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Oxidative Phosphorylation
Pratt and Cornely, Chapter 15
Goal: ATP Synthesis
Overview
• Redox reactions
• Electron transport chain
• Proton gradient
• ATP synthesis
• Shuttles
Standard Reduction Potential
½ O2 + 2 e + 2 H+ H2O
• “potential to be reduced”
• Listed according to
oxidized compound being
reduced
• High RP means that the
compound is a strong
oxidizer (ie O2)
• Its conjugate is a strong
reducing agent (ie NADH)
Half Reactions
• Reduction potential
written in terms of
a reduction half
reaction
• Aox  Ared
• Example: Calculate
DEo for reduction of
FMN by NADH,
given the Eo of
FMN to be -0.30V
• Then calculate DGo’
NADH + FMN  NAD+ + FMNH2
Redox reactions: electricity
•
•
•
•
NAD+: Eo’ = -.32
FMN: Eo’=-.30
DEo’ = +0.02V
Calculate DGo’:2 etransfer
• DGo’ = -nFDEo’
= -2(96485 J/mol
V)(0.02V)
= -3.9 kJ/mol
Numerous Redox Substrates
• Chain of increasing
reduction potential
(potential to accept e-)
• Mobile carriers vs.
within Complex
• Types of redox groups
– Organic cofactors
– Metals (iron/sulfur
clusters)
– Cytochromes
– O2
Overall chain from NADH
•
•
•
•
•
•
•
NADH
Complex I
Q
Complex III
Cytochrome c
Complex IV
O2
Within Complexes
• FAD/FMN
– Prosthetic group
– Bridge from 2 e- donors
to 1 e- donors
• Iron-sulfur clusters
Coenzyme Q: Mobile Carrier
• 1 or 2 e- carrier, 1 eacceptor/donor
• Ubiquinone is a mobile
carrier
• Can diffuse through
nonpolar regions easily
• “Q pool” made by
Complex I and Complex II
(and others)
Cytochrome c
• Mobile carrier
• Protein/heme
• 1 electron carrier
Oxygen: the final electron acceptor
• Water is produced—has very low reactivity, very
stable
• Superoxide, peroxide as toxic intermediates
• Overall reaction
NADH + H+ + ½ O2  NAD+ + H2O
Flow Through Complexes
Compartmentalization
Protonmotive Force
• NADH + H+ + ½ O2  NAD+ + H2O + 10 H+ pumped
• succinate + ½ O2  fumarate + H2O + 6 H+ pumped
Complex I
• NADH  Q through
– FMN
– Iron-sulfur clusters
• “Q pool”
• 4 protons pumped
– Proton wire
Complex III
•
•
•
•
QH2  cytochromes
4 protons pumped
Through Q cycle
Problem 10: An ironsulfur protein in Complex
III donates an electron to
cytochrome c. Use the
half reactions below to
calculate the standard
free energy change. How
can you account for the
fact that this process is
spontaneous in the cell?
FeS (ox) + e-  FeS (red)
Eo’ = 0.280 V
Cyt c (Fe3+) + e-  cyt c (Fe2+) Eo’ = 0.215 V
Complex IV
• Cytochromes  O2
• Stoichiometry of half of
an oxygen atom
Complex II (and others)
• Non-NADH sources
– Complex II (part of the
citric acid cycle)
– Fatty acid oxidation
– Glycolysis NADH shuttle
(Glycerol-3-phosphate)
• Bypasses Complex I
– Loss of 4 protons
pumped
Two paths of NADH into Matrix
• NADH of glycolysis
must get “into” matrix
• Not direct
• Needs either
– malate-aspartate
shuttle (liver)
– Glycerol-3-phosphate
shuttle (muscle)
• Costs 1 ATP worth of
proton gradients, but
allows for transport
against NADH
gradient
Glycerol-3-phosphate Shuttle
• Glycerol phosphate
shuttle (1.5 ATP/NADH)
• Produces QH2
• Operational in some
tissues/circumstances
Net ATP Harvest from Glucose
• Glycolysis = 2 ATP
– Plus 3 or 5 ATP from
NADH
– What leads to
difference in this case?
• Pyruvate DH = 5 ATP
• Citric Acid Cycle = 20
ATP
• Total: 30-32
ATP/glucose
Overall
• Chemiosmosis
• 10 protons shuttled
from matrix to
intermembrane space
• Makes pH gradient and
ion gradient
Protonmotive Force and
Oxidative Phosphorylation
• Flow of electrons is
useless if not
coupled to a useful
process
– Battery connected
to wire
• Proton gradient
across
mitochondrial
membrane
Proton Gradient
• Gradient driven by concentration difference +
charge difference
– Assume DpH 0.5 and 170mV membrane potential
Using the Gradient
• Coupled to ATP synthesis
• Uncouplers used to show
link of oxygen uptake and
ATP synthesis
Uncouplers
• “Uncouple”
protonmotive force
from ATP synthase
– DNP pKa / solubility
perfectly suitable
Respiratory Poisons
• Other respiration
poisons
– Cyanide—binds Complex
IV in place of oxygen
Complex V: ATP Synthase
• Molecular motor
• Rotor: c, g, e
– Proton channel
Proton Channel
• Protons enters channel
between rotor and
stator
• Rotor rotates to release
strain by allowing
proton to enter matrix
• 8- 10 protons = full
rotation
– Species dependent
• “Stalk” (g) moves
inside the“knob”—
hexameric ATP
synthase
• Knob held
stationary by “b”
Hexameric Knob
Binding-Change Mechanism
• Stalk causes ATP synthase to
have three different
conformations: open, loose,
tight
• In “tight” conformation,
energy has been used to cause
an energy conformation that
favors ATP formation
Problem 39
• How did these key experiments support the
chemiosmotic theory of Peter Mitchell?
– The pH of the intermembrane space is lower than
the pH of the mitochondrial matrix.
– Oxidative phosphorylation does not occur in
mitochondrial preparations to which detergents
have been added.
– Lipid-soluble compounds inhibit oxidative
phosphorylation while allowing electron transport
to continue.
Energy Accounting
• ATP costs 2.7 protons
– 8 protons produces 3 ATP
• NADH pumps 10 protons when 2 e- reduce ½ O2
– 4 protons in Complex I, 4 protons in Complex III, and 2
protons in Complex IV
• P/O ratio--# of phosphorylation per oxygen atom
– 10H+/NADH (1 ATP/2.7 H+) = 3.7 ATP/NADH
– 6H+/QH2 (1 ATP/2.7 H+) = 2.3 ATP/QH2
• In vivo, P/O ratio closer to 2.5 and 1.5 due to other
proton “leaking”
– i.e. importing phosphate
Active Transport of ATP
• ATP must go out, ADP and Pi must go in
• Together, use about 1 proton of protonmotive
force
Regulation of Oxidative
Phosphorylation
• Electron transport is
tightly coupled to ATP
production
– Oxygen is not used
unless ATP is being made
– Avoid waste of fuels
– Adding ADP causes
oxygen utilization
– Respiratory control
Problem 47
• A culture of yeast grown under anaerobic
conditions is exposed to oxygen, resulting in
dramatic decrease in glucose consumption.
This is called the Pasteur effect. Explain.
• The [NADH]/[NAD+] and [ATP]/[ADP] ratios
also change when an anaerobic culture is
exposed to oxygen. Explain how the ratios
change and what effect this has on glycolysis
and the citric acid cycle in yeast.