Transcript document

Terminal Oxidative
Phosphorylation
NAD+ and NADH





Nicotinamide adenine dinucleotide is a central intermediate
Previously encountered as a product of glycolysis
NADH is formed by the addition of a hydrogen nucleus and 2 electrons (hydride ion)
to NAD+
Nicotinamide ring has reduced stability when it accepts the hydride ion since it is no
longer stabilized by resonance.
Consequently the electrons (i.e., hydride ion) of NADH can be readily transferred
Example of a reaction involving NAD+ and NADH

Oxidation of an alcohol: two hydrogen atoms are lost. One is added as a hydride ion
(H. ) to NAD+ the other is released into solution as a proton (H+)
Another view of oxidative phosphorylation







The energetically favorable reaction
H2 + 1/2 O2 ---> H2O
is made to occur in many small steps.
This allows the energy to be stored
rather than released as heat.
Electron
transport
involves
a
respiratory chain (or electron transport
chain).
Respiratory chain consists of protein
pumps that harness energy from
electron transfer to perform work.
Electrons ultimately are passed to
oxygen.
The carriers are transmembrane protein
complexes.
The work done is to pump H+ across
the inner membrane. This has 2 results:
• Low [H+] in the mitochondrial
matrix
• Voltage gradient - matrix side
more negative than outside
Another overview of oxidative phosphorylation




The electrons from H2 (or NADH) are
used to reduce O2.
It is evident that the reduction potential
of NADH must be higher than the next
step in the respiratory chain (or
electron transport chain). Only then the
acceptor of the electrons can be
reduced (accept the electrons).
The same applies to the next steps until
the electrons are passed to oxygen
which has the lowest reduction
potential (but the highest oxidation
potential).
In order to better understand the flow of
electrons through the respiratory chain
it is good to measure the redox
potential.
The redox potential (E)





An electrochemical concept that measures the affinity of a substance for electrons
Consider a substance that can exist in an oxidized form X and a reduced form X(redox couple).
The reduction potential of this couple can be determined by measuring the
electromotive force (voltage) generated by a sample half-cell connected to a standard
reference half-cell
The sample half-cell consists of an electrode immersed in a solution of 1 M oxidant
(X) and 1 M reductant (X-). The standard reference half-cell consists of an electrode
immersed in a 1 M H+ solution that is in equilibrium with H2 gas at 1 atmosphere
pressure.
The electrodes are connected to a voltmeter, and an agar bridge establishes electrical
continuity between the half-cells.
The redox potential (E)

Electrons then flow from one half-cell to the other. If the reaction proceeds in the
direction
the reactions in the half-cells (referred to as half-reactions or couples) must be


Thus, electrons flow from the sample half-cell to the standard reference half-cell, and
the sample-cell electrode is taken to be negative with respect to the standard-cell
electrode. The reduction potential of the X:X-couple is the observed voltage at the
start of the experiment (when X, X-, and H+ are 1 M). The reduction potential of the
H+:H2couple is defined to be 0 volts.
In the example, the reduction potential of the X:X-couple is higher (more negative E)
than the reduction potential of the H+:H2couple. The oxidation potential of the X:Xcouple is lower (more negative E) than the reduction potential of the H+:H2couple.
Standard reduction potentials of some reactions

If we know the redox potentials of half-reactions, we can predict how a redox reaction
will proceed.
Reduction potentials and free energy


The redox potential of reactants/products determines in which direction redox
reactions will proceed – but there is also a relation between the difference of the
redox potentiaIs of a redox pair and the free energy change:
ΔG°’= -nFΔE°’
where n is the number of electrons involved and F is the Faraday constant (23
kcal/mol V).
Thus, one can even predict the energy liberated or needed for a reaction.



The redox potential of the NADH/NAD+ pair is –0.32
The redox potential of the H2O/ ½O2+H+ pair is +0.82
nFΔE°’ = 1.14 V
• ΔG°’ = - (2)(23 kcal/mol V)(1.14 V)
•
= -52 kcal/mol


ΔG°’ for the formation of ATP is +8 kcal/mol.
Thus, if electron transport chain is 100% efficient it could be predicted that (52/8) or
≈6 ATP would be formed per NADH oxidized.
The highest actual values measured are around 3, or 50% efficiency which is pretty
good.

A little history
• Once the relationship between the redox potentials of
the respiratory chain was worked out, it was easy to
identify steps in the process where there was enough
energy to phosphorylate ADP, and it was assumed that
at these ‘phosphorylation sites’, phosphorylation was
coupled directly to the corresponding redox reaction.
• So it was possible to calculate a reasonable
stoichiometry for the whole process that related Pi/O and
thus ATP/O2. The numbers for this were well accepted
by 1960.
• However, it was seldom possible to obtain the calculated
numbers with isolated mitochondria, and never possible
to get phosphorylation to happen in fragmented
mitochondria or in a soluble system.
From Baldwin, Dynamic
Aspects of
Biochemistry, 1957
Continued…
• In 1961 Peter D. Mitchell published a hypothesis
for the nature of the coupling between electron
transport and phosphorylation – the
chemiosmotic hypothesis.
• This hypothesis is universally accepted now, and
Mitchell received the Nobel Prize for it in 1978.
• However, many textbook authors have still not
figured out the implications of this mechanism
for the yield of ATP from complete oxidation of
glucose.
Morals of the story
• Much of what you learn in school is not
true
• Science progresses by revolution rather
than steady evolution
• Scientists are, on balance, not better
about changing their views than nonscientists

The electron transport chain in the inner
mitochondrial membrane
Electron flow through 4 complexes.
Some things to take note of:
The NADH that feeds into
this process can come from
glycolysis, lipolysis and/or the
TCA cycle.
At some points in the
sequence that starts with
NADH, some of the free
energy is used to transport
H+ across the inner
mitochondrial membrane
FADH2 derived from the
succinate-fumarate step of
the TCA cycle has only
enough free energy to
energize two of these H+
sites.
Coupling between TCA cycle and oxidative
phosphorylation


The reduction equivalents NADH and FADH2 couple the TCA cycle to the oxidative
phosphorylation.
NAD+ and FAD+ are reduced in the TCA cycle and their oxidized forms are regenerated
in terminal oxidative metabolism, with oxidative phosphorylation of ADP.
A different view

Electron transport energizes H+ transport from the matrix into the intermembrane
space.
1 NADH
= 10 H+
1 FADH2
= 6 H+

FADH2 results in less H+ transport.
Generation of a proton motive force






The osmotic and electrical effects of the proton gradient result in a proton-motive
force (PMF or Δp).
pH gradient across the membrane is ΔpH = 1.4
Voltage across the membrane is 0.14 V (outside positive)
magnitude of Δp can be measured in volts
Δp = 0.14 V - (2.3RT/F)(ΔpH) = 0.224 V
This Δp corresponds to a ΔG of 5.2 kcal per mol of protons
Use of the proton-motive force

ATP generation by ATP synthase or F0F1-ATPase.
•
•

F1 unit contains the catalytic site for ATP synthesis
Flow of 3 protons through ATP synthase leads to phosphorylation of 1 ADP
Drive transport of necessary/useful substances.
Transport across inner mitochondrial membrane

ADP and ATP need to be exchanged across the inner mitochondrial membrane

Other transporters:
Cytosolic NADH enters mitochondria via shuttles

Glycerol 3-Phosphate Shuttle
This one costs some energy – the
difference between an NADH and a
FADH2 – and the cytosolic NADH does
not literally enter the mitochondria

This one operates for free
– swapping a NADH in the
mito for a NAD+ in the
cytoplasm
Malate-Aspartate
Shuttle
What is the ATP yield from oxidative
phosphorylation?
•
•
•
Yield of ATP (or GTP) from glycolysis or citric acid is unequivocally known
For oxidative phosphorylation, stoichiometry of proton pumping, ATP synthesis, and
metabolite transport
• need not be integers or have fixed values.
What is the ATP yield based on current estimates?
•
•
•
•
•
NADH oxidation ---> 10 H+ pumped
FADH2 oxidation ---> 6 H+ pumped
3 H+ flow in per 1 ATP produced
ATP/ADP translocase costs 1 H+ per ADP
Therefore 2.5 ATP per NADH, 1.5 ATP per FADH2
•
If NADH originated from glycolysis (cytosol), it must be transported into
mitochondrial matrix – this may or may not cost something
Pyruvate is transported into the matrix by an OH- coupled transporter, so it is driven
by the H+ gradient – this reduces the yield
So ultimately, yield is around 1.5 ATP per cytosolic NADH
•
•
What is the actual yield of ATP from oxidative
phosphorylation?
2 NADH from glycolysis (about 1.5 ATP each)
3
2 NADH from pyruvate dehydrogenase (about 2.5 ATP each)
5
2 FADH2 from citric acid cycle (about 1.5 ATP each)
6 NADH from citric acid cycle (about 2.5 ATP each)
3
15
Total from gradient-mediated phosphorylation
26
2 ATP equivalents (GTP) from substrate-level
phosphorylation in TCA cycle
Total for oxidative metabolism only
2
28
(older calculations gave 36 or 38 ATP)
+2 ATP from substrate level phosphorylations in glycolysis
This is the approximate total yield of ATP starting with 1 glucose –
remember, it’s not a hard number or necessarily an integer
30
Overview of stage 3
Control of terminal oxidative metabolism by
ADP
• At low activity levels, when ADP levels are low, the
proton electrochemical gradient approaches its
maximum possible value, so electron flow through the
ETC slows, oxygen uptake drops, and levels of reduced
coenzyme build up. High ATP levels, low levels of
oxidized coenzyme, and low levels of ADP feed back to
the substrate-level reactions, slowing them.
• If activity levels are increased, ADP arriving at the
phosphorylation sites starts the F1 F0 ATPase spinning
and as the proton gradient runs down somewhat, it is
easier for electrons to flow from reduced coenzymes to
oxygen. Levels of reduced coenzymes drop, oxygen
consumption rises, and inhibition of the substrate-level
reactions is relieved.
Fate of C-skeleton of amino acids



Amino acids that are degraded to acetyl CoA or acetoacetyl CoA are termed ketogenic amino
acids (yellow), because they can give rise to ketone bodies or fatty acids.
Amino acids that are degraded to pyruvate, a-ketoglutarate, succinyl CoA, fumarate, or
oxaloacetate are termed glucogenic amino acids. The net synthesis of glucose from these amino
acids is feasible, because these citric acid cycle intermediates and pyruvate can be converted into
phosphoenolpyruvate and then into glucose.
Mammals lack a pathway for the net synthesis of glucose from acetyl CoA or acetoacetyl CoA.
Another thought question
• There are animals – such as the housefly
– that use oxidative phosphorylation to fuel
flight
• When a fly takes off, it has to rapidly kick
its mitochondria into high gear
• Fly blood has high levels of various amino
acids, especially proline – how could this
relate to the ability to take off quickly?
Oxidative Phosphorylation, Natural Doping,
Ecology and Global Warming
• The omega-3 forms of eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) greatly increase capacity
for oxidative metabolism and exercise tolerance.
• The mechanism for this involves both a general increase
in membrane unsaturated fatty acids and an increase in
the expression of enzymes involved in oxidative
metabolism and fat metabolism.
• The increase in oxidative capacity that results from this
diet alone (roughly 20% in a sedentary bird model – the
quail) is comparable to what can be achieved with
extreme regimes of exercise.
• The semipalmated sandpiper
migrates from the Canadian and
Alaskan Arctic to South America.
• In preparation for their trip across
the Atlantic, the sandpipers stop
off in staging areas on the
Canadian east coast, where they
gorge themselves on a particular
species of amphipod, Corophium
volutator. In the process, they
double their weight in about 2
weeks. The crustaceans they
eat contain record amounts of
DHA and EPA fatty acids. This
dietary strategy apparently
makes transoceanic flight
possible.
• Global warming is causing the natural ranges of
the crustaceans and molluscs normally
consumed by the birds to shift northward. As a
result, the migration on which this species
depends may become impossible.