Summary of electron transport Unfortunately, oxygen is not just a ...

Summary of electron transport
Unfortunately, oxygen is not just
a terminal electron acceptor
• There can be branches, at terminal electron
acceptor, at terminal oxidase, at entry point
of NADH (ie. hot stinking plants)
NADH, a great source of energy
• NADH + 11 H + + ½ O 2 NAD + + 10
H + + H 2 O
• Highly exergonic; ∆G o = -220 kJ/mol
• Actually in cell, much NADH than NAD,
making the available free energy more
negative
• Much of this energy is used to pump
protons out of the matrix
Pumping protons lowers the pH and
generates an electrical potential
Generation of a proton-motive
force
• In an actively respiring mitochondria, the pH is
~0.75 units lower outside than in the matrix
• Also generates an electrical potential of 0.15 V
across the membrane, because of the net
movement of positively charged protons outward
across the membrane (separation of charge of a
proton without a counterion)
• The pH difference and electrical potential both
contribute to a proton motive force
Really, what does that mean?
• Energy from electron transport drives an
active transport system, which pumps
protons across a membrane. This action
generates an electrochemical gradient
through charge separation, and results in a
lower pH outside rather than in. Protons
have a tendency to flow back in to equalize
the pH and charge. This flow is coupled to
ATP synthesis.
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Measuring the proton motive
force
∆µ H = ∆ψ – 2.3RT∆pH/F
(different in Lehninger)
µ H is the resulting proton motive force
(sometimes p)
ψ is the electrochemical membrane potential
Don’t get bogged down in the
math, but …
• (under standard conditions) ∆µ H = 0.224 V
Plug into ∆G o = -nF∆E o and ∆G o = ~20
kJ/mole H +
The bottomline
pH has a negative value, thus contribution is
positive in this equation
The proton motive force bottom
line
Two components to energy derived from
electron transport, pH and electrical
potential. The electrical potential is the
primary contributor to free energy.
So what is the proton motive
force used for?
Most of the energy from oxidation of
NADH is conserved in the proton gradient
Introducing ATP synthase
Electron transfer and ATP
synthesis are coupled
• ATP synthesis occurs only if electron transfer
does, and vice-versa
• When isolated mitochondria are suspended in
buffer containing ADP, Pi and an oxidizable
substrate (succinate) three things happen
– Substrate is oxidized
– Oxygen is consumed
– ATP is synthesized
2

All components are essential
• If ADP were omitted, no ATP synthesis
would occur and electron transfer to oxygen
does not proceed, as well.
Black – oxygen
consumption
Red – ATP
synthesis
There are compounds that can inhibit
ATP synthesis
• The antibiotic oligomycin binds to ATP synthase
and inhibit it’s action.
• By stopping ATP synthesis, this compound also
stops electron transport.
• Because oligomycin is specific for ATP synthase
and not the various electron carriers, this
inhibition supports the coupling of ATP synthesis
to electron transport
There are compounds that can uncouple
ATP synthesis from electron transport
Evidence for uncoupling
• DNP and FCCP block ATP synthesis, while
permitting continued electron transport to oxygen
– they are uncouplers
• They pick up protons from the outside, diffuse in
(they are hydrophobic so can pass through the
membrane), and release proton back inside.
• Electrons are still passed through the electron
transport chain, but the proton gradient is
destroyed.
ATP synthase – A molecular
machine
Something we’ll cover when we
talk about enzymes in detail:
• ATP synthase stabilizes ATP relative to ADP + P i
by binding ATP more tightly, this results in a free
energy change that is near zero
• This is an important point, but ignore for the most
part now as we will cover this in detail later
• What’s important now is that this reaction ATP
synthesis from ADP and Pi occurs without a huge
input of energy – you’ll see it is just mechanical
energy.
3

ATP synthase has two functional
domains
• This enzyme has two distinct parts, one a
peripheral membrane protein (F 1 ) and one a
integral membrane protein (F o ) ( the o
stands for oligomycin sensitive)
• These parts can be separated biochemically,
and isolated F 1 catalyses ATP hydrolysis (it
has the site for ATP synthesis and
hydrolysis)
The F 1 component
• This component is made up of nine proteins
of five different types with a composition
of: α 3 β 3 γδε
• Each of the three β subunits have a catalytic
or “active” site where the reaction occurs
– ADP + Pi ATP + H 2 O
The α and β subunits make a
cylinder with the γ subunit as an
internal shaft
Conformational changes
• Although the β subunits have the exact
same amino acid sequence and composition,
they are in different conformations due to
the γ subunit.
• These conformational differences affect
how the enzyme binds ATP and ADP
The F o component forms a proton
pore in the membrane
Rotation of the γ subunit by H +
translocation drives ATP synthesis
• Passage of protons through the F o component
causes γ to rotate in that internal chamber
• Each rotation of 120 o causes γ to contact another β
subunit, this contact forces β to drop ATP and stay
empty
• The three β subunits interact so that when one is
empty, one has ADP and P i , while another has
ATP.
4

Proton transfer is converted to
mechanical energy, then chemical energy
ATP synthase – at work
• http://nature.berkeley.edu/~hongwang/Proje
ct/ATP_synthase/
• http://www.sciencemag.org/feature/data/10
45705.shl
ATP exits the mitochondria
through active transport
Regulation of ETC
•P
Side
N
Side
• Rate of mitochondrial respiration controlled
by ADP availability ([ATP]/[ADP][Pi])
•IF 1 can bind and block ATP synthase at low
pH
• Hypoxia influences gene expression
Coordinated regulation – more on this
later, but think about global effects
5

What happens when…
• Cells increase NADH oxidation using
alternative NADH oxidase?
• Cells using lots of ATP
6