle: 2 H+
cyt c
intermembrane space

As depicted above, 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 Q. QH2 QH2
QH2 gives up
one e that is cyt bH
Complex III
transferred via

hemes bL & bH to e
cyt bL 
a bound Q on the 
e
Q Q·  Fe-S cyt c1
other side of the
2 H+
membrane. intermembrane space
cyt c

Loss of one e to the b hemes, and release of 2 H+ to the

intermembrane space, generates a Q· radical in the site
adjacent to the intermembrane space.
Q· becomes Q as it gives up a second e to the Rieske
iron-sulfur center (Fe-S).
 matrix 2 H+
Q Q. QH2 QH2
Fe-S is
reoxidized by cyt bH
electron transfer Complex III
to cytochrome e
c1, which passes cyt bL 
e
 
the electron out Q Q· Fe-S cyt c1
of the complex 2 H+
to cytochrome c.  intermembrane space cyt c

Some evidence suggests instead a concerted reaction in

which e transfer from QH2 to Fe-S & cytochrome bL is
essentially simultaneous.
But there is agreement about the overall reaction cycle.
 matrix 2 H+
Q Q. QH2 QH2

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

It takes 2 cycles for CoQ bound at a site hear the matrix to

be reduced to QH2, as 2e are transferred 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 e
complex III, cyt bL 
e
 
including net Q Q· Fe-S cyt c1
inputs & outputs 2 H+
of the Q cycle : cyt c
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.
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 2 CoQ
binding sites, one near

membrane
heme bH
heme bH & the other near
heme bL. heme bL
The b hemes are
Fe-S
positioned to provide a
pathway for electrons heme c1
across the membrane.
 PDB Complex III
1BE3
The Rieske iron-sulfur (bc1 Complex)
center (Fe-S) has a
flexible link to the rest
of the complex.
It changes position
during e transfer.

membrane
heme bH
Fe-S extracts an efrom
CoQ, & then moves heme bL
closer to heme c1, to
which it transfers the e. Fe-S
(Fe-S protein in green.) heme c1
Complex III is an PDB-1BGY Complex III
homo-dimer
obligate homo-dimer.
Fe-S in one half of the
dimer interacts 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

++
Simplified cyt c
+ +
animation 4H 4H 2H+
depicting: 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.
 matrix

A conventional view inter-

of mitochondrial cristae membrane
space
structure is
represented here. 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 actually 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.
See also Wadsworth Ctr.
website.
Apparently tubular cristae connect to the inner membrane
via narrow passageways that may limit the rate of proton
equilibration between the lumen of cristae & the
intermembrane space.
 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, states
that 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+
Animation
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?
 Problem
For, summing up synthesis of ~P bonds via oxidative
phosphorylation, assume:
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 (electrons transferred via FAD to
coenzyme Q).
1.5 ~P bonds synthesized during oxidation of FADH2
produced in Krebs Cycle (Succinate Dehydrogenase –
electrons transferred to coenzyme Q).
 All Quantities Per Glucose
~P bonds ~P bonds
FADH2 ~P bonds
NADH 1.5 or 2.5 1.5 per Total ~P
Pathway produced ATP or
produced per NADH FADH2 in bonds
(QH2) GTP direct
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.