Download studies on the mitochondrial electron transport and atp synthesis

Department of Biochemistry and Molecular Biology
Faculty of Medicine
University of Debrecen
BIOCHEMISTRY PRACTICE
STUDIES ON THE
MITOCHONDRIAL ELECTRON
TRANSPORT AND ATP SYNTHESIS
Theoretical Background
János Kádas, Ph.D.
2015
The development of this curriculum was sponsored by TÁMOP 4.1.1.C-13/1/KONV-20140001. The project is supported by the European Union and co-financed by the European
Social Fund.
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STUDIES ON THE MITOCHONDRIAL ELECTRON TRANSPORT AND ATP
SYNTHESIS – THEORETICAL BACKGROUND
The structure and function of the mitochondria
Mitochondria are double membrane bounded organelles mostly found in all eukaryotic
cells. Structurally in the mitochondria the outer membrane; inner membrane with the
convolutions (cristae); intermembrane space is bordered with the two membranes, and the
mitochondrial matrix is bordered with the inner membrane can be perceptible. The outer
membrane structure is simple; the inner membrane is more complex which can be
characterized by about 75% protein content.
The outer membrane is permeable to small molecules and ions. Transmembrane
channels composed of porin proteins allow smaller molecules to pass through the membrane.
Translocase systems are responsible for the transportation of higher molecules into the
mitochondria. The intermembrane space contains high number of cytochrome c molecules
and has a role in forming a proton gradient. The inner membrane is highly impermeable to
all (small) molecules and ions (including protons), for the passage through specific
transporters are required. The surface of the inner membrane is very large due to the
membrane convolutions. These convolutions are the cristae. The inner membrane contains the
mitochondrial electron transport chains, adenosine nucleotide (ADP-ATP) translocases and
the ATP synthase complexes. Inner membrane of the liver mitochondria may have more than
10.000 sets of electron transport systems. Besides the several metabolic intermediates,
mitochondrial DNA, and ribosomes, the mitochondrial matrix contains the pyruvate
dehydrogenase enzyme complexes, the enzymes of citric acid cycle reactions, amino acid and
fatty acid oxidation.
Primarily the mitochondrion is responsible for the energy production of the cells
since the center of energy production tightly connected to other metabolic pathways. In
addition the mitochondria are also involved in signaling, calcium storage, heat production,
differentiation and cell growth, cell death, hem synthesis, steroid synthesis and aging
processes (Figure 1.).
Figure 1. Structural elements of the mitochondrion.
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The biological oxidation
Carbon atoms of organic molecules are oxidized during the biological oxidation,
while oxygen is reduced. Carbon atoms are oxidized to carbon dioxide (CO2), the oxygen is
reduced to water during the terminal oxidation. Catabolism of nutrients from food intake and
mobilized storages occurs in the three stages of the cellular respiration (Figure 2.).
In the first stage the oxidation of glucose, fatty acids and amino acids yields acetylCoA. In the citric acid cycle (TCA or tricarboxylic acid cycle), during the second stage the
acetyl group of acetyl-CoA is oxidized to carbon dioxide, and the released energy is
conserved in reduced electron carrier molecules (NADH and FADH2). The third stage is the
oxidation of reduced electron carrier molecules in the terminal oxidation leading to the
release of protons and electrons. In the last stage the electrons reduce oxygen to water; the
released energy drives the ATP synthesis during the process of oxidative phosphorylation.
Figure 2. Stages of biological oxidation in the cell.
Reduced electron carrier molecules ultimately release their hydrogens and electrons in
the terminal oxidation. While reduced electron carrier molecules are formed in the citric acid
cycle and during the reaction catalyzed by pyruvate dehydrogenase enzyme directly oxidized
in the mitochondrion; for the transportation to the mitochondrial matrix of NADH is produced
in the glycolysis in the cytosol during the reaction catalyzed by glyceraldehyde-3-phosphate
dehydrogenase specific transport mechanism is necessary. Primarily the malate – aspartate
and glycerol phosphate shuttle are responsible for the mitochondrial transport.
With the help of malate – aspartate shuttle the NADH formed in the cytosol
connected to the electron transport chain in a form of mitochondrial NADH. During the
process the malate dehydrogenase enzyme converts malate to oxaloacetate in the
intermembrane space in addition to the oxidation of NADH. The resulting malate is
transferred to the mitochondrial matrix by the involvement of malate – α-ketoglutarate
antiporter. In the matrix the malate is transformed to oxaloacetate with the reduction of NAD
cofactor of the malate – dehydrogenase enzyme. Aspartate, which is formed in a transaminase
reaction (with a conversion of glutamate to α-ketoglutarate) from oxaloacetate, is transferred
to intermembrane space with the involvement of glutamate – aspartate antiporter.
Oxaloacetate reproduced due to the repeated transaminase reaction in the intermembrane
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space from the aspartate, can be involved into subsequent transportation cycle of the NADH.
The reduction reaction is catalyzed by the malate-dehydrogenase enzyme (Figure 3.).
Figure 3. The malate - aspartate shuttle.
In case of the glycerol-3-phosphate dehydrogenase shuttle the glycerol-3-phosphate
dehydrogenase converts dihydroxyacetone phosphate to glycerol-3-phosphate in the cytosol,
while the NADH is oxidized. After access to the mitochondria, the glycerol-3-phosphate is
converted back to dihydroxyacetone phosphate by the mitochondrial isoform of the enzyme
with the reduction of its FAD cofactor. With this shuttle mechanism the NADH produced in
the cytosol enters into the respiratory chain as FADH2 (Figure 4.). Both shuttle mechanisms
are suitable for the transportation of reducing equivalents. While the malate – aspartate shuttle
is reversible, and is activated in case of high cytosolic NADH concentration, the operation of
glycerol-3-phosphate dehydrogenase shuttle is irreversible and independent from the
concentration conditions.
Figure 4. The glycerol - 3 - phosphate shuttle.
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During the biological oxidation, oxidation of one NADH molecule is resulted in the
phosphorylation of 3 ADP molecules, while oxidation of one molecule succinate generates 2
molecules of ATP. The two NADH molecules formed during the glycolysis can be utilized for
the synthesis of 4 or 6 ATP depending on which transport system is used for entering into the
mitochondrial matrix. Under aerobic conditions total oxidation of one molecule of glucose
can lead to 36 or 38 ATP formation. It is important to note that the under anaerobic
circumstances the pyruvate is utilized for the synthesis of lactate, and only 2 molecules of
ATP are synthesized.
The mitochondrial electron transfer chain
In terminal oxidation, on the mitochondrial electron transfer chain the hydrogen is
oxidized and the oxygen is reduced to water. Connected to the process an oxidative
phosphorylation occurs whereby the ADP is phosphorylated to ATP. The terminal oxidation
and the oxidative phosphorylation are spatially and temporally linked processes.
The mitochondrial electron transport system is a complex structure of four
supramolecular organizations connected to the fifth element of the ATP synthase complex,
ubiquinone and cytochrome c are also part of the system. The oxidation of reduced electron
carrier molecules occurs via a coordinated action of the four respiratory complexes (Figure
5.).
Figure 5. Elements of the mitochondrial electron transfer chain and the ATP synthase.
The Complex I. is the NADH - ubiquinone oxidoreductase or also known as the
NADH dehydrogenase. Proteins containing iron – sulfur (Fe-S) or flavin mononucleotide
(FMN) prosthetic groups are involved as a part of the complex. The complex oxidizes the
NADH, and carries the electrons to ubiquinone, while pumps protons into the intermembrane
space.
Next member is the Complex II., succinate-ubiquinone oxidoreductase complex a
flavin adenine dinucleotide (FAD) prosthetic group containing enzyme complex. In fact, it is
a combination of succinate dehydrogenase and a hydrogen transferase. During the operation
the ubiquinone is reduced by this complex. Electrons released from succinate – fumarate
conversion are transferred to FAD of succinate dehydrogenase in the first step, and then enter
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into the electron transport chain and are delivered to ubiquinone. The succinate
dehydrogenase also a part of the citrate cycle as the one membrane-bound enzyme of the
system. The complex II., in contrast with the other complexes, has no proton pump activity.
Ubiquinone is the oxygen supplier in the respiratory chain. It is long, hydrophobic
molecule consists of isoprene subunits, is located in the membrane and it is diffusible. It is an
important electron transporter of the respiratory chain, which is not bounded to other proteins,
but moving inside the membrane and connects the first and second complexes with the
respiratory complex III.
The Complex III. is the ubiquinone-cytochrome c oxidoreductase, the second
enzyme complex, which pumps protons into the intermembrane space. Electrons that are
carried by the ubiquinone are moved to cytochrome c through the complex III. The
cytochrome c transfers the electrons to complex IV. The complex contains cytochrome b and
c1 molecules with heme prosthetic groups.
Cytochrome c is not a membrane bounded protein. It has heme prosthetic group, and
it is moving between the two membranes and connects to complex III. and IV.
Last element of the mitochondrial electron transport system is the IV., Cytochrome
oxidase complex. Contains cytochrome a and a3, proteins with heme – iron groups. In
addition, two copper ions can be found in the complex. The cytochrome oxidase oxidizes the
cytochrome c and reduces oxygen to water molecules in the matrix. The complex works as a
proton pump too (Figure 6.).
Figure 6. Complexes of the mitochondrial electron transfer chain.
Mechanism of the ATP synthesis
Electron transfer components of terminal oxidation are localized in the inner
membrane of the mitochondrion, and interconnected spatially and temporally to structural
components that are responsible for the ATP synthesis. Since the ATP synthesis is closely
related to terminal oxidation it is called oxidative phosphorylation.
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The chemiosmotic theory
The mitochondrial electron transport is connected to proton transportation, and
produces both chemical and an electrical gradient. In the respiratory chain a proton gradient is
generated between mitochondrial matrix and intermembrane space due to the operation of the
complexes with proton pumping activity.
Figure 7. Schematic representation of the electron transfer, and the ATP synthesis.
According to Peter Mitchell’s chemiosmotic theory during mitochondrial electron
transport the energy of electrons is transformed into the generation of H+ concentration
difference (proton gradient) between two surfaces of the inner membrane. Complexes are
pumping protons into the intermembrane space during their operation and make difference in
proton concentration and that is resulted in pH change. Membrane potential is also generated,
and the matrix will be negatively charged, while the intermembrane space becomes positively
charged. The proton motive force that drives the ATP synthesis is provided by
electrochemical potential and hydrogen concentration difference (Figure 7.).
The oxidative phosphorylation
Energy source of the ATP synthesis is the proton gradient connected to the electron
flow and electrochemical potential difference that leads to the formation of ATP macroergic
phosphate bounds.
For the protons that have been pumped out into intermembrane space, specific
channels (F0) of ATP synthase complex provides opportunity to flow back into the
mitochondrial matrix. Energy from the concentration difference and membrane potential is
utilized for ATP synthesis in the equalization. The ATP synthase complex contains F1 and F0
units. Four integral protein subunits of the F0 unit build the proton channel, while the matrixfacing F1 unit is responsible for the ATP synthesis.
ATP molecules that have been synthetized in the mitochondrion are exported by the
ADP-ATP translocase, while an inner membrane transport system carries the ADP and
anorganic phosphate that are necessary for ATP synthesis in the matrix.
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The acceptor control and P/O ratio
The electron transport system and the oxidative phosphorylation processes are tightly
connected. This phenomenon can be explained by the acceptor control regulatory
mechanism. Electrons and hydrogens that are necessary for the oxidative phosphorylation are
provided by reduced electron carrier molecules, while anorganic phosphate and ADP are also
required for the generation of ATP. The concentration of ADP is decisive in the aspect of
ATP synthesis. At high concentrations of ADP the ATP synthesis is increased. The
accelerating effect of ADP on reaction speed is called acceptor control or respiratory
control (RC).
In the absence or at a low level of ADP the proton flow rate is decreased due to the
decreased speed of the phosphorylation. If the proton flow through the F1-F0 complex into the
matrix is inhibited, the proton concentration is increased significantly in the intermembrane
space and an increased energy is required for keeping the proton pumping ability of the
complexes against the increased gradient. This energy exceeds the energy that is released
during the electron transfer and it consequently stops the electron flow. The increased proton
gradient stops not only the terminal oxidation, but also inhibits the citric acid cycle due to the
increased NADH concentration.
Quantity of the anorganic phosphate incorporation for the use of one oxygen atom, i.e.
production of one molecule of water in the terminal oxidation can be experimentally
determined, and this value is the P/O ratio.
In the course of succinate - fumarate conversion, the Complex II. (succinateubiquinone oxidoreductase) transfers the hydrogens to FAD of the succinate dehydrogenase
(FADH2 is formed), and then reduces the ubiquinone, and the end of the electron transfer the
ATP synthesis will be started. The malate can get through the inner membrane by the help of
malate α-ketoglutarate transporter. It is converted into oxaloacetate by malate-dehydrogenase
enzyme with the generation of NADH+H+. The NADH+H+ is oxidized by the Complex I.
(NADH-ubiquinone oxidoreductase), which subsequently reduces the ubiquinone.
Synthesis of one molecule ATP requires 4 protons. In case of an oxidation of one
NADH+H+, 10 protons are pumped out into the intermembrane space due to the proton pump
activity of the mitochondrial electron transport complexes , and 6 protons are pumped out
during one FADH2 oxidation. Thus, if 4 protons are necessary for the synthesis of one
molecule ATP, the P/O ratio is 2.5 (10/4) for NADH+H+, while 1.5 (6/4) for FADH2. In case
of NADH+H+ the 3 and in case of FADH2 the 2 is commonly used as P/O value in the
literature.
Inhibitors of the mitochondrial electron transfer chain and ATP synthesis
Functionality and the operational order of the elements of electron transport chain,
terminal oxidation and oxidative phosphorylation can be tested and verified with experimental
methods.
The different reactions that are catalyzed by each complex can be determined and can
be measured by the examination of the fractions containing different respiratory chain
complexes that were isolated from mitochondrial fraction with chromatography methods.
The operation sequence of the elements of the electron transport chain can be
determined by examining the effects of the different electron transfer inhibitors on the
oxidation state and kinetics of each transporter. Different steps of the electron transport can be
blocked specifically with various inhibitors (Figure 8.).
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Figure 8. Inhibitors of the mitochondrial electron transfer chain and ATP synthesis.
Some well-known inhibitors and effects on the terminal oxidation and oxidative
phosphorylation (Figure 9.):
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Cyanid (CN-) and Carbon monoxide (CO): inhibitors of the cytochrome oxidase
(Complex IV.), inhibit Cytochrome a and a3 oxidation, and electron flow to the
oxygen.
Antimycin A: inhibits the electron transition between Cytochrome b and Cytochrome
c1 in the Complex III.
Malonate: competitive inhibitor of the succinate – dehydrogenase (Complex II.).
Rotenone: inhibits the electron transfer from Fe-S center to ubiquinone in NADH –
ubiquinone oxidoreductase (Complex I.).
Oligomycin: specific inhibitor of the ATP synthase, it is responsible for the inhibition
of F0 and CF1.
Other agents that interfere with oxidative phosphorylation:
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Dinitrophenol (DNP): protonophore, uncoupling agent, which makes the membrane
permeable to protons, so the protons will not flow back through the ATP synthase to
the matrix. It uncouples the electron transport chain and oxidative phosphorylation.
Atractyloside: specific inhibitor of ATP-ADP (adenine nucleotide) translocase
inhibits adenine nucleotide exchange between the two sides of the inner membrane.
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Figure 9. Effect of different inhibitors on the electron transport chain.
In the presence of a given inhibitor all components of the chain prior to the inhibited point
become reduced, while all subsequent components are remained in oxidized form, and the
electron transport and oxygen uptake are stopped (Figure 10.).
Figure 10. Blocking effect of different inhibitors on the electron transport chain.
In certain physiological conditions uncoupling of the ATP synthesis (oxidative
phosphorylation) and electron transport chain may be biologically useful. The Thermogenin
is a physiological uncoupling protein (UCP) typically occurs in inner membrane of brown
fat tissue mitochondria. Due to the operation of thermogenin, the protons that flow back into
the matrix flow through thermogenin pores instead of channel F0, which results in
thermogenesis instead of ATP production (Figure 11.).
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Figure 11. Role and function of the uncoupling protein (UCP, Thermogenin).
Measurement of the oxygen consumption in practice, determination of P/O ratio
Under adequate experimental conditions, terminal oxidation, the properties of the
electron transport chain and the effect of various agents can be tracked by measuring oxygen
consumption using isolated mitochondrial suspension.
Determination of dissolved oxygen concentration with Clark-type polarographic
oxygen electrode is a suitable method for the monitoring of oxygen consumption (Figure
12.). This device is a bipolar electrochemical oxygen sensor composed of a platinum cathode
and a larger silver anode covered with silver-chloride. Electrodes are immersed in saturated
potassium chloride (KCL). The reaction vessel is separated from the electrodes by a specific
membrane which permits dissolved gases, like oxygen but impermeable for components of
the reaction solution (e.g. water, ions, etc.). Polarization voltage between the two electrodes is
0.6-0.7 V. Due to the voltage that is applied on the electrode, the platinum electrode (cathode)
surface becomes negatively charged, oxygen diffuses to the cathode through the membrane,
which is permeable for dissolved gases and reduced; the silver is oxidized on the anode and
silver chloride (AgCl) is precipitated. Current generated by the electrode processes becomes
directly proportional to the concentration of oxygen reduced at the cathode.
Figure 12. Elements of the equipment are used during the practical experiment.
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The mixture in the reaction vessel should be maintained at a constant temperature,
since changes in temperature affect the solubility of oxygen. Continuous mixing of the
reaction mixture with magnetic stirrer is needed for equipartition of the mitochondrial
suspension, substrates and the inhibitors in the reaction chamber. No air bubbles are allowed
during the addition of reagents in order to avoid additional oxygen dissolution the reaction
chamber should be closed. This ensures the constant oxygen concentration; besides
application of permanent voltage the oxygen amount measured by the electrode will be
proportional to the concentration of the oxygen in the reaction sample. Since the membrane is
not permeable to the dissolved substances, such as corresponding substrates and inhibitors
that were added to the mitochondria suspension, therefore these materials do not affect
processes on the electrodes. When the inhibitor affect is formed after the addition of agent,
restart of the respiration can be checked with application of uncoupling agent (DNP).
Relatively to a given temperature, the saturation concentration of oxygen can be
determined (the temperature should be kept at an approximately constant rate). A zero oxygen
concentration can be also determined by the addition of sodium dithionite, which removes
oxygen from the reaction mixture and it also helps to demonstrate that the respiration is not
slowed or stopped due the lack of oxygen.
During experiments oxygen consumption is monitored as a function of time and the
changes in the current is recorded using computer system. Current recorded at these
conditions is used to calculate oxygen level during respiration (Figure 13 and 14.).
Figure 13. Recorded curves in different buffers during the experiment.
Effects on the oxygen consumption.
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Figure 14. Effects of different compounds on the oxygen consumption during the experiments.
Due to the operation of the respiratory chain ATP synthesis occurs, the released
amount of oxygen can be determined experimentally and the experimental data can be used to
calculate the P / O ratio. In the absence of ADP the rate respiration is slow and the O2
consumption is low, while the addition of ADP increases O2 consumption. After adding of
ADP parallel with the O2 uptake a stoichiometric amount of ATP is generated, and this
coefficient gives the P/O ratio. The P/O ratio is the number of ATP synthesis / consumed
oxygen atoms, namely the number of ATP molecules generated after every 2 electrons that
are transferred from NADH to O2 (synthesized ATP molecules / 2 electrons).
The P/O ratio can be determinable based on the known saturation concentration of
oxygen and the amount of ADP used in the reaction (Figure 15.).
Figure 15. Calculation of the P/O ratio.
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References
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Imre Törő: Az élet alapjai (in Hungarian). Gondolat Kiadó, Budapest 1989.
Studies on the Coupling of Mitochondrial Electron Transport by Proton Motive Force
to ATP Synthesis. Medical Biochemistry and Molecular Biology: Practical Manual.
Semmelweis University, Department of Medical Biochemistry, Budapest, 2014.
Biochemistry of the Mitochondria, Biochemistry I. lecture presentation. Department
of Biochemistry and Molecular Biology, Faculty of Medicine, University of
Debrecen, Debrecen, 2014.
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