Molecular Biochemistry I
Oxidative Phosphorylation
Copyright © 1999-2007 by Joyce J. Diwan.
All rights reserved.
 Conventional view of
mitochondrial
structure is at right.
 Respiratory chain is
in cristae of the inner
membrane.
matrix
cristae
intermembrane
space
inner
outer
 Spontaneous electron
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
cristae
intermembrane
space
inner
membrane mitochondrion
outer
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
2
e
––
through each
I
Q
III
IV
of complexes
I, III, & IV is
++
+
coupled to H
cyt c
+
+
+
4H
4H
2H
ejection from
Intermembrane Space
the matrix.
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+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
++
4H+
4H+
cyt c
2H+
Intermembrane Space
Complex I (NADH Dehydrogenase) transports
4H+ out of the mitochondrial matrix per 2etransferred from NADH to CoQ.
Peripheral domain of a bacterial Complex I
NAD+
NADH
A
FMN
peripheral
domain
 FMN
B
 FMN
matrix
inner mitochondrial
membrane
membrane domain
Complex I
membrane
domain

 N2
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+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
++
+
4H
+
4H
cyt c
2H+
Intermembrane Space
Complex III (bc1 complex):
H+ transport in complex III involves coenzyme Q (CoQ).
O-
O
CH3O
CH 3
CH 3
CH3O
(CH 2 CH
O
C
e-
CH 2)nH
CH3O
CH 3
CH 3
CH3O
(CH 2 CH
O
coenzyme Q
C
CH 2)nH
coenzyme Q •-
e- + 2 H+
OH
CH3O
CH 3
CH 3
CH3O
(CH 2 CH
OH
C
CH 2)nH
coenzyme QH2
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
Q
2 H+
Q.-
QH2
QH2
cyt bH
Complex III
cyt bL e-
One version
of Q Cycle:
Q
e- 
Q·
2 H+
intermembrane space
Fe-S
cyt c1
cyt c
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
QH2 gives up 1eto the Rieske
iron-sulfur center,
Fe-S.
Q
2 H+
Q.-
QH2
QH2
cyt bH
Complex III
Fe-S is reoxidized
cyt bL eeby transfer of the
- 
Q
Q·
Fe-S
e to cyt c1, which
+
2
H
passes it out of the
intermembrane
space
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·.
cyt c1
cyt c
e-
matrix
2 H+
A 2nd is
transferred from
Q
Q.QH2
QH2
the semiquinone to
cyt bH
cyt bL (heme bL)
Complex III
which passes it via
cyt bH across the
cyt
b
e
L
e
membrane to
- 
Q
Q·
Fe-S
cyt c1
another CoQ
bound at a site on
2 H+
cyt c
intermembrane
space
the matrix side.
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
Q
2 H+
Q.-
QH2
QH2
cyt bH
Complex III
cyt bL eQ
e- 
Q·
2 H+
intermembrane space
Fe-S
cyt c1
cyt c
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
Q
2 H+
Q.-
QH2
QH2
cyt bH
Complex III
cyt bL eQ
e- 
Q·
2 H+
intermembrane space
Fe-S
cyt c1
cyt c
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, 2QH2 enter the pathway & one is regenerated.
matrix
Animation
Q
2 H+
Q.-
QH2
QH2
cyt bH
Overall reaction
catalyzed by
complex III,
including net
inputs & outputs
of the Q cycle :
Complex III
cyt bL eQ
e- 
Q·
2 H+
intermembrane space
Fe-S
cyt c1
cyt c
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
Q
2 H+
Q.-
QH2
QH2
cyt bH
Complex III
cyt bL eQ
e- 
Q·
2 H+
intermembrane space
Fe-S
cyt c1
cyt c
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+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
++
4H+
4H+
cyt c
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:
Half of the homodimeric
structure is shown.
PDB
1BE3
Complex III
(bc1 Complex)
Not shown are the CoQ
binding sites near heme
bH and near heme bL.
The b hemes are
positioned to provide a
pathway for electrons
across the membrane.
membrane
Approximate location of
the membrane bilayer is
indicated.
heme bH
heme bL
Fe-S
heme c1
Fe-S changes position
during e- transfer.
After Fe-S extracts an efrom QH2, it moves
closer to heme c1, to
which it transfers the e-.
View an animation.
membrane
The domain with
attached Rieske Fe-S has
a flexible link to the rest
of the complex.
(Fe-S protein in green.)
PDB
1BE3
Complex III
(bc1 Complex)
heme bH
heme bL
Fe-S
heme c1
This would help to
prevent transfer of the
2nd electron from the
semiquinone to Fe-S.
membrane
After the 1st e- transfer
from QH2 to Fe-S, the
CoQ semiquinone is
postulated to shift position
within the Q-binding site,
moving closer to its eacceptor, heme bL.
PDB
1BE3
Complex III
(bc1 Complex)
heme bH
heme bL
Fe-S
heme c1
Complex III is an
obligate homo-dimer.
PDB-1BGY
Complex III
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
• heme c1 in the half of
complex with proteins
colored blue or green.
Fe-S
heme c1
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
Complex IV
(Cytochrome
Oxidase):
2H+ + ½ O2 H2O
––
III
IV
++
4H+
4H+
cyt c
2H+
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+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
++
4H+
4H+
cyt c
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 sidechains 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 halfwire (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+
2 eQ
I
Simplified
animation
depicting:
2H+ + ½ O2 H2O
––
III
IV
++
4H+
4H+
cyt c
2H+
Intermembrane Space
Ejection of a total of 20H+ from the matrix per 4etransferred from 2 NADH to O2 (10H+ 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 a3b3gde.
 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:
 DG for ATP synthesis under cellular conditions (free
energy required)
 DG for transfer of each H+ into the matrix, given the
electrochemical H+ gradient (energy available per H+).
ADP + Pi ATP
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
Fo
++
4H+
F1
4H+
cyt c
2H+
3H+
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.
ADP + Pi ATP
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
Fo
++
4H+
F1
4H+
cyt c
2H+
3H+
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 (DY, negative
in matrix) and a pH gradient (DpH, alkaline in matrix).
ADP + Pi ATP
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
Fo
++
4H+
F1
4H+
cyt c
2H+
3H+
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
ATP4-
matrix
lower [H+]
__
++
3 H+
ATP4- ADP3- H2PO4- H+
energy
requiring
reactions
ADP + Pi
higher [H+]
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
ATP4-
matrix
lower [H+]
__
++
Animation
3 H+
ATP4- ADP3- H2PO4- H+
energy
requiring
reactions
ADP + Pi
higher [H+]
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+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
++
4H+
4H+
cyt c
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+
For, summing up
synthesis of ~P
bonds via ox
phos, assume:
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
++
4H+
4H+
cyt c
2H+
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
Pathway
Glycolysis
Pathway
Pyruvate
Dehydrogenase
Krebs Cycle
Sum of
Pathways
NADH
produced
QH2
~P bonds
produced
ATP or
(via
GTP direct
FADH2)
~P bonds ~P bonds
1.5 or 2.5
1.5 per
per NADH QH2 in
in oxphos oxphos
Total ~P
bonds
An oxygen electrode
may be used to record
[O2] in a closed vessel. [O2]
Electron transfer, e.g.,
NADH  O2, is
monitored by the rate
of O2 disappearance.
a
c
ADP added
b
ADP all
converted
to ATP
time
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
[O2]
c
ADP added
b
ADP all
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
e- transfer (NADH O2)
H+ ejection from matrix
e- transfer with H+ ejection
DG
negative value*
positive; depends on H+
gradient**
algebraic sum of above
*DGo' = -nFDEo' = -218 kJ/mol for 2e- NADH O2.
**For ejection of 1 H+ from the matrix:
DG = RT ln ([H+]cytosol/[H+]matrix) + FDY
DG = 2.3 RT (pHmatrix - pHcytosol) + FDY
ADP + Pi ATP
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
Fo
++
+
4H
F1
+
4H
cyt c
+
2H
3H+
Intermembrane Space
With no ADP, H+ cannot flow through Fo. DpH & DY are
maximal. As respiration/H+ pumping proceed, DG 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.
ADP + Pi ATP
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
2H+ + ½ O2 H2O
––
III
IV
Fo
++
4H+
F1
4H+
cyt c
2H+
3H+
Intermembrane Space
When ADP is added, H+ enters the matrix via Fo, as ATP
is synthesized. This reduces DpH & DY.
DG 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+
2 eQ
I
+
4H
2H+ + ½ O2 H2O
III
IV
+
4H
cyt c
2H+
uncoupler
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 DpH or DY.
Matrix
H+ + NADH NAD+ + 2H+
2 eQ
I
+
4H
2H+ + ½ O2 H2O
III
IV
+
4H
cyt c
2H+
uncoupler
H+
Intermembrane Space
With uncoupler present, there is no DpH or DY.
 DG for H+ ejection is zero
 DG 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
+
3H
matrix
Fo
intermembrane
space
ATPase with H+ gradient dissipated
 DG 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. DG 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.