Chapter 21: The Proton Motive Force
Problems: 1,3,5,6,8,10,12-14, 17, 19-20, 23-24, 28, 30, 32-34.
ETC:
NADH + ½O2 + H+
ATP Syn: ADP + Pi + H+
H2O + NAD+
ATP + H2O
G = -220.1 kJ/mole
Proton
Gradient
G = + 30.5 kJ/mole
Proton motive
force
21.1 A proton gradient powers the
synthesis of ATP.
21.2 Shuttles allow movement
mitochondrial membranes.
21.3 Cellular respiration is regulated
by the need for ATP
21.1 A Proton Gradient Powers the
Synthesis of ATP
The Chemiosmotic Theory
• Proposed by Peter Mitchell
in the 1960’s
(Nobel Prize 1978)
The proton gradient generated by the
oxidation of NADH and FADH2 is
called the proton-motive force.
The proton-motive force powers the
synthesis of ATP.
Heterologous experimental systems
confirmed that proton gradients can
power ATP synthesis.
Coupled nature of respiration
in mitochondria
Oxidation of substrates is
coupled to the
phosphorylation of ADP
Respiration (consumption
of oxygen) proceeds only
when ADP is present
(a) O2 consumed only with ADP,
excess Pi.
(b) (+) Uncoupler DNP (O2
consumed without ADP).
The amount of O2
consumed depends upon
the amount of ADP
added
Fig 14.4 2,4-Dinitrophenol: an
uncoupler
•Uncouplers stimulate the oxidation of substrates in the absence of ADP.
•Uncouplers are lipid-soluble weak acids.
•Both acidic and basic forms can cross the inner mitochondrial membrane.
•Uncouplers deplete any proton gradient by transporting protons across the
membrane.
Mitchell’s postulates for chemiosmotic
theory
1. Intact inner mitochondrial membrane is required
(to maintain a proton gradient).
2. Electron transport through the ETC generates
a proton gradient (pumps H+ from the matrix to
the intermembrane space).
3. The membrane-spanning enzyme, ATP synthase,
catalyzes the phosphorylation of ADP in a reaction
driven by movement of H+ across the inner membrane
into the matrix.
The Protonmotive Force
•Protonmotive force (p) is the energy of the proton
concentration gradient .
1. Chemical contribution
Gchem = nRT ln ([H+]in / [H+]out)
n = number of protons
translocated)
2. Electrical contribution:
=membrane potential
(in – out)
Gelect = zF
(z = charge (1.0 for H+),
F =96,485 JV-1mol-1 )
p =  - (0.059 V) pH
p = - 0.17 - 0.03 = - 0.20 V
ATP synthase
• F0F1 ATP Synthase uses the proton
gradient energy for the synthesis of ATP
• An F-type ATPase which generates ATP
• Composed of a “knob-and-stalk”
structure
• F1 (knob) contains the catalytic subunits
• F0 (stalk) has a proton channel which
spans the membrane.
• There are 3 active sites, one in each 
subunit
• The c-- unit forms a “rotor”
• Passage of protons through the Fo
(stalk) into the matrix is coupled to
ATP formation
• Estimated passage of 3 H+ / ATP
synthesized
• Rotation of the  subunit inside the 33
hexamer causes domain movements in the •
-subunits, opening and closing the active
sites
• The a-b--33 unit is the “stator” (the
Fo channel is attached to 33 by the ab- arm)
Passage of protons through the Fo
channel causes the rotor to spin in
one direction and the stator to spin
in the opposite direction
Binding-change mechanism of ATP synthase
 The binding change mechanism
accounts for the synthesis of ATP
in response to proton flow.
 The three catalytic β subunits of
the F1 component can exist in three
conformations:
 In the O (open) form, nucleotides
can bind to or be released from the
β subunit.
 In the L (loose) form, nucleotides
are trapped in the β subunit.
 In the T (tight) form, ATP is
synthesized from ADP and Pi.
The rotation of the γ subunit
interconverts the β subunits.
H+ H+ H+
Three protons = one complete rotation = one ATP
Binding-change mechanism of ATP synthase
1. ADP, Pi bind to an open site.
2. Inward passage of protons, conformation change, ATP
synthesis from ADP and Pi.
3. ATP released from open site, ADP and Pi form ATP in
the tight site.
www.mech.northwestern.edu/courses/389.S02/intro.html
Proton flow around the “c” ring
The number of c rings determines the number of
protons required to synthesize a molecule of
ATP.
The c ring of vertebrates consist of 8 subunits,
making vertebrate ATP synthase the most
efficient known.
Overview of Oxidative Phosphorylation
21.2 Shuttles are Necessary for Aerobic
Oxidation of Cytosolic NADH
• Cytosolic NADH must enter the mitochondria to fuel oxidative
phosphorylation, but NADH and NAD+ cannot diffuse across the
inner mitochondrial membrane
• Two shuttle systems for reducing equivalents:
(1) Glycerol phosphate shuttle: insect flight muscles
(2) Malate-aspartate shuttle: predominant in liver and other
mammalian tissues
The Glycerol 3-phosphate shuttle
(prominent in muscle)
For each mitochondrial NADH, approximately 2.5 ATPs are made; however
for cytoplasmic NADH entering via the G3P shuttle, only 1.5 ATPs are made.
Why?
The Malate-aspartate shuttle
(prominent in liver and heart)
H+ +
+ H+
Entry of ADP into Mitochondria and Exit of ATP
ADP3-
- charge in matrix is depleted
cyto
+ ATP4-matrix
ADP3-matrix + ATP4-cyto
- charge in cytoplasm is increased
ATP-ADP translocase
Cyto.
++++++ -
Matrix
------ +
p
decreases during
ADP/ATP exchange
1 proton transferred from cytoplasm per ATP
Numerous Mitochondrial Transporters
+ ATP synthase = ATP synthasome
21.3 Cellular Respiration is Regulated by the
Need for ATP
Of the 30 molecules of ATP formed by the
complete combustion of glucose, 26 are formed in
oxidative phosphorylation.
The metabolism of glucose to two molecules of
pyruvate in glycolysis yields the remaining four
ATP.
When glucose undergoes fermentation, only two
molecules of ATP are generated per glucose
molecule.
Calculating the Amount of ATP/Glucose
Complex
#H+ translocated/2e-
I II III
4 0
4
IV
2
Since 4 H+ are required for each ATP synthesized:
For NADH: 10 H+ translocated / O (2e-)
ATP/2e- = (10 H+/ 4 H+) = 2.5
For FADH2 = 6 H+/ O (2e-)
ATP/2e- = (6 H+/ 4 H+) = 1.5
O
H3C
NADH + H+
Pyruvate
COOH
Pyruvate dehydrogenase
NAD
CO2
Rate of Ox/Phos is Determined by the Need for ATP
Respiratory Control of Ox/Phos
Electron flow to O2 requires ADP
Energy Charge and Ox/Phos
• Low ADP = low FAD, NAD+,
and CAC.
• Increased ADP = increased
FAD, NAD+, and active CAC.
Electrons do not flow to O2
unless ATP needs to be
synthesized.
Uncoupling ETC from ATP synthesis leads to heat
generation
NADH + ½O2 + H+ → H2O + NAD+ + 52.6 kcal/mol
UCP-1
thermogin
brown fat
Non-shivering thermogenesis
(humans, but not pigs)
ATP
Heat
Inhibition of ETC and Ox/Phos
Inhibitors of ETC
Inhibitors of:
ATP synthase
ATP export
Oligomycin,
dicyclohexylcarbodiimide (DCCD),
DNP
Atractyloside, bongkrekic acid
Mitochondrial Diseases
10 to 15/100,000
Complex I functioning (NADH, etransfer to Q)
Implications for aging, cancer,
degenerative disorders, ROS
generation, nervous system,
heart
General Uses of Proton Gradents
(Cellular Power Generation)