Oxidative Phosphorylation

General Considerations
• How do we define oxidative
phosphorylation?
– formation of ATP using the energy released by
the transfer of electrons from NADH and
FADH2 through a series of electron carriers
• What couples the formation of ATP to the
transfer of electrons?
– a proton gradient
 General Considerations
• Where in the cell does oxidative
phosphorylation take place?
– inner mitocondrial membrane
• What do we know about the mitocondrial
membranes?
– outer membrane – reasonably permeable
• contains porins – VDAC
– inner membrane – relatively impermeable
 Origin of Mitocondria
• What is the believed origin of mitocondria?
– endosymbiosis
• What evidence supports this idea?
– mitcondrial DNA
– machinery for transcription and translation
– similarity of genome to bacteria
 Redox Potentials and Free
Energy Changes
• How does one determine the redox potential of a
substance?

standard reference half-cell

sample half-cell
 Redox Potentials and Free
Energy Changes
• What is the relationship between change in redox
potential and change in free energy?
– G01 = -nF E10
• n = number of electrons transferred
• F =faraday (constant, 23.06 kcal/mole/volt)
– Can calculate free energy change from reduction
potentials of the reactants
• By knowing the electron transfer potential of
NADH relative to O2 one can calculate the amount
of free energy released when O2 is reduced by
NADH.
 Redox Potentials and Free
Energy Changes
• One can also quantify the energy associated
with a proton gradient.
– G = RTln(c2/c1) + ZF V
• c2 = concentration on one side of membrane
• c1 = concenetration on other side of membrane
• Z = electrical charge of transported material
• F = Faraday constant (23.06 kcal/mole/volt)
 Electron Transport
• What determines the rate of electron transport?
– distance between donor and acceptor
 Electron Transport
– driving force or free energy change
 Electron Transport
• What are the
complexes making up
the respiratory chain?
– three proton pumps
– one link to citric acid
cycle
 Electron Transport
• What is the role of ubiquinone or coenzyme Q?
 Electron Transport
• What happens to the electrons from NADH?
– enter ETS at NADH-Q oxidoreductase
 Electron Transport
• Initial step is transfer of electrons to FMN a
prosthetic group of the enzyme
 Electron Transport
• Electrons are then transferred to iron-sulfur
clusters another prosthetic group
 Electron Transport
• Electrons from clusters transferred to
coenzyme Q
– as a result of electron transfer four protons are
pumped out of mitocondrial matrix

• Reaction summarized:
– NADH + Q + 5H+matrix  NAD+ + QH2 + 4H+cytosol
 Electron Transport
• Coenzyme Q also serves as entry point for
electrons from FADH2 from oxidation of
succinate
– succinate-Q reductase complex
• inner mitocondrial membrane
• FADH2 transfers electrons to iron-sulfur
clusters then to Q
– no protons are pumped
 Electron Transport
• Q-cytochrome c oxidoreductase catalyzes
the transfer of electrons from Q to
cytochrome c
– What is a cytochrome?
• electron transferring protein with heme prosthetic
group
• transfers only electrons
• iron in heme goes between Fe+2 and Fe+3
 Electron Transport
• Q-cytochrome c
oxidoreductase
contains 3 hemes and
a iron-sulfur cluster
 Electron Transport
• What is the Q cycle?
– mechanism of coupling of electron transfer from Q to
cytochrome c to proton transport
 Electron Transport
• What is the function of cytochrome c
oxidase?
– reduction of oxygen to water
• What are the major prosthetic groups of this
complex?
– CuA/CuA
– heme a
– heme a3-Cub
Cytochrome c Oxidase
 Cytochrome c Oxidase
• Mechanism of action
 Cytochrome c Oxidase
• cytochrome c oxidase
pumps four additional
protons from matrix
for a total of eight
protons removed from
matrix
Electron Transport System
Electron Transport System
 Electron Transport
• Toxic derivatives of molecular oxygen may
be formed by partial reduction
_ _
O2  O2  O2 2

superoxide peroxide
anion
 Electron Transport
• How does the cell protect itself against
these reactive oxygen species?
– makes use of superoxide dismutase and catalase
_
– 2O2 + 2H+  O 2 + H2O2
– 2H2O2  O2 + 2H2O
 ATP Synthesis
• What is the chemiosmotic hypothesis?
– ATP synthesis and electron transport are coupled by
proton gradient across mitocondrial membrane
 ATP Synthesis
• What is ATP synthase
and what do we know
about its structure?
– consists of F1 and F0
– F1 has 5 types of
polypeptide chains
• 3,3,,,
– F0 contains proton
channel
• 10-14 c subunits
• a,b2 subunits
 ATP Synthesis
• How is ATP synthesized?
 ATP Synthesis
• What is the role of the proton gradient in ATP synthesis?
– part of binding-change mechansm
• 3  subunits promote ADP & P binding, ATP synthesis, ATP release
 ATP Synthesis
• How does proton flow
through F0 drive the
rotation of the 
subunit?
– each c subunits
consists of 2  helices
with one helix
containing an aspartic
acid residue
– a subunit contains two
proton half channels
 ATP Synthesis
• Proton enters half-channel, neutralizes charge on aspartate
• c can rotate clockwise
• proton can move into matrix
 ATP Synthesis
• Since c ring is linked to  and  subunits, as
c turns these subunits rotate
– rotation protmotes synthesis of ATP via
binding-change mechanism
– each 3600 rotation of  subunit leads to
synthesis of 3 ATP’s
• 10 protons generate 3 ATP’s
• each ATP requires transport of about 3 protons
 Mitocondrial Shuttles
• Reoxidation of
cytosolic NADH
requires shuttle
mechanism
– glycerol 3-phosphate
shuttle
• found in muscle
 Mitocondrial Shuttles
• malate-aspartate shuttle
– heart and liver
 Mitocondrial Shuttles
• What is an ATP-ADP translocase?
– transport protein allowing ATP to exit mitocondrion and
ADP to enter
– result in moving one negative charge out of matrix
• decreases proton motive force
 Mitocondrial Shuttles
• Other mitocondrial transport proteins act as
shuttles
 Regulation of Respiration
• Energy formed from
oxidation of glucose
• 3 protons = 1 ATP
• 1 proton used to move
ATP
• one pair of electons
from NADH = 2.5
molecules of ATP
 Regulation of Respiration
• What controls rate of electron transport?
 Regulation of Respiration
• Oxidative
phosphorylation can
be inhibited by many
substances
 Regulation of Respiration
• What are uncoupling agents?
– transport protons across mitocondrial membrane
 Regulation of Respiration
• Does uncoupling serve any useful purpose?
– body heat generation