FUNDAMENTALS 1 11:00-12:00
THURSDAY, AUGUST 27TH, 2010
POPOV
I.
OXIDATIVE PHOSPHORYLATION
Scribe: BRANDON TERRY
Proof: TERRI CALL
Page 1 of 6
Oxidative Phosphorylation [S1]:
a. Oxidative Phosphorylation is the last step in degradation of metabolic fuels.
b. This is the process where electrons that have been obstructed from metabolic fuels will be used to reduce
oxygen.
c. During this process, energy will be synthesized in the form of ATP. This process is different from substrate
level phosphorylation.
II. Overview Slide [S2]
a. The important aspects to focus on here are mitochondria structure, the electron transport process, the actual
process of oxidative phosphorylation, and the role that mitochondria play in our physiology.
III. Figure, Biochemical Anatomy of a Mitochondrion [S3]
a. Mitochondria are specialized organelles that exist in the majority of cells (but not all cells).
b. They are surrounded by two membranes: an outer membrane and an inner membrane. The inner membrane
forms structures called cristae. These cristae are needed to increase the surface area of the inner membrane.
This is needed because most of oxidative phosphorylation takes place in the membrane. This large surface
area is needed for the many enzymes that will work here.
c. Various processes take place in different parts of the mitochondria. The majority of enzymes in the TCA cycle
are in the mitochondrial matrix. For oxidative phosphorylation, its enzymes are all located in the inner
membrane.
d. Since mitochondria are self-replicating, there are other enzymes in the mitochondria as well such as DNA
polymerase, RNA polymerase, ribosomes, etc.
e. The outer mitochondrial membrane is very permeable. It contains specialized proteins called porins that make
large holes in the membrane that let ATP or ADP pass through.
f. The inner mitochondrial membrane is virtually impermeable to even small molecules such as protons.
g. From a functional point of view, if you want to bring something into or take something out of the mitochondria,
you must have a specialized transporter protein.
IV. Figure, Electron Coenzyme Q (Ubiquinone) [S4]
a. The electron transport chain is built of electron carriers. These include NADH, FADH2, FMN, etc. In the
mitochondrial inner membrane, there are additional electron carriers.
b. One important electron carrier is Coenzyme Q (ubiquinone). It is a very hydrophobic isoprenoid molecule that
is lipid soluble. It carriers electrons inside the mitochondrial membrane.
c. The working part of the molecule is the quinone ring. It can be in a completely oxidized form, or it can be in a
completely reduced form. Similarly to FAD, it can transport one electron at a time or two electrons at the
same time. It can work with many electron carriers in the electron transport chain.
d. QUESTION: Does it carry one electron or two? ANSWER: It can either carry two electrons at a time or just
one electron.
V. Figure, Prosthetic Groups of Cytochromes [S5]
a. Cytochromes are proteins that have color. They have color because all of them have the prosthetic group
Heme. This is similar to coenzyme B12.
b. Iron is the electron carrier in cytochromes. Iron can either be in the Fe2+ state or the Fe3+ state. Because of
this, all cytochromes are single electron carriers.
c. There are several types of Heme such as A Heme, B Heme, C Heme, etc. Depending on which Heme is
attached to a particular protein will determine whether it is cytochrome A, cytochrome B, cytochrome C, etc.
VI. Figure, Absorption Spectra of Cytochrome C [S6]
a. Many properties of cytochromes are not dependent only on properties of the Heme attached, but are
dependent on the protein component of the molecule. Very often, these proteins will be given names such as
cytochrome B562 or cytochrome B566. What is being referred to is the protein that carries the Heme type B.
Its characteristic feature is unique because the name comes from the fact that oxidized cytochromes absorb
light at just one wavelength.
b. When cytochromes are reduced, they can absorb light at a maximum value. Each cytochrome has its unique
absorption maximum. For example, cytochrome B562 will absorb its maximum amount of light at a
wavelength of 562 nm.
VII. Figure, Iron-Sulfur Centers [S7]
a. Iron-sulfur proteins are another large group of electron carriers. They are similar to cytochromes as they all
have iron as an electron carrier. However, in these proteins, the iron is attached directly to the protein through
its interaction with cysteine residues.
b. These are single electron carriers like cytochromes regardless of the protein’s size.
VIII. Table, Standard Reduction Potentials of Respiratory Chain and Related Electron Carriers [S8]
a. Many electron transfer components sit in the membrane.
FUNDAMENTALS 1 11:00-12:00
Scribe: BRANDON TERRY
THURSDAY, AUGUST 27TH, 2010
Proof: TERRI CALL
POPOV
OXIDATIVE PHOSPHORYLATION
Page 2 of 6
b. In order to get an idea of how electrons are moving from one carrier to another, one can compare the
standard reduction potentials of the various carriers.
c. In reality, these parameters are virtually equivalent to delta G.
d. Strong oxidants like oxygen have a large positive reduction potential. Strong reducing agents like NADH have
a large negative reduction potential.
e. Electrons move from strong reducing agents to strong oxidizing agents. In other words, from a negative
standard reduction potential to a positive standard reduction potential.
f. If you compare the parameters for the different components of the electron transport chain, it predicts the
order in which the electron carriers are supposed to operate in the electron transport chain. This order can be
checked experimentally.
IX. Table, Agents That Interfere with Oxidative Phosphorylation [S9]
a. Over the years, people discovered a large number of chemical compound that kill different components of
the electron transport chain or enzymes that make ATP. These can be used to see what happens to the
oxidation and reduction states of different carriers.
X. Figure, Method for Determining the Sequence of Electron Carriers [S10]
a. Antimycin A is a compound that kills electron transport in cytochrome B and cytochrome C1. This causes all
carriers past this point to become oxidized. All of the preceding electron carriers will be reduced.
b. Different inhibitors allow you to predict in what order the electron carriers separate from each other.
XI. Figure, Separation of Functional Complexes of the Respiratory Chain [S11]
a. The electron transport chain can be purified by taking mitochondria, breaking them open, and isolating their
inner membranes. If NADH and oxygen are added to the membranes, the membranes will be oxidizing
NADH. This tells us the electron transport chain is functional in the inner membrane.
b. The membranes can then be solubilized and then they cannot catalyze the complete reaction anymore.
c. If NADH and Coenzyme Q are added, electrons will go from NADH to Coenzyme Q. If you add a reduced
Coenzyme Q and cytochrome C, electrons will flow from Coenzyme Q to cytochrome C. If you add a reduced
cytochrome C, you will see electrons flow from it to oxygen. This tells us there are parts of the electron
transport chain that organize in large complexes (which have been purified and well characterized).
XII. Table, The Protein Components of the Mitochondrial Electron-Transfer Chain [S12]
a. The major components of the electron transport chain are complex I, complex II, complex III, and complex
IV.
b. Cytochrome C is not usually considered to be part of the complex itself, but as a soluble electron carrier like
NADH that carries electrons in the intermembrane space of the mitochondria.
c. The large proteins of the pathway have many different electron carriers and many subunits. This tells us the
mechanisms involved are very complex.
XIII. Figure, Pathways in the Mitochondrial Electron Transport [S13]
a. NADH and FADH2 generated in the TCA cycle are not the only ways to feed electrons into the electron
transport chain.
b. There are other pathways that involve oxidation of fatty acids and one pathway from 3-phosphoglycerol.
c. All of the electrons eventually end up in the form of reduced Coenzyme Q. There is then a single process that
brings electrons from it to oxygen.
d. Complexes I and II (and a few others) take up electrons, Coenzyme Q is oxidized by complex III, and
cytochrome C is oxidized my complex IV.
XIV.
Figure, Electrons Move Downhill [S14]
a. All electron transfers go downhill.
b. Oxygen is the strongest electron acceptor, and NADH is the strongest electron donor.
XV. Figure, NADH: Ubiquinone Oxidoreductase (Complex I) [S15]
a. Complex I abstracts electrons from NADH that has been made in the TCA cycle. It then donates the electrons
to flavin mononucleotide (FMN). This is because FMN can accept two electrons from NADH at the same
time.
b. FMN is able to feed electrons to electron carriers downstream to iron-sulfur proteins one electron at a time.
c. Eventually, the electrons travel through complex I and are used to reduce Coenzyme Q. When Coenzyme Q
is reduced, it also picks up protons from the inner surface of the membrane.
d. The overall chemical reaction of this complex is the oxidation of NADH and production of reduced Coenzyme
Q. However, this complex can also use energy that is released from the flow of electrons to physically pump
protons from the inner space of the mitochondria into the intermembrane space. It uses the power of this
electric current to power a proton pump. For every two electrons that pass through the chain, complex I will
pump out four protons. The outcome of this is a proton gradient across the inner mitochondrial membrane.
FUNDAMENTALS 1 11:00-12:00
Scribe: BRANDON TERRY
THURSDAY, AUGUST 27TH, 2010
Proof: TERRI CALL
POPOV
OXIDATIVE PHOSPHORYLATION
Page 3 of 6
XVI.
Figure, Succinate Dehydrogenase (Complex II) [S16]
a. Complex II is the same Succinate Dehydrogenase that is found in the TCA cycle. It is responsible for the
oxidation of succinate and its conversion to fumarate.
b. Two electrons are abstracted and are fed to FAD and through a series of electron carriers that are once again
used to reduce Coenzyme Q.
c. Protons are also picked up here from the inner space of the mitochondria, but unlike complex I, complex II
does not pump protons across the membrane.
XVII. Figure, Cytochrome bc1 Complex (Complex III) [S17]
a. Complex III is the enzyme that catalyzes the Q cycle.
XVIII. Figure, The Q Cycle [S18]
a. The substrate for complex III is reduced Coenzyme Q that comes from different parts of the electron transport
chain.
b. Complex III oxidizes Coenzyme Q and in the process, it pumps protons across the membrane. These protons
also catalyze the reduction of cytochrome C.
c. For complex I, we don’t actually know how it pumps protons across the membrane, but for complex III we
have a good idea of how it works.
d. Complex III has two active sites for Coenzyme Q. One reduced Coenzyme Q comes in and it loses its
electrons. This active site is located on the outer surface of the membrane. When Coenzyme Q loses
electrons, protons are released on the outer surface of the membrane. Protons are being pumped out of the
mitochondria by being oxidized on the outer surface of the membrane.
e. Coenzyme Q transports two electrons while electron carriers downstream are single electron carriers.
f. The first electron that is released goes through an iron-sulfur cluster to cytochrome C1 and then to
cytochrome C. When cytochrome C becomes reduced, it dissociates and leaves looking for complex IV.
g. The second electron cannot be held at complex III because semi-oxidized Coenzyme Q is very reactive. After
time, it can react with oxygen to produce reactive oxygen species. To prevent this, the second electron is
taken to cytochrome B (with two types of Heme), and fed to another Coenzyme Q binding site that is located
on the inner surface of the membrane. In this site, the oxidized form of Coenzyme Q comes and accepts the
electron. During this oxidation, it will also pick up protons from the inside.
h. One electron goes to reduce cytochrome C, and another electron is used to reduce Coenzyme Q.
i. During the next step of the cycle, a fresh molecule of reduced Coenzyme Q comes and the cycle begins
again. The overall outcome here is that per pair of electrons that go through this complex, four protons are
moved across the membrane. This is done by what is called a ligand driven proton pump. Every time
Coenzyme Q is oxidized on the outer surface, protons are released to the outside. When Coenzyme Q is
reduced, it is reduced on the inner surface. By doing this, protons are picked up from the inner space.
j. A proton gradient is physically being created here. The stoichiometry here helps us tell how many molecules
of ATP are being made per molecule of NADH.
XIX.
Figure, Path of Electrons through Complex IV [S19]
a. Complex IV is a large protein. Its primary function is to oxidize cytochrome C.
b. Cytochrome C is reduced by complex III and then travels to complex IV and loses its electrons.
c. Electrons from cytochrome C go through several electron carriers and end up in an iron-copper center. This
is complex IV’s active site where electrons are used to reduce oxygen.
d. For each oxygen, four electrons and four protons are needed. Complex IV also picks up protons from the
inner membrane, and this also contributes to the proton gradient. The proton pump here is called a direct
pump because this enzyme has a channel that goes all the way across the membrane. This channel
organizes in a way that when electrons go from outside to the inside active site, it pushes the protons to move
through the channel from the inside out.
e. The overall stoichiometry here is that for each two electrons that go into complex IV, two protons go out of
complex IV.
XX. Figure, Reaction Sequence for the Reduction of O2 by the Cytochrome C Oxidase [S20]
a. Semi-reduced oxygen is a very nasty molecule. It is highly reactive and can destroy nucleotides, lipids, etc.
b. Particular care is exercised is the active site of complex IV to make sure that semi-reduced oxygen is not
leaving the molecule. At every step of reduction, oxygen is physically attached to the copper or iron of the
reactive site. This prevents formation of reactive oxygen species and helps us to survive without extensive
oxidative damage.
XXI.
Figure, The Flow of Electrons and Protons through the Respiratory Chain (Proton-motive Force and
Chemiosmotic Model) [S21]
a. What the electron transport chain does is it takes electrons from NADH and succinate and uses them to
reduce Coenzyme Q. Then Coenzyme Q is used to reduce cytochrome C, and cytochrome C is used to
reduce oxygen, the final acceptor of electrons. This is the actual reason why we have to breath in oxygen.
FUNDAMENTALS 1 11:00-12:00
Scribe: BRANDON TERRY
THURSDAY, AUGUST 27TH, 2010
Proof: TERRI CALL
POPOV
OXIDATIVE PHOSPHORYLATION
Page 4 of 6
b. The net outcome of all this activity is that protons are pumped across the inner mitochondrial membrane. For
a pair of electrons, complex I pumps four protons, complex III pumps four protons, and complex four pumps
two protons. What this does is it creates a proton gradient across the inner membrane.
c. If a hole is punched in the membrane, protons will go down their concentration gradient. This gives up
potential energy that we can use to make ATP.
d. The enzyme that utilizes this energy is proton-dependent ATP synthase. It lets protons back in the membrane
and uses the energy released during the passage of protons to create ATP.
e. Another thing about the proton gradient to consider is the fact that these protons are charged. Every time a
proton is removed from the inside, one positive charge is carried from the inside out. Consequently, there are
more positive charges on the outside and more negative charges on the inside.
f. It is this electrochemistry that drives the synthesis of ATP.
XXII. Figure, Chemical Uncouplers of Oxidative Phosphorylation [S22]
a. Over the years, evidence was found that supported the idea of chemiosmotic coupling. The first evidence for
this came from simple experiments with organic acids. If a chemical agent is introduced that disperses this
gradient, the cell will no longer make ATP.
XXIII. Figure, Coupling of Electron Transfer and ATP Synthesis [S23]
a. If mitochondria is purified and given ADP and Pi, and you start measuring ATP synthesis and oxygen
consumption, nothing will be happening. In order to do something, the mitochondria need a source of
electrons.
b. If you give them a substrate like succinate, you will see mitochondria start consuming oxygen and making
ATP at the same time.
c. If you kill the electron transport chain with cyanide, you will not only stop the electron transport chain, but you
will also stop ATP synthesis. This tells you that one process cannot go without the other one.
d. This experiment can also be repeated in the opposite way. If you give mitochondria just succinate, nothing
will happen. IF you then give it ADP and Pi, oxygen will begin to be consumed and ATP will begin to be
made.
XXIV. Figure, Coupling of Electron Transfer and ATP Synthesis [S24]
a. Oligomycin can be used to specifically kill just the enzymes responsible for ATP synthesis. However, at the
same time you also kill the activity of the electron transport chain. This is because the proton gradient
eventually becomes so large that it blocks the ability of the electron transport chain to pump protons.
b. If at this time you treat the mitochondria with dinitrophenol (DNP), the proton gradient is destroyed and
mitochondria become uncoupled. The electron transport chain operates fine, but the mitochondria cannot
make ATP because there is no proton gradient.
XXV. Figure, Evidence for the Role of a Proton Gradient in ATP Synthesis [S25]
a. The proton gradient is used as a source of energy to make ATP.
XXVI. Figure, Catalytic Mechanism of F1 [S26]
a. ATP is a high energy compound because energy released during its hydrolysis reaction is very high. This
happens because a molecule of ATP is charged. When you break it apart, you separate these charges. When
you hydrolyze ATP, ADP and Pi immediately dissociate.
b. If you put ATP in an organic solution, the synthesis reaction of ATP is virtually irreversible. The high energy
properties of ATP come from the fact that everything is happening in a water solution.
c. What ATP synthase is actually doing is taking ADP and Pi, binds them, and completely shields them from the
water environment. This makes the thermodynamic rules not applicable because there are no factors that
make hydrolysis favorable.
d. Inside of the enzyme, ATP synthesis is at equilibrium. This can be demonstrated by treating ATP with
radioactively labeled water. The radioactive oxygen from the water will be incorporated into the Pi group. This
tells us that the enzyme can make ATP, but it can’t release it because that would break the laws of
thermodynamics.
e. ATP Synthase makes ATP in an equilibrium reaction. The energy of the proton gradient is used to release
ATP from the enzyme’s active site.
XXVII. Skipped Slides [S27, S28]
XXVIII. Figure, Rotation of Fo and gamma [S29]
a. ATP synthase looks like a lollipop sticking down from the inner mitochondrial membrane. The lollipop-looking
part is the part of the enzyme that contains the active site for the synthesis of ATP. It is a six subunit complex
which is anchored to the membrane through what is called the Fo component.
b. The Fo component consists of one C ring of subunits and an AB subunit. The F1 (gamma) subunit is the
lollipop-looking portion.
XXIX. Skipped Slide [S30]
FUNDAMENTALS 1 11:00-12:00
Scribe: BRANDON TERRY
THURSDAY, AUGUST 27TH, 2010
Proof: TERRI CALL
POPOV
OXIDATIVE PHOSPHORYLATION
Page 5 of 6
XXX. Figure, A Model of the FoF1 Complex, a Rotating Molecular Motor [S31]
a. All synthesis reactions happen in the F1 component that is made out of alpha and beta subunits.
b. It is attached to the membrane through delta, B2, and A subunits. It is immobile.
c. The ring of C10 subunits is like an electric rotor of water. It rotates inside of the membrane relative to the A
subunit.
d. There are two other subunits, epsilon and gamma, that are physically attached to the rotor. The rotor causes
the gamma subunit to rotate inside of the lollipop-looking part. This rotation is driven by the passage of
protons through the C and A subunits.
e. The protons enter the C chain from the A subunit. It interacts here with an acidic residue of serine. When this
happens, the charges neutralize. This causes an imbalance of charges that moves the entire ring of the C
subunit by one step (one-tenth of a turn). When this happens, it brings another C subunit on the other side of
the ring in alignment with an exit channel also located in the A subunit. This subunit will lose its proton and
become charged. This causes another charge imbalance, and causes the ring to turn again. There is now an
aspartate residue aligned to pick up another proton.
f. Ten protons pass through the system, and this causes one turn of a cycle. A consequence of this is the
gamma subunit rotates inside the lollipop periodically interacting with different components of the ATP
synthesizing apparatus.
XXXI. Figure, Binding-Change Model for ATP Synthase [S32]
a. Depending on what set of subunits in the lollipop the gamma subunit interacts with, it causes simultaneous
changes in conformation of all the subunits associated with the lollipop.
b. The lollipop is classified as a trimer of dimers. Each dimer catalyzes the synthesis of one molecule of ATP.
c. There are always three different conformations for each dimer. One conformation is empty (the dimer
physically attached to the gamma subunit), one conformation is bound ADP and Pi, and the last conformation
is bound ATP where ATP is synthesized and shielded from water.
d. The gamma subunit is continuously rotating. This is driven by the passage of protons through the C rings. As
the protons pass, the gamma subunit will rotate by about 120 degrees and will come in touch with the empty
dimer subunit. This causes the enzyme to physically change its entire conformation.
e. During the conformation change, ATP is released. The subunit with ADP and Pi assumes the ATP
conformation, and the empty subunit now binds ADP and Pi.
f. As more protons pass, the gamma subunit rotates another 120 degrees and causes another conformational
change in the enzyme. This process repeats over and over.
g. The passage of about 9 protons through the C subunit is sufficient to synthesize 3 molecules of ATP. This is
one complete cycle.
h. The energy of the passage of protons is used to drive coordinated changes of conformation in synthase that
allow the enzyme to release ATP.
XXXII. The P/O is an Index of the Efficiency of Coupling [S33]
a. The P/O ratio is used to determine the energetic output of various processes.
b. It can be measured directly. You add ADP and Pi, then measure how much ATP is made and how much
oxygen is consumed.
c. When this is done with NADH as a source of electrons, 2.5 molecules of ATP are made. When this is done
with FADH2 as a source of electrons, 1.5 molecules of ATP are made. This makes sense because when
NADH is used as a substrate, you pump 10 protons across the membrane (complexes I,II, and IV). When
FADH2 is used as a substrate, you pump 6 protons across the membrane (complexes II and IV).
d. These numbers are used to calculate ATP tables. However, there are other processes that occur that actually
use the energy of the gradient to make ATP.
XXXIII. Figure, Adenine Nucleotide and Phosphate Translocases [S34]
a. In order to make ATP in the mitochondria, you have to bring in ADP and Pi.
b. The uptake of ADP is an energetically favorable process as 4 negative charges go out while 3 negative
charges go in. Remember, the inside is more negative since protons are being taken out.
c. The uptake of Pi is energetically unfavorable since an excess negative charge is being brought in. To make it
favorable, the transporter also brings in a proton. This process is known as symport.
d. You waste one proton for each ATP that is synthesized; this decreases the yield of the synthesis of ATP.
XXXIV. Figure, Glycerol 3-Phosphate Shuttle [S35]
a. A lot of NADH is made in the cytosol. Normally, most of it will be oxidized through the mitochondria.
b. However, NADH by itself cannot cross the inner membrane. There are a couple of shuttle that exist for this
purpose.
c. One of these is the glycerol 3-phosphate shuttle that takes NADH and uses it to reduce dihydroxyacetone
phosphate to glycerol 3-phosphate. The glycerol 3-phosphate is then oxidized in a membrane enzyme and
carries electrons through FADH2.
FUNDAMENTALS 1 11:00-12:00
Scribe: BRANDON TERRY
THURSDAY, AUGUST 27TH, 2010
Proof: TERRI CALL
POPOV
OXIDATIVE PHOSPHORYLATION
Page 6 of 6
XXXV. Figure, Malate-Aspartate Shuttle [S36]
a. The other NADH shuttle is the malate-aspartate shuttle. It takes electrons from NADH and uses them to
synthesize malate, brings malate in, and oxidizes inside producing NADH.
b. This shuttle is more energy efficient than the glycerol 3-phosphate shuttle since it is producing NADH.
XXXVI. Table, ATP Yield from Complete Oxidation of Glucose [S37]
a. All of the aforementioned processes combined is how the final ATP yield from the oxidation of glucose is
determined.
XXXVII. Skipped Slides [S38, S39, S40, S41, S42]
a. ADVICE: YOU SHOULD STILL READ OVER THESE SLIDES, BECAUSE HE COULD PULL QUESTIONS
FROM THEM EVEN THOUGH WE DIDN’T GO OVER THEM IN CLASS.
[End 49:48 mins]