Transcript Electron Transport System

Electron Transport System:
The Chemiosmotic Theory, redox reactions of the
electron transport system, NAD/ATP exchange ratio
Bioc 460 Spring 2008 - Lecture 29 (Miesfeld)
Passing the baton is
analogous to passing
along the e- in the ETS
Rotenone (rat poison) blocks etransport through FeS clusters
Hydrogen cyanide is a deadly
gas that blocks e- transport from
complex IV to O2 in the ETS
Key Concepts in the Electron Transport System
• The Electron Transport System converts redox energy into protonmotive force. In this “pathway” the oxidation of NADH and FADH2 is
coupled to the reduction of O2 to form H2O. The proton motive force is
used to induce conformational changes in the ATP synthase complex.
• The Chemiosmotic Theory states that energy from redox reactions is
translated into vectorial energy by coupling electron transfer to
membrane bound proton pumps that transverse a proton impermeable
membrane and thereby establish an electrochemical proton gradient.
• The ATP currency exchange ratios of NADH and FADH2 reflect ATP
synthesis in response to H+ movement through the ATP synthase
complex. It takes 4 H+ to synthesize 1 ATP, and since NADH oxidation
pumps across 10 H+, the exchange ratio is 2.5 ATP/NADH. However,
FADH2 oxidation only results in 6 H+ being pumped across the
membrane, and therefore the exchange ratio is 1.5 ATP/ FADH2.
The Electron Transport
System (ETS) is intimately
linked to the process of
oxidative phosphorylation,
both of which take place
within the mitochondrial
matrix.
Photosynthesis also uses a
form of electron transport
that is driven by light
absorption rather than
redox energy.
Peter Mitchell's Chemiosmotic Theory
•
Oxidation of NADH and FADH2 in the mitochondrial matrix by the
electron transport system links redox energy to ATP synthesis.
•
Chemiosmosis involves the outward pumping of H+ from the
mitochondrial matrix through three protein complexes in the
electron transport system (ETS complexes I, III, IV).
•
H+ flow back down the proton gradient through the membranebound ATP synthase complex in response to a chemical (H+
concentration) and electrical (separation of charge) differential.
Overview of Chemiosmotic Theory
Electron Transport
System
FADH2
Ox Phos
ATP synthase
complex
Energy Conversion Requires the Proton Circuit
Basic Components of the Chemiosmotic Theory
Basic Components of the Chemiosmotic Theory
• Energy from redox reactions or light is translated into
vectorial energy and a proton circuit.
• Vectorial H+ pumping results in chemical gradient (pH)
and a membrane potential ΔΨ (Δpsi)
• Separation of charge is due to build-up of positivelycharged protons (H+) and negative hydroxyl ions (OH-)
Basic Components of the Chemiosmotic Theory
• In mitochondria, the contribution of ΔΨ (ΔV) to ΔG is actually
greater than that of ΔpH (the ΔpH across the mitochondrial
membrane is only 1 pH unit)
• In chloroplasts, the ΔpH contribution to ΔG is much more
significant with ΔpH close to 3 pH units
• Change in free energy (ΔG) for a membrane transport process is the
sum of the ion concentration (RT·ln(C2/C1)) and the membrane
potential (ZFΔV)
• In mitochondria, the ZFΔV term makes a larger contribution than
does RT·ln(C2/C1).
The Mitochondrion, the Powerhouse of the Cell
A critical feature of the mitochondrion is the extensive surface area of
the inner mitochondrial membrane which forms the proton-impermeable
barrier required for chemiosmosis.
Peter Mitchell - Eccentric Scholar
He established the Glynn Research Institute in the early 1960s with a
research staff of less than twenty, and remained a private research
institution for almost 30 years. Mitchell's uncle was Sir Godfrey Mitchell
who owned George Wimpy and Company Limited, the largest
construction company in England at the time.
How was Mitchell’s idea proven?
Using biochemical approaches:
1. "inside-out" submitochondrial
membrane vesicles that could be
shown to pump protons into the
interior of the vesicle when
oxidizable substrate was made
available.
2. artificial vesicles containing
bacterial rhodopsin protein were
exposed to light
•
•
proton pumping by the
bacteriorhodopsin protein resulted in
both inward proton pumping
ATP synthesis on the vesicle surface
The Nobel Prize in Chemistry 1978
Peter Mitchell's speech at the Nobel Banquet, December 10, 1978:
The philosopher Karl Popper, the economist F. A. Hayek, and
the art historian K. H. Gombrich have shown that the creative process
in science and art consists of two main activities: an imaginative
jumping forward to a new abstraction or simplified representation,
followed by a critical looking back to see how nature appears in
the light of the new vision. The imaginative leap forward is a
hazardous, unreasonable activity. Reason can be used only when
looking critically back. Moreover, in the experimental sciences, the
scientific fraternity must test a new theory to destruction, if possible.
Meanwhile, the originator of a theory may have a very lonely time,
especially if his colleagues find his views of nature unfamiliar, and
difficult to appreciate.
Pathway Questions
1. What does the electron transport system/oxidative
phosphorylation accomplish for the cell?
– Generates ATP derived from oxidation of metabolic fuels
accounting for 28 out of 32 ATP (88%) obtained from glucose
catabolism.
– Tissue-specific expression of uncoupling protein-1 (UCP1) in
brown adipose tissue of mammals short-circuits the electron
transport system and thereby produces heat for
thermoregulation.
2. What is the overall net reaction of NADH oxidation by the
coupled electron transport and oxidative phosphorylation
pathway?
2 NADH + 2 H+ + 5 ADP + 5 Pi + O2 → 2 NAD+ + 5 ATP +2 H2O
Pathway Questions
3.
What are the key enzymes in the electron transport and oxidative
phosphorylation pathway?
ATP synthase complex – the enzyme responsible for converting protonmotive force (energy available from the electrochemical proton
gradient) into net ATP synthesis through a series of proton-driven
conformational changes.
NADH dehydrogenase – also called complex I or NADH-ubiquinone
oxidoreductase. This enzyme catalyzes the first redox reaction in the
electron transport system in which NADH oxidation is coupled to FMN
reduction and pumps 4 H+ into the inter-membrane space.
Ubiquinone-cytochrome c oxidoreductase - also called complex III,
translocates 4 H+ across the membrane via the Q cycle and has the
important role of facilitating electron transfer from a two electron carrier
(QH2), to cytochrome c, a mobile protein carrier that transfers one
electron at a time to complex IV.
Cytochrome c oxidase - also called complex IV pumps 2 H+ into the
inter-membrane space and catalyzes the last redox reaction in the
electron transport system in which cytochrome a3 oxidation is coupled
to the reduction of molecular oxygen to form water ( O2 + 2 e- + 2 H+ →
H2O).
Pathway Questions
4. What are examples of the electron transport system and
oxidative phosphorylation?
Cyanide binds to the heme
group in cytochrome a3 of
complex IV and blocks the
electron transport system
by preventing the reduction
of oxygen to form H2O.
Hydrogen cyanide gas is
the lethal compound
produced in prison gas
chambers when sodium
cyanide crystals are
dropped into sulfuric acid.
The Electron Transport System Is A Series Of
Coupled Redox Reactions
The electron transport system consists of five large protein
complexes:
1. Complex I; NADH-ubiquinone oxidoreductase (NADH
dehydrogenase
2. Complex II; succinate dehydrogenase (citrate cycle
enzyme
3. Complex III; Ubiquinone-cytochrome c oxidoreductase
4. Complex IV; cytochrome c oxidase
5. F1F0 ATP synthase complex consisting of a "stalk" (F0)
and a spherical "head" (F1)
It was possible to order the four electron transport system
complexes because of:
• Specific redox reaction inhibitors
(such as rotenone, antimycin A and cyanide)
• Known reduction potentials (Eº') of conjugate redox pairs
Electrons flow spontaneously in this direction
FADH2
The stoichiometry of "proton pumping" is:
4 H+ in complex I
4 H+ in complex III
2 H+ in complex IV
(10 H+/NADH and 6 H+/FADH2)
Metabolic Fuel for Electron Transport
• NADH and FADH2 feed into the electron transport system from the
citrate cycle and fatty acid oxidation pathways.
• Pairs of electrons (2 e-) are donated by NADH and FADH2 to
complex I and II, respectively
• Pairs of electrons flow through the electron transport system until
they are used to reduce oxygen to form water (O2 + 2 e- + 2 H+ →
H2O).
• The two mobile electron carriers in this series of reactions are
coenzyme Q (Q), also called ubiquinone, and cytochrome c which
transfer electrons between various complexes.
How is the energy released by redox reactions used to
"pump" protons into the inter-membrane space?
• a redox loop mechanism
– Q cycle in complex III
• redox-driven conformational changes : “proton pump”
– complexes I and IV
Separation of the H+ and e- on opposite sides of the membrane
The Q cycle in complex III uses this mechanism to translocate protons
across the membrane
Redox-driven conformational changes in the protein complex
"pump" protons across the membrane by altering pKa values of
functional groups located on the inner and outer faces of the
membrane.
Complex I: NADH-ubiquinone oxidoreductase
Complex 1 passes along 2 e- obtained from the oxidation of NADH
to Q using a coupled reaction mechanism that results in the net
movement of 4 H+ across the membrane.
Complex II: Succinate dehydrogenase
The 2 e- extracted from succinate in the citrate cycle is passed through
the other protein subunits in the complex to Q as shown below. No
protons are translocated across the inner mitochondrial membrane by
complex II.
Complex III: Ubiquinone-cytochrome c oxidoreductase
Note the relative position of the electron carriers and the presence of two distinct
binding sites for ubiquinone called QP and QN, which play a crucial role in
diverting one electron at a time to cytochrome c via the Q cycle.
The terms QP and QN refer to the proximity of the sites to the positive (intermembrane space) and negative (matrix) sides of the membrane.
The Q Cycle
• Functions as both a mobile electron carrier and a
"transformer" that converts the 2 e- transport system
used by complexes I and II, into a 1 e- transport system
required by cytochrome C.
• The Q cycle requires that 2 QH2 molecules get oxidized
by complex III, with one of QH2 molecule being reformed by reduction to give a net oxidation of 1 QH2
molecule.
Four Steps of
the Q Cycle
Converts a 2 e- carrier
(QH2) into a 1 e- carrier
(cytochrome c).
This requires that 2
QH2 molecules be
oxidized, with 1 QH2
being reformed.
Note that the Q cycle
reactions require that 2H+
from the matrix be used to
regenerate QH2, even though
4H+ are translocated.
However, this apparent
imbalance of 2H+ is
corrected by the redox
reactions of complex IV
where 2H+ are required to
reduce oxygen to water
and 2H+ are pumped across
the membrane.
Therefore, the net
translocation of protons
across the membrane in the
combined redox reactions
of complexes III and IV
becomes 6 H+N → 6 H+P.
2 H +P
2 H +P
2 H+N
To see how the Q cycle accomplishes the 2 e- → 1 e- + 1 e- conversion
process, write out two separate QH2 oxidation reactions and then sum them
to get the net reaction for complex III:
QH2 + Cyt c (oxidized) → Q•- + 2 H+P + Cyt c (reduced)
QH2 + Q•- + 2 H+N + Cyt c (oxidized) → Q + QH2 + 2 H+P + Cyt c (reduced)
QH2 + 2 H+N + 2 Cyt c (oxidized) → Q + 4 H+P + 2 Cyt c (reduced)
Why are 2 QH2 required to transfer 2 e- to 2 cytochrome c molecules?
What happens to the “extra” QH2 molecule in step 4 of the Q cycle?
Cytochrome C
Cytochrome c (Cyt c) is responsible
for transporting one electron at a time
from complex III to complex IV using
an iron-containing heme prosthetic
group.
Oxidized Cyt c contains ferric iron
(Fe3+) in the heme group, reduced
Cyt c contains ferrous iron (Fe2+).
mystery
protein
revealed
Complex IV: Cytochrome c oxidase
2 H+P
Complex IV accepts electrons one
at a time from Cyt c and donates
them to oxygen to form water.
In the process, 2 net H+ are
pumped across the membrane
using a conformational-type
mechanism similar to complex I.
In addition, 2 H+ from the matrix
side are used to reduce 1/2 O2 to
produce 1 H2O.
Combining proton movement
across the membrane in complex
III and IV, results in 6 net H+
4 H+N
ATP Currency Exchange Ratios of NADH and FADH2
Experimental measurements demonstrate 3 H+ are required to
synthesize 1 ATP when they flow back down the electrochemical proton
gradient through the ATP synthase complex, and 1 H+ is needed to
transport each negatively-charged Pi molecule into the matrix.
ATP Currency Exchange Ratios of NADH and FADH2
Taking into account the requirement of 3 H+/ATP synthesized,
and the use of 1 H+ to translocate ADP, the total is 4 H+/ATP.
We can now see where the ATP currency exchange ratios of
~2.5 ATP/NADH and ~1.5 ATP/FADH2 come from:
Oxidation of NADH starting at complex I yields:
10 H+/4 H+ = 2.5 ATP
Oxidation of FADH2 starting at complex II yields:
6 H+/4 H+ = 1.5 ATP for FADH2