How ATP is Synthesized at “Energy-Coupling” Membranes:Chemiosmotic Coupling

Module 0210101:

Molecular Biology and Biochemistry of the Cell

Lecture 13

Dale Sanders

23 February 2009

Objectives

1. the notion that NADH oxidation can be coupled to ATPsynthesis via a transmembrane electrochemical potentialfor protons (or protonmotive force, PMF);

2. how the energy stored in transmembrane solute potentialdifferences and ion potential differences can be quantifiedin terms of kJ/mol or mV;

3. how uncouplers increase the rate of electron transport;

4. that the PMF is used in a wide array of biological systemsto transduce free energy from one form to another.

By the end of the lecture you should understand…

Reading

Voet & Voet (2004) Biochemistry (3rd Ed.) Chapter22 and especially pp. 827-835

As in previous lectures, all the topics today are wellcovered by the standard big biochemistry textbooks. Anexample is

Also useful for a more in-depth treatment is

Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3pp. 46 & 47 and Chapter 4

How is ATP Synthesis Coupled to theRedox Reactions?

NADH

NAD+

½O2 + 2H+

H2OADP+ Pi

ATP

Phosphorylation is energized indirectly,

via generation of a H+ potential across

the inner mitochondrial membrane:

Redox

Phosphorylation

The chemiosmotic hypothesis (Peter Mitchell, 1960s):

1. The coupling complexes in the redox chain are H+ pumps.

2. Low membrane permeability to H+ allows build up of transmembrane H+

potential.

3. Passive return of H+ across membrane energises ATP synthesis.

Cytosol Mito matrix

Note: Redox enzymes+ ATP synthase arediscrete elements inmembrane

UQ

Cyt c

Innermembrane

A hydraulic analogy of chemiosmotic coupling

EnergyinputMainselectricitye- transport

PumpH2O pumpH+ pump

Energy outputLightATP

Couplingenergy:Potentialenergydifferencefor H2O H+

High potential H2O H+

Low potentialH2OH+

Energy transducerWater wheel/dynamoATP synthase

http://www.antonine-education.co.uk/physics_gcse/Unit_1/Topic_4/hydroelectric_dam.jpg

Economic Exploitation of Hydraulic EnergyCoupling

biologicinstitute.org/2008/04/03/perspectives/

The F-Type ATP Synthase

Twosectors

ADPbindingsite

Rotation

Quantifying the Free Energy inTransmembrane Solute Potentials

(i) Uncharged solute:

[S]o [S]i

membrane

Chemical potential of S = μs = μos + RT ln [S] (1)

Chemical potential difference, inside with respect tooutside is

ΔμS = (μS)i – (μS)o = RT ln [S]i (2)

[S]o

ΔμS = (μS)i – (μS)o = RT ln [S]i (2)

[S]o

= RT x 2.303 log10 [S]i[S]o

e.g. if [S]i = 10 mM, [S]o = 1 mM

ΔμS = 8.314 x 298 x 2.303 x1 = +5.7 kJ/mol

Note: 1. Units are those of Gibbs free energy.

2. Polarity: + ve value says that reaction

So Si

is “uphill”

conversely, Si So is “downhill”

Will release 5.7 kJ/mol

Quantifying the Free Energy inTransmembrane Solute Potentials

(ii) Charged solute (ion):

[Sz]o [Sz]i

membrane

Electrochemical potential of Sz = μs = μos +RT ln[Sz] + zF

(3)

Electrochemical potential difference, inside with respect tooutside is

ΔμS = (μS)i – (μS)o = RT ln [Sz]i + zF (4)

[Sz]o

z = valence

= electrical potential

i -o = io

ΔμS = (μS)i – (μS)o = RT ln [Sz]i + zF (4)

[Sz]oUnits of Eq. 4: J/mol.

To convert to volts (recall ΔG Em): ÷ F. Thus

ΔμS = RT (2.303) log10 [Sz]i + z (5)

F [Sz]oFor the special case of the proton (H+), z = +1 ;

log10 [H+]i = -pHi ; log10 1/[H+]o = pHo ;

RT (2.303) = (8.314)(298)(2.303) = 0.059 V = 59 mV

Thus can rewrite Eq 5 as

ΔμH = 59 (pH0 – pHi) + (6)

F

96500F

F

Electrochemical Potential Difference of the Proton

ΔμH/F is Known as The Protonmotive Force(PMF)

From Eq 6 we can write

PMF = 59 (pHo – pHi) + (7)

Units: mV

A measure of the free energy in the electrochemicalpotential difference for protons across a membrane;

An important biological parameter because so much ofenergy transduction in biology is through the PMF

pHcytosol = pH0 = 7.5 ; pHmatrix = pHi = 8.0

= -170 mV (inside negative)

From Eq 7,

PMF = 59 (7.5 – 8.0) + (-170)

= -30 – 170 = -200 mV

THE PMF IN COUPLED MITOCHONDRIA

Proton flows, stoichiometries and energetics

Note:1. Total of 10 H+ pumped out per

2e– flowing through redoxchain

2. 4 mol H+ used directly foreach mol ATPsynthesised

Requirement for > 1 mol H+/mol ATP

Energy available for ATP synthesis is

ΔμH = (PMF)(F) = (-0.2)(96500) = -19.3 kJ/mol

But ΔG’ATP = -48.8 kJ/mol (Lecture 6)

i.e. for ATP synthesis, +48.8 kJ/mol is required

Reaction is

nH+o + ADP + Pi ↔ nH+

i + ATP

Energetically, this reduces to

nF(PMF) + ΔG’ATP < 0 for ATP generation.

By coupling ATP synthesis to translocation of 4 H+/ATP,

(-19.3)(4) = -77.2 kJ is available for each mol of ATP

This is more than enough energy!!

-

H+ Flows and Impact on Redox-Phosphorylation Coupling

1. Why do redox reactions speed up when ADP isadded? [See slide 20, Lecture 12]

Answer:

ADP facilitates flow of H+ through ATP synthase.

This decreases the PMF slightly.

Decrease in opposing driving force enables fasteroperation of redox coupled H+ pumps.

2. The action of uncouplers

[A brilliant prediction of the chemiosmotic hypothesis]

Some compounds (eg 2, 4 dinitrophenol)

(i) abolish ATP synthesis “uncouple”

(ii) speed up respiration

Chemiosmotic explanation:

Uncouplers catalyse passive H+ flow:

They are “protonophores”: abolish PMF

Thus (i) no driving force for ATP synthesis

(ii) no force opposing redox reactions

H+ Flows and Impact on Redox-Phosphorylation Coupling

How Uncouplers Work

UncouplerUncoupler

ATP synthase

Respiratorycomplexes

Membrane-bound ATP Synthase is Ubiquitous atEnergy-Coupling Membranes

No resp. pumps:

ATPase inhydrolytic mode

Evolved as H+ pump??

cc

compared with mitosC.

ATP Synthase and the PMF:

Recurring Themes in Bioenergetics

From previous slide…

Although energy sources and mechanisms of H+

pumps are variable,

presence and mechanism of ATP synthase isconserved through evolution.

Furthermore (next slide)…

Bacteria have evolved alternative physiological usesfor the PMF…

Solute uptake

PMF

ChampionSprinter

Summary

1. NADH oxidation is coupled to ATP synthesis viageneration of a PMF.

2. The energy stored in ion potential differencescan be expressed in terms of kJ/mol or mV andcomprises electrical and chemical components.

3. For H+, PMF = 59(pH0 - pHi) + Δψ (in mV).

4. Uncouplers (and ADP to some extent) tend toincrease the rate of e- transport by dissipating thePMF.

5. The PMF is also used to generate ATP atbacterial cell membranes and thylakoids….and…..

6. …powers bacterial solute uptake andswimming.