Lecture 6 BCHM2971
Biochemical thermodynamics:
ATP and redox reactions.
Oxygen’s double edged sword
Thermodynamics and mechanisms of
storing and spending energy
Proton
gradient
Krebs
WORK
store
C02
Glycolysis
spend
ADP
spend
NAD NADH
release
store
fuel
ATP
e- transport chain
Oxidative
Free energy DG
Redox and E
phosphorylation
coupling
Plan for today’s lecture
1. Free-energy currency is "spent" to drive
nonspontaneous reactions
•
DG and coupling
2. Why is ATP the currency of free-energy?
3. Redox cycles of e- and H+ transfer:
•
redox potentials (DE )
4. Mechanism of e- and H+ transfer:
•
Complex 4 of the electron transfer chain
5. Oxygen as the final acceptor of electrons
Why eat?
• most metabolic reactions are not spontaneous
• require a source of free energy = DG
• Energy released from food is eventually
‘saved’ in ATP
 ‘spent’ to drive energetically unfavourable
reactions
Free energy change (DG)
• Free energy change (DG) of a reaction
determines its spontaneity
• negative DG  spontaneous ( products)
ie: G products < G reactants
For a reaction A + B  C + D
[C] [D]
DG = DG ' + RT ln
[A] [B]
o
R = gas constant; T = temp.
For a reaction A + B  C + D
[C] [D]
DG = DG ' + RT ln
[A] [B]
o
standard free energy change
reactants & products = 1M
DG
free energy change of reaction
under ‘other’ conditions (eg in
the cell)
pH 7 ([H+] = 10-7M)
Value depends on actual [products] and [reactants]
Hydrolysis of ATP
•
•
useful free-energy ‘currency’
dephosphorylation reaction is very
spontaneous
ATP  ADP + Pi
(DGo' = -31 kJ/mol) DG<0
Spontaneous?
• Spontaneous does not indicate how
quickly a reaction occurs
• ATP (and pals) are kinetically stable
(usually have free energies of activation)
• Rate low without enzyme
Activation energy
energy
-ve DG
reaction
Spontaneous?
Why doesn’t ATP explode??
• Spontaneous does not indicate how
quickly a reaction occurs
• ATP (and pals) are kinetically stable
(usually have free energies of activation)
• Rate low without enzyme
Activation energy
(lowered by enzyme)
energy
-ve DG
reaction
Spontaneous?
• Kinetic stability essential:
• reaction energy is then
 Controllable by catalysis
 Can be coupled to useful reactions
Activation energy
(lowered by enzyme)
energy
-ve DG
reaction
What makes the bonds in ATP
‘high-energy”?
• Phosphoanhydride bonds tend to have a large
negative DG (-30.5 kJ.mol-1)
• NB: bond energy is not necessarily high, just
the free energy of hydrolysis.
Phosphoester
Phosphoanhydride
bonds
gP
O
bP
O
Adenine
bond
aP
CH2
Ribose
ATP
1. PhAnH bond has less stable
resonance than its product
• Two strongly ewithdrawing groups
compete for p e- of
the bridging oxygen
hydrolysis
• No such competition
in the hydrolysis
product more stable
2. PhAnH bond has greater
electrostatic repulsion than its product
• At pH 7, ATP has 3 –ve
charges
hydrolysis
• Repulsion is relieved by
hydrolysis
 more stable
3. Solvation energy
• Phosphoanhydride bond has smaller
solvation energy than product
 favours hydrolysis
Phosphoryl group-transfer potential
• Measure of tendency
of compound to
transfer ~P to H20
• ATP is intermediate!
• Can accept ~P from
compounds above
• Or donate ~P to
compounds below
Other high energy compounds
•Other phosphorylated compounds
–Phosphocreatine
•Thioesters
–CoenzymeA (you will meet this in
other lectures)
Phosphocreatine
• Higher P-group transfer potential than ATP
• ‘reservoir’ of ~P for rapid ATP regeneration
Maintains constant level of ATP by swapping ~P
=reversible ‘substrate-level phosphorylation’ in tissues with
high need (muscle, nerve)
When  ATP 
P phosphocreatine
ADP
creatine
ATP P
When  ATP
When ATP is low,
phosphocreatine
can lend a P to ADP
to make ATP.
When ATP is
replenished by
catabolism, P is
‘paid back”.
Why create high energy
compounds?
• spontaneous reactions DG<0 are often
coupled with non-spontaneous
reactions (DG>0) to drive them forward.
• The free-energy change (DG) for coupled
reactions is the sum of the free-energy
changes for the individual reactions.
DGcoupled = DG reaction 1 + DG reaction 2
• Thus, ATP  ADP +Pi (DG<0) is coupled
with non-spontaneous reactions (DG>0)
to drive them forward.
Glucose
glucose-6-P + H20
DG = 13.8 kJ.mol-1
hexokinase
DG = -30.5 kJ.mol-1
ATP +H20
ADP +Pi
DG = -16.3 kJ.mol-1
Glucose + ATP
Overall: spontaneous!
glucose-6-P + ADP
Energy coupling with ion gradient
Energy can also be stored as an ion gradient
ADP
+ve DG
-ve DG
• eg oxidative
Proton
phosphorylation
gradient
• Spontaneous H+
movement against
gradient coupled to
ATP synthesis
ATP
How does energy from food get
transferred to ATP for storage?
Controlled
cycles of
oxidation and
reduction
e- H
glucose
CO2
OXIDATION
REDUCTION
e-e-
NAD+
NADH
Sequential transfer of H: (2e- and H) from
fuels indirectly provides free energy for
production of ATP. What causes transfer of eand H+? How does this release energy to
create an ion gradient?? Remember redox
potentials?
e-eH2O
O2
OXIDATION
REDUCTION
H
I
e- e-
Cyt C
Q
III
IV
Electron transport chain (ETC)
Aoxidised
A reduced
OXIDATION
REDUCTION
B oxidised
gain electrons,
gain H
lose O
e-
B reduced
The tendency of a substance to undergo reduction
= E°’ (reduction potential)
E°’ =  Affinity for electrons
DE °' = E °‘ (acceptor) – E °‘ (donor)
Reduction Potential and
Relationship to Free Energy
DE °' = E °'(acceptor) – E °'(donor)
o
DG '
**Don’t learn these
equations! Just
understand the
implications of +ve or
–ve values
= – nFDE °'
# electrons
transferred
Faraday
constant
o
DG '
= – nFDE °
'
• An electron transfer reaction is
spontaneous (-ve DG)
if DE°‘ is +ve
ie: when E °' of the acceptor >
E °' of the donor
Electrons spontaneously flow from low  high
reduction potentials
Spontaneous if...
Aoxidised
A reduced
OXIDATION
REDUCTION
B oxidised
acceptor has higher DE
e-
B reduced
thermodynamics of the ETChain
• NAD accepts e- and H+ from fuel NADH
• NADH donates e- and H+ to ETC
Hydride ion =
2e + H+
Oxidised
Accepts e- from fuel
In ETC
reduced
NADH oxidation is spontaneous
and releases free energy
E°’ = -0.3 V
NAD+ + H+ + 2e-
NADH
reduced
oxidised
E°’ = +0.8 V
½ O2 + 2H+ + 2e-
H2O
DE °' = E °'(acceptor) – E °'(donor)
DE °‘ = 0.8 – (-0.3) = 1.13V
O2 has greatest affinity for eNADH becomes the e- donor
NADH oxidation is spontaneous
and releases free energy
NAD+ + H+ + 2e-
NADH
reduced
oxidised
OXIDATION
REDUCTION
½ O2 + 2H+ + 2e-
H2O
DE °‘= 1.13V
o
DG '
-
ve
= – nFDE °
‘
+ve
electrons are not transferred
directly from NADH to O2
• rather pass through a series of
intermediate electron carriers
• Why? This allows energy released to be
coupled to protons pump.
• ultimately responsible for coupling the
energy of redox to ATP synthesis.
Electrons spontaneously flow from
low to high reduction potentials
Increasing E
One example in more detail:
Complex IV (cytochrome c oxidase)
Transmembrane
spanning a-helices
Complex IV
(cytochrome c oxidase)
• Catalyses final reduction in the ETC
• O2 + 4 H+ + 4 e-  2 H2O (irreversible)
• The four electrons are transferred into the
complex one at a time from cytochrome c.
• Results in pumping of 4 H+ across the
membrane.
Has 4 metal ‘redox centers’
• CuA (=2 Cu atoms)
• haem a (Fe)
• haem a3, (Fe)
• CuB
Ions in close proximity
= binuclear complex
FIRST: 2e- passed from cytC by
haem a-CuA to binuclear center
eCyt C
• e- are
passed
one at a
time
So far…
Fully oxidised
H O-
Fe3+
H O
e- e-
H+
Fully reduced
e-
Cu2+
Fe2+
Tyr
H O
e-
Cu+
Tyr
H O
H
• 2e- were passed from
cytC by haem a-CuA
to fully reduce Fe and
Cu in the binuclear
center
• H+ from matrix and
hydroxyl from
binuclear center 
H2O
Then, O2 binds
e- eH O-
Fe3+
H O
H+
Fully reduced
O
e-
Cu2+
Fe2+
Tyr
O
H O
e-
Cu+
Tyr
O
e-
2+
O Fe
H O
e-
Cu+
Tyr
H O
H
This O2 is going to become O22It’s going to need 4 e-
The tricky bit!!
Fully oxidised
H O-
Fe3+
H O
e- e-
O
H+
O
e-
Cu2+
Fe2+
Tyr
H O
e-
Cu+
Tyr
O
e-
2+
O Fe
H O
e-
Cu+
Tyr
H O
H
• 4e- are rearranged
• Only 3e- can be donated by the
metal ions (see why?)
• So 1e- ALSO must be donated
temporarily from tyrosine
•  OXYFERRYL complex
Fe2+ - 2e-  Fe4+
Cu + - 1e-  Cu2+
Tyr-OH - 1e- -H+  Tyr-O.
ee O2- e Fe4+
O
e- H
2+ O
Cu
Tyr
O22- shared between Cu and Fe
1 more e- passed in via haem3-CuA to
binuclear complex  Reconverts tyrosine
Fully oxidised
H O-
e- e-
H O
O
e-
Cu2+
Fe3+
O
H+
Fe2+
Tyr
H O
e-
O
Cu+
Tyr
e-
2+
O Fe
H O
e-
Cu+
Tyr
H O
H
H O
H
ee O2- e Fe4+
H O
ee O2- e Fe4+
Cu2+
Tyr
And more H+  H2O
H
H
e-
O
e- H
2+ O
Cu
Tyr
4th e- passed via h3CuA
Regenerates Fe3+: Completed cycle!
Fully oxidised
H O-
e- e-
H O
O
e-
Cu2+
Fe3+
O
H+
Fe2+
Tyr
H O
e-
O
Cu+
Tyr
e-
2+
O Fe
H O
e-
Cu+
Tyr
H O
H
eH
H O
H
And one more H+
ee O2- e Fe4+
H O
ee O2- e Fe4+
Cu2+
Tyr
O
eH
H
e- H
2+ O
Cu
Tyr
H+
H+
H+
H+
Meanwhile pumps
4 H+ were pumped
to proton
gradient
H+
H+
O
H+
H+
O
O2 as final e- acceptor
• Strong e- acceptor (high E)
Provides  thermodynamic force
• Also, controllable: reacts slowly
unless catalysed by enzyme
Disadvantages
•
•
•
•
O2 + 4 e-  safe 2H20
BUT partial reduction  DANGER!!!
O2 + e-  O2 – (superoxide)
Can extract e- from other molecules 
‘free radicals’
• Oxidisation of membranes, DNA, enzymes
• Implicated in Alzheimers, Parkinsons,
aging
Summary
• Hydrolysis of ATP is spontaneous (–ve DG)
• Free energy of ATP coupled to nonspontaneous reactions
• Phospho-anhydride bond is ‘high energy’
• Electrons spontaneously flow from low to
high E
Food NAD e- transfer chain O2
• Free energy used to create proton gradient
that is then ‘spent’ to make ATP
The individual reactions are:
• oxidation
NADH  NAD+ + H+ + 2e-
Do NOT learn these values!
Just know which are +ve or
–ve/ spontaneous or
not…understand concept of
coupling!!
DGo= -158.2 kJ
spontaneous
• reduction
½ O2 + 2H+ + 2e- H2O
DGo= -61.9 kJ
• phosphorylation
ADP  ATP
DGo= +30.5 kJ
spontaneous
nonspontaneous
• The net reaction is obtained by summing the coupled reactions,
ADP + NADH + ½ O2 + 2H+ 
ATP + NAD+ + 2 H2O
DGo= -189.6 kJ
spontaneous
Coupled non-spontaneous work