Lecture 2 - April 8, 2004
Lecture 2 Slides (PDF)
1. The Krebs
Citric Acid Cycle and production of reducing equivalents, NADH AND
FADH2.
Acetyl CoA formation (cytosol)
Citric Acid Cycle (mitochondrial matrix)
Reduction of NAD+
2. Components
of The Electron Transport Chain (ETS)
COMPLEX I (NADH-ubiquinone oxidoreductase)
COMPLEX II (Succinate-ubiquinone oxidoreductase)
CoQ (Ubiquinone)
COMPLEX III (Ubiquinol-cytochrome c oxidoreductase)
Cytochrome c
COMPLEX IV (Cytochrome c oxidase)
3. Energetics of
electron transport (respiration).
Electron transport is a series of oxido-reduction
(redox) steps. The energy drops for each step have been
measured.
But also, the over all redox energy can be calculated by knowing the
redox energies at the beginning and end. This is
because ΔE, the redox energy, is a free energy, which is a state
function. Thus, the total free energy depends only on
the initial and final steps.
The redox span of the ETS from NADH to O2 is 1.14
Volts.
Relationship between ΔE and ΔG
The free energy drop from NADH to O2 is - 216 kJ/mol.
4. Energetic
relationship between NADH oxidation and ATP synthesis.
The free energy of respiration is very large.
We know energy is conserved in mitochondria via the synthesis of ATP -
what
happens to this energy if it is not conserved? Remember the
definition of energy - if it is neither expended as work or
conserved as potential energy in the form of ATP, it must be given off
as heat. This is very important: In most bioenergetic
reactions, the free energy given off by one reaction (negative ΔG) is
passed on to another reaction with positive ΔG.
Thus most reactions are coupled. The energy-consuming
reactions of the cell (muscle contraction, synthesis of
macromolecules - proteins, lipids, polysaccharides, ion transport
across membranes, cell signaling via protein kinases)
normally use the energy contained in ATP. In these final steps,
the energy is converted to chemical, electrical or
mechanical work.
5. How is the
energy of ET coupled to ATP synthesis? The coupling mechanism was
the holy grail of
biochemistry during the 1950s and 60s.
Chemical coupling. Substrate-level
phosphorylation is a mechanism in which negative free energy of a
chemical reaction is
coupled to ATP synthesis. It was thought that electron transport
led to synthesis of a “high-energy intermediate” and that the
energy conserved in this molecule was then passed on to ADP + Pi to
make ATP. There was an energetic search for the
high-energy intermediate.
Chemiosmotic coupling. Peter Mitchell.
6. The Four
Postulates of the Chemiosmotic Theory
Peter Mitchell proposed that nature uses protonic
batteries to drive ATP synthesis and that biological energy
conservation is
essentially a problem in membrane transport (see chemiosmotic
theory diagram). The chemiosmotic theory consists of four
postulates:
1. The inner membrane contains electron
transport enzymes which are vectorially oriented so that the energy of
electron transport
drives ejection of protons outward across the membrane. The
energy of substrate oxidation is thereby converted to and stored as
a proton electrochemical potential gradient, called the protonmotive
force.
2. The inner membrane contains a reversible,
proton-translocating ATPase, which is also vectorially oriented so that
the energy of
ATP hydrolysis will drive protons outward across the inner
membrane. The ATPase is reversible, so that protons driven inward
through the enzyme by the redox-generated protonmotive force will cause
ATP synthesis.
3. The inner membrane must have a low
diffusive permeability to ions in general and to protons in
particular. Otherwise, ion leaks
would short-circuit the protonmotive batteries, and ATP would not be
synthesized.
4. The inner membrane was postulated to
contain exchange carriers in which anion entry is effectively coupled
to proton entry.
This provides a thermodynamically favorable pathway for substrate
anions to reach enzymes witnin the electro-negative matrix.
The membrane was also postulated to contain exchange carriers in which
cation exit is coupled to proton entry. This provides
a
thermodynamically favorable pathway for removal of cations that entered
the matrix by diffusion down the very large electrical
gradient caused by outward proton pumping.
The chemiosmotic theory begins by describing the
mechanism of coupling between substrate oxidation and
phosphorylation.
It goes on to describe the membrane properties that are required in
order for mitochondria to provide ATP to the cell and,
indeed, to survive within the cell. The chemiosmotic theory was
presented as an hypothesis far in advance of experimental
evidence, and it stands as a monument to the scientific method.
For this extraordinary achievement, Peter Mitchell was
awarded the Nobel Prize in Chemistry in 1978.
7. Protonmotive
Force
Electron transport through complexes I, III, and IV
is coupled to electrogenic proton ejection across the inner
membrane.
The protonmotive force (Δp) is the free energy per mol for moving
protons outward across the membrane. It is simply the
sum of the work done against the electrical force and the work done
against the proton concentration difference.
xx free energy section
Δp is defined as the electrochemical proton gradient
divided by the Faraday constant
(- ΔH+/F); therefore,
Δp = ΔΨ –
ZΔpH (1)
where Z ≡ (RTln10)/F = 59 mV at 25°C, and ΔΨ is
the membrane potential (inside minus outside). ΔΨ and ΔpH can be
estimated from equilibrium distributions of cationic dyes and weak
acids, respectively. It is customary in bioenergetics to
drop the negative signs of ΔΨ and Δp. Commonly observed values in
isolated , non-phosphorylating mitochondria are
190 mV for ΔΨ, 0.3 units for ΔpH, resulting in a Δp of about 208 Mv.
8.
Stoichiometries
In the final step of electron transport, the
dioxygen molecule (O2) is reduced to water by four electrons (e-/O =
2).
When a pair of electrons moves from NADH to oxygen, it is estimated
that 10 protons are ejected across the inner
membrane (H+/O = 10).
9. Respiratory
Control
Respiration can readily be measured as oxygen uptake
by isolated mitochondria. The typical traces in Fig. 3 illustrate
the principle of respiratory control, which is that respiration
increases if the proton back-flux across the inner membrane
is facilitated, either through the ATP synthase or by
proton-translocating drugs or proteins.
10. Behavior of
the Protonmotive Circuit
The chemiosmotic theory identifies the electron
transport system (ETS) as a protonmotive cell. It is an
electrical circuit,
as shown in the circuit diagram. We note four salient aspects of
this circuit: (1) The electron current is measured as
respiration (as shown “respiratory control”). (2) The current is
determined entirely by the external resistances, and the
battery will deliver increased current only when Re or RATP are
decreased. (3) The battery will respond the same
whether current is drawn through Re or RATP. (4) As increasing
current is drawn from the battery, the voltage will
decrease, due to the internal resistance, Ri. Thus, respiration
is driven by the free energy contained in the redox drop,
and it is controlled by the proton back-flux through leak pathways and
the ATP synthase.
The experiment labeled “the mitochondrial battery
curve” was performed by measuring respiration and membrane
potential (ΔΨ) in respiring mitochondria. Electron current was
progressively increased by adding a protonophore that
decreases external resistance (Re) to H+ ions. The resulting
increase in respiration causes ΔΨ to fall gradually until the
Vmax of the ETS is reached. Δp = ZΔpH - ΔΨ; however ΔpH was held
constant in this experiment, so only ΔΨ is plotted.
What is being measured in such experiments is
evident from the circuit diagram.
Δp = Δpo – Ri *
VO (2)
where VO is the respiration rate. The slope,
Ri, is the internal resistance of the ETS, representing the weighted
sum of frictional
coefficients of the reactions leading to proton ejection. The
intercept is Δpo, the theoretical open-circuit voltage of the system,
whose value is given by
Δpo = (2/nH) ΔE
(3)
where nH is the H+/O stoichiometry, and ΔE is the
redox span being studied. The total redox potential, ΔE, for a
pair of
electrons moving from NADH/NAD+ to oxygen is about 1.16 volts.
Therefore, if nH is 10, Δpo = 232 mV.
Decreasing Re to increase proton back-flux may be achieved by
ionophores, by uncoupling protein, or by futile Ca2+
or K+ cycling. Decreasing RATP to increase proton back-flux may
be achieved by adding ADP and phosphate so that
current is drawn via the ATP synthase. Careful measurements
show that all methods of increasing respiration yield points
that fall on the same battery curve as illustrated in the figure.