Lect

ure

6 B

CH

M29

71

Biochemical thermodynamics: ATP and redox reactions.

Oxygen’s double edged sword

Thermodynamics and mechanisms of storing and spending energy

fuel

release

C02

stor

e

Proton gradient

spend

NA

DN

AD

H

ADP

ATP

store

spen

d

WO

RK

e- transport chain

Redox and E

Glycolysis

Krebs

Oxidative

phosphorylation

Free energy G

coupling

Plan for today’s lecture1. Free-energy currency is "spent" to drive

nonspontaneous reactions• G and coupling

2. Why is ATP the currency of free-energy?

3. Redox cycles of e- and H+ transfer:• redox potentials (E )

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 = G

• Energy released from food is eventually ‘saved’ in ATP

‘spent’ to drive energetically unfavourable reactions

Free energy change (G)• Free energy change (G) of a reaction

determines its spontaneity

• negative G spontaneous ( products)ie: G products < G

reactants

For a reaction A + B C + D

G = Go' + RT ln[C] [D][A] [B]

R = gas constant; T = temp.

For a reaction A + B C + D

G = Go' + RT ln[C] [D][A] [B]

standard free energy change

pH 7 ([H+] = 10-7M)

reactants & products = 1Mfree energy change of reaction under ‘other’ conditions (eg in the cell)

Value depends on actual [products] and [reactants]

G

Hydrolysis of ATP • useful free-energy ‘currency’ • dephosphorylation reaction is very

spontaneousATP ADP + Pi

(Go' = -31 kJ/mol) G<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

reaction

energy-ve G

Activation energy

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

reaction

energy-ve G

Activation energy

(lowered by enzyme)

Spontaneous?

• Kinetic stability essential:

• reaction energy is then Controllable by catalysis

Can be coupled to useful reactions

reaction

energy-ve G

Activation energy

(lowered by enzyme)

Adenine

P P P

Ribose

What makes the bonds in ATP‘high-energy”?

Phosphoanhydride bonds

O O CH2

• Phosphoanhydride bonds tend to have a large negative G (-30.5 kJ.mol-1)

• NB: bond energy is not necessarily high, just the free energy of hydrolysis.

ATP

Phosphoester

bond

1. PhAnH bond has less stable resonance than its product

• Two strongly e- withdrawing groups compete for e- of the bridging oxygen

• No such competition in the hydrolysis product more stable

hydrolysis

2. PhAnH bond has greater electrostatic repulsion than its product

• At pH 7, ATP has 3 –ve charges

• Repulsion is relieved by hydrolysis

more stable

hydrolysis

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 phosphorylated compounds–Phosphocreatine

•Thioesters–CoenzymeA (you will meet this in other lectures)

Other high energy compounds

Phosphocreatine

phosphocreatine creatine

ADP ATP

When ATP P

• 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

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 G<0 are often coupled with non-spontaneous reactions (G>0) to drive them forward.

• The free-energy change (G) for coupled reactions is the sum of the free-energy changes for the individual reactions.

Gcoupled = G reaction 1 + G reaction 2

hexokinase

• Thus, ATP ADP +Pi (G<0) is coupled with non-spontaneous reactions (G>0) to drive them forward.

Glucose glucose-6-P + H20

G = 13.8 kJ.mol-1

ATP +H20 ADP +Pi

G = -30.5 kJ.mol-1

Glucose + ATP glucose-6-P + ADPOverall: spontaneous!

G = -16.3 kJ.mol-1

Energy coupling with ion gradientEnergy can also be stored as an ion gradient

• eg oxidative phosphorylation

• Spontaneous H+

movement against gradient coupled to ATP synthesis

Proton gradient

-ve G

ATP

+ve

GADP

How does energy from food get transferred to ATP for storage?

Controlled cycles of

oxidation and reduction

Electron transport chain (ETC)

OXIDATION

REDUCTION

NAD+ NADHe-

OXIDATION

glucose CO2

e-H

IQ III IV

H2OO2

e-

REDUCTION

e-

H

e-

e-e-

Cyt C

Sequential transfer of H: (2e- and H) from fuels indirectly provides free energy for production of ATP. What causes transfer of e- and H+? How does this release energy to create an ion gradient?? Remember redox potentials?

REDUCTION

B reduced

e-

OXIDATION

A reducedAoxidised

B oxidised

The tendency of a substance to undergo reduction

= E°’ (reduction potential)

E°’ = Affinity for electrons

E °' = E °‘ (acceptor) – E °‘ (donor)

gain electrons, gain Hlose O

Reduction Potential and Relationship to Free Energy

E °' = E °'(acceptor) – E °'(donor)

Go' = – nFE °'

Faraday constant

# electrons transferred

**Don’t learn these equations! Just understand the implications of +ve or –ve values

Go' = – nFE °'

• An electron transfer reaction is spontaneous (-ve G) if E°‘ is +ve

ie: when E °' of the acceptor > E °' of the donor

Electrons spontaneously flow from low high reduction potentials

REDUCTION

B reduced

e-

OXIDATION

A reducedAoxidised

B oxidised

acceptor has higher E

Spontaneous if...

Oxidised reduced

Hydride ion = 2e + H+

Accepts e- from fuel

thermodynamics of the ETChain

In ETC

• NAD accepts e- and H+ from fuel NADH• NADH donates e- and H+ to ETC

NADH oxidation is spontaneous and releases free energy

E °' = E °'(acceptor) – E °'(donor)

E °‘ = 0.8 – (-0.3) = 1.13V

NAD+ + H+ + 2e- NADH

H2O½ O2 + 2H+ + 2e-

E°’ = -0.3 V

E°’ = +0.8 V

reduced

oxid

ised

O2 has greatest affinity for e-NADH becomes the e- donor

NADH oxidation is spontaneous and releases free energy

NAD+ + H+ + 2e- NADH

H2O½ O2 + 2H+ + 2e-

reduced

oxid

ised REDUCTION

OXIDATION

E °‘= 1.13V

Go' = – nFE °‘

- ve +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 -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’

• haem a3, (Fe)

• CuB 

• CuA (=2 Cu atoms)

• haem a (Fe)

Ions in close proximity

= binuclear complex

FIRST: 2e- passed from cytC by haem a-CuA to binuclear center

Cyt C

e-

• e- are passed one at a time

Fe3+ Cu2+ Fe2+ Cu+

Fully oxidised Fully reduced

• H+ from matrix and hydroxyl from binuclear center H2O

• 2e- were passed from cytC by haem a-CuA to fully reduce Fe and Cu in the binuclear center

e- e-

Tyr

H+e-e-

O-H

OH TyrOHOHH

So far…

Fe3+ Cu2+ Fe2+ Cu+

Fully reduced

Then, O2 binds

e- e-

Tyr

H+e-e-

O-H

OH TyrOHOHH

Fe2+ Cu+e- e-

TyrOH

OO

OO

This O2 is going to become O22-

It’s going to need 4 e-

e-

Fe3+ Cu2+ Fe2+ Cu+

Fully oxidised

e- e-

Tyr

H+e-e-

O-H

OH TyrOHOHH

Fe2+ Cu+e- e-

TyrOH

OO

OO

Fe4+ Cu2+e-

e-

TyrO

O2- O-e-

The tricky bit!!

• 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

H

Fe2+ - 2e- Fe4+ Cu + - 1e- Cu2+

Tyr-OH - 1e- -H+ Tyr-O.

O22- shared between Cu and Fe

e-

Fe3+ Cu2+ Fe2+ Cu+

Fully oxidised

e- e-

Tyr

H+e-e-

O-H

OH TyrOHOHH

Fe2+ Cu+e- e-

TyrOH

OO

OO

Fe4+ Cu2+e-

e-

TyrO

O2- O-e- H

e-H

H

e- Fe4+ Cu2+e-

Tyr

O2-e-

OHH

OH

1 more e- passed in via haem3-CuA to binuclear complex Reconverts tyrosine

And more H+ H2O

e-

Fe3+ Cu2+ Fe2+ Cu+

Fully oxidised

e- e-

Tyr

H+e-e-

O-H

OH TyrOHOHH

Fe2+ Cu+e- e-

TyrOH

OO

OO

Fe4+ Cu2+e-

e-

TyrO

O2- O-e-

4th e- passed via h3CuA Regenerates Fe3+: Completed cycle!

HAnd one more H+

e-

H H

e- Fe4+ Cu2+e-

Tyr

O2-e-

OHH

OH

e-

H

OO

H+

H+H+

H+

H+

H+

H+

H+

Meanwhile pumps 4 H+ were pumped

to proton gradient

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 G)• Free energy of ATP coupled to non-

spontaneous reactions• Phospho-anhydride bond is ‘high energy’• Electrons spontaneously flow from low to

high EFood 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- Go= -158.2 kJ

spontaneous

• reduction  ½ O2 + 2H+ + 2e- H2O Go= -61.9 kJ

spontaneous

• phosphorylation  ADP   ATP Go= +30.5 kJ

nonspontaneous

• The net reaction is obtained by summing the coupled reactions,

ADP + NADH + ½ O2 + 2H+        ATP + NAD+ + 2 H2OGo= -189.6 kJspontaneous

Coupled non-spontaneous work

Do NOT learn these values! Just know which are +ve or –ve/ spontaneous or not…understand concept of coupling!!