Chapter 11

Respiratory Complexes and ATP

Synthesis
 SLO’s for chapter 11
• 11.1 Define and use correctly the following terms: respiration, electron
transport chain, respiratory chain, proton pump, coenzyme Q, oxidative
phosphorylation, heme-iron, ATP synthase, F0-F1 ATPase, electrochemical
potential, proton gradient, uncoupling agent etc.
• 11.2 Describe the organization of respiratory complexes,
• 11.3 List the respiratory complexes (identify which of them are proton
pumps) in appropriate order, along with intermediate electron carriers.
• 11.4 Compare electron entry via complex I and complex II and in relation to
cellular ATP production.
• 11.5 Explain chemiosmotic model of oxidative phosphorylation, proton
gradients and the role of F0-F1 complex.
• 11.6 Explain the roles of uncoupling agents in ATP generation and
thermogenesis.
 Overview of Oxidative
Phosphorylation
• Oxidative phosphorylation (ox phos): The passage
of electrons from NADH or FADH2 to O2 is tightly
coupled to the phosphorylation of ADP to make
ATP.
• The inner membrane of mitochondria is the site of
oxidative phosphorylation.
• Respiration (the set of redox reactions involving the
electron transfer chain, or ETC) is different from
phosphorylation, but the two kinds of reaction are
coupled through gradients in pH and in electrical
voltage.
Flow of Electrons in Respiration
• Electrons are transferred through a series of
carriers, including flavins, non-heme iron
compounds, hemes, and quinones.
• Overall, electrons are transferred from NADH,
through a variety of intermediates, onto O2, while
the NADH is oxidized to form NAD+.
Electron transfer chain (ETC) coupled to
Oxidative Phosphorylation
 NAD+ NADH (From glycolysis)
Peripheral membrane protein

FAD+
Interstitial space Mito-
G3PDH
FADH2

Inner mitcohondrial
membrane FAD+ FADH2

TCA cycle Succinate dehydrogenase

 Coenzyme Q
• Quinone structure.
• Abbreviations: Q, QH2, etc.
• Known also as "ubiquinone" because it is ubiquitous
in cells.
• Allows one-electron transfers but overall the
compound can carry two electrons.
• Hydrophobic tail, an "anchor" to hold it in a lipid
bilayer.
– Allows considerable lateral mobility in the bilayer,
for easy transport of reducing equivalents.
 Cytochromes
• Family of proteins containing heme prosthetic
groups.
• Ubiquitous 1-electron transfer agents.
• Involved in respiratory complexes, drug
metabolism, etc.
• The mechanism by which electrons pass to
and from the heme group is still uncertain.
 Iron-Sulfur (Fe-S) Complexes
• Prosthetic groups that contain one, two, or four
Fe atoms in complex with sulfides. The sulfides
may be of inorganic type, or from the sulfhydryl
group of Cys residues.
• The iron atoms cycle between +2 and +3
oxidation states; one electron is transferred at a
time.
• These clusters are usually buried inside
proteins, well away from contact with solvent
water.
 NADH-Q reductase -- "Complex I"
• Transfers electrons from NADH to coenzyme Q,
forming NAD+ and QH2
• Pumps protons out of the mitochondria.
• Net reaction:
 More Complete Representation
• Four protons are expelled for each molecule of NADH that
is oxidized to NAD+, while five protons are taken up from
the matrix during the process of reducing coenzyme Q and
oxidizing NADH.
• The inner mitochondrial membrane is typically polarized,
with negative (N) and positive (P) sides. There is a
transmembrane electrical potential of around 0.2 volts.
• Protons on the negative (or matrix) side of the membrane
have a subscript N, while those outside the matrix, on the
positive side, have a subscript P.
 Features of Complex I
• A flavoprotein that uses FMN.
• Also contains non-heme iron centers (Fe-S
complexes).
• Multiple subunits (42 polypeptide chains), and
some of these are buried in the lipid bilayer of
the inner mitochondrial membrane.
• Complex I helps to make ATP by coupling
proton pumping to the phosphorylation reaction
performed by a distinct ATP synthase complex,
but complex I itself does NOT make ATP.
 Complex II
• Transfers electrons, but it does not act as a proton
pump.
• Contains prosthetic groups: FAD, FeS centers, and
a b-type cytochrome with a heme group.
• There is no coupling to ATP synthesis here because
this complex is not a proton pump.
• A connection here of respiration to the TCA cycle;
this is the same enzyme as in the TCA cycle.
• The overall reaction is:
The Reaction Cycle for Complex II.
• Flavin group is re-generated after each cycle of
electron transfers.
• A similar cycle operates in the flavoenzyme that
converts glycerol 3-phosphate to
dihydroxyacetone phosphate, in the glycerol
phosphate shuttle system.
 "Complex III"
• Multiple subunits.
• Embedded in inner membrane.
• Contains b- and c-type cytochromes, and an iron-
sulfur center. It also has a binding site for the
protein cytochrome c, located on the outer side of
the mitochondrial membrane.
• This respiratory complex is a proton pump.
• Net reaction:
 "Complex IV"
• Transfers electrons and pumps protons
• The last ("terminal") complex in the respiratory
chain
• Membrane-embedded
• Multiple subunits
• Prosthetic groups include two different
cytochromes (cyt a and cyt a3) and two copper
ions. It also has a binding site for the protein
cytochrome c,
 Features of Complex IV
• Ultimate electron acceptor is O2.
– H2O is a reduced form of oxygen, since it has
gained electrons.
• Catalyzes a four-electron reduction of O2 (highly
unusual).
• 4 separate molecules of reduced cytochrome c bring
in those four electrons.
• Mechanism of the reduction/oxidation reaction is not
understood completely.
• The net reaction is:
 ATP Synthase
(F0-F1 Complex, Complex V)
• This is the enzyme that is mainly responsible for
ATP synthesis from ADP and Pi in metabolism.
• ATP synthesis is tightly coupled to the flow of
electrons down to O2 through the respiratory
complexes.
• The ATP synthase has two major domains and a
multitude of polypeptide chains.
• The enzyme’s structure has been highly conserved
in evolution; bacterial and eukaryotic forms are very
similar.
 Proton Free Energy and ATP
Synthesis
• Free energy is released as electrons pass along the
respiratory chain.
• This is used to pump protons out of the matrix (an
“uphill” process).
• This creates a concentration gradient, and a voltage
gradient, across the membrane.
• The protons “outside” are at a higher free energy
than those “inside”.
• When those outside protons move back inside, they
give up that free energy.
• This release of free energy is what drives the
unfavorable phosphorylation of ADP to ATP.
 Uses for the Electrochemical
Potential of Protons

• Heat production
• Flagellar rotation
• Active transport of a variety of substances across
membranes (sugars, amino acids, ions, etc.
• NADPH synthesis
• ATP synthesis.
 Overall ATP production
• Oxidation of each NADH from the TCA cycle yields
2.5 ATP.
• Oxidation of each FADH2 from the TCA cycle yields
1.5 ATP.
• Oxidation of each NADH from glycolysis yields 1.5
ATP due to G3PDH shuttle cost.
 Proton Pumping Summary

• Complex I ejects 4 protons as it oxidizes one

molecule of NADH.
• Complex II is not a proton pump.
• Complex III ejects 4 protons also, as it oxidizes one
molecule of QH2.
• Complex IV ejects 2 protons per atom of oxygen
(not per molecule of O2 !) that is used to make one
molecule of water.
• The ATP synthase is a distinct protein complex,
different from all the respiratory complexes. It uses
incoming proton flow to drive ATP synthesis.
 Poisons of Respiration
• Block electron flow: Rotenone and amytal inhibit
electron transfer to CoQ in complex I. Antimycin
acts on Complex III and inhibits it. CN-, N3-, and CO
act on Complex IV; they bind to heme and inactivate
it.

• Inhibit phosphorylation: Oligomycin blocks the F0F1

ATPase by binding to a particular. Arsenate
substitutes for phosphate in the phosphorylation
reaction, but is spontaneously and rapidly
hydrolyzed after being joined to ADP.
 Ischemia and ATP Hydrolysis
• Ischemia: lack of O2 for the cell.
• Anaerobic metabolism will now be used to produce
ATP. This will generate lactate and pyruvate.
• If these acids build up, the proton-motive force
across the mitochondrial membrane will collapse.
Cell now needs to shut down the ATP synthase, so
that it will not reverse itself and become an ATPase.
• There is a special inhibitory protein, IF1, that binds
to and inhibits two ATP synthase complexes,
simultaneously. It acts this way as a dimer, and it
only dimerizes when the pH drops below about 6.5
in the matrix.
 Brown Fat
• Brown fat is a type of adipose tissue that serves to
generate heat without shivering.

• The brown color comes from the large numbers of

mitochondria, with heme groups, in the cells.
 Brown Fat cont.
• Heat is generated by uncoupling respiration from ATP
synthesis in these cells. They contain a special
uncoupling protein, thermogenin.
– Thermogenin is a transmembrane protein extending
across the inner mitochondrial membrane. It serves
as a channel for protons to enter the mitochondrial
matrix without having them first pass through the
ATP synthase complex.
• The bypass of the ATP synthase releases free energy,
in the form of heat. This raises the temperature of the
cell, and helps newborns to maintain homeostasis with
respect to temperature.