Tema 3. Membrane BioenergeticsBioenergetics

The proton potentialCap. 3 pages 83-119

Chemiosmotic theoryStates that energy-transducing membranes pump protonsacross the membrane, thereby generating an electro-chemicalgradient of protons across the membrane that can be used to do useful work when the protons return across the membraneto the lower potential.

Membranes are energized by proton currents

The return of the protons should by through special proton conductors that coupleThis movement to useful cellular work.

These include:Solute transporters, ATPase, flagellar motility.

1

2

H+

H+

H+

Redox reactionsATP drive proton pump Extrusion of Na ions,

solute transport, flagellarotation, and synthesisof ATP.

Electrochemical energy

Work

Energy

-

+ +∆µ = µin - µout

For the proton it would be ∆µH+

∆µH+= F∆Ψ + RT ln[H+]in / [H+]out Joules

Faraday constant

Membrane potential

Chemical energy

Faraday constant~96,500 C

∆p = ∆µH+/F = ∆Ψ – 60∆pH mV

In millivolts

Proton motive force which is the potential energy in the electrochemical proton gradient

X ∆p of a bacteria is -140 to -200 mV

Contributions of the ∆Ψ and ∆pH to ∆p

Neutrophilic bacteria

∆Ψ contributes 70-80%

∆pH contributes 30-20%

Acidophilic bacteria (pH =1 - 4)

∆Ψ is positive and contributes 0%

∆pH contributes 100%

Thiobacillus ferroxidanspH in = 6.5pH out = 2∆p = ∆Ψ – 60∆pH mV

∆p = 10 – 60(4.5)= -260 mV

Alkilophilic bacteria is the opposite

K+

Valinomycin could create ordisrupt ∆Ψ

Ionophores

R-

H+

gramicidin

Collapse both∆Ψ and ∆pH

IN

OUT

FCCP collapse both

H+

K+

Nigericincould collapse ∆pH

But not the ∆Ψ

H+

Na+

monensin

They perturb ion gradients

H+

R-

R-

dinitrophenol

Collapse both∆Ψ and ∆pH

The ATP synthase

FoF1

3H+

ADP + Pi

IN OUT

Polypeptides a1, b2 and c10

ATP + H2O

Polypeptides α3, β3, γ1, δ1, and ε1

The amount of energy to synthesize an ATP∆Gp = 518 mV or the ∆p must be -173 mV

The ATP synthase

Polypeptides α3, β3, γ1, δ1, and ε1

T

L O

ATP

Polypeptides

a1, b2 and c10

L O

ADP + Pi

3 conformational changes

A= 3 mM,pH=5

B= 0.4mM, pH=6

C = 3 mM, pH=6Intr

acel

lula

r AT

P (

mM

)

A

B

C

Pro

ton

entr

y (m

M)

Streptococcus lactis

How the cells create ∆p In the presence of Valinomycin

C = 3 mM, pH=6

Minutes

Intr

acel

lula

r AT

P (

mM

)

Minutes

Pro

ton

entr

y (m

M)

A B C

∆pH= 75 mV 15 mV 15 mV∆Ψ = 125mV 185 mV 125 mV∆p = 200mV 200 mV 140 mV

Proton influx and ATP synthesis depend upon the ∆p rather than on the individualvalues of the ∆Ψ or the ∆pH.

How the cells create ∆p

Exergonic reactions∆G = yF∆p where ∆p = ∆G /yF

We first must understand the redox potentialwhich is the tendency of a molecule to accept an electron from another

molecule. The symbol is E.

Eh = E0 + [RT/nF] ln [ox]/[red]

Std cond1M, 1atm e transferred

Faraday cGas cActual electrodepotential

STD potentials at pH =7Are denoted E’0

∆G = -nF∆Eh

Work done per n moles of electrons

Free energy∆Eh = Eh acceptor- Eh donor

∆G = -nF∆Eh = yF∆p

y = 4 protonsn = 2 electrons∆Eh = 0.2 V

Substituting in the Eq.

∆G/F = -n∆Eh = y∆p

∆p = -2(0.2) / 4∆p = -2(0.2) / 4∆p = - 0.1V = -100 mV

When 2e travel down a ∆Eh of 200 mVAnd 4 protons are translocated -100 mV Is stored in the ∆p.

Respiration coupled to sodium ion efflux

Vibrio alginolyticus is halophilic marine bacterium that uses a ∆µNa+ forsolute transport, flagella rotation, and ATP synthesis.

At pH= 6.5 the organism generates a ∆µH+ which drives a H+ - Na+

antiporter that creates a ∆µNa+.

At pH= 8.5 ∆µNa+ is created directly by a Na+ dependant At pH= 8.5 ∆µNa+ is created directly by a Na dependant NADH-quinone reductase.

Desulfovibrio salexigensBacillus sp. (alkilophilic)

Use ∆µNa+ for most solute transport and flagellarotation but the ATP synthase is H+ dependant

Creating a ∆p in fermenting bacteria

A major energy source for the creation of the ∆p is:ATP hydrolysis

How much potential could we generate per ATP hydrolyzed?

∆G = yF∆p V∆Gp = yF∆p V

y = 3

∆p = - 173 mV

Other mechanisms for creating a ∆p or a ∆Ψ

Organisms:Vibrio alginolyticus that creates a ∆µNa+ as explain earlier.

Strategies include the decarboxylation of organic acids coupledto sodium ion efflux.

Veillonella alcalescens and Propionigenium modestumVeillonella alcalescens and Propionigenium modestumUse a methylmalonyl-CoA decarboxylase

Acidaminococcus fermentansUse a glutaconyl-CoA decarboxylase

Klebsiella pneumoniae and Salmonella typhimuriumUse a oxaloacetate decarboxylase

Propionigenium modestumAnaerobe isolated form marine and fresh water mud, and from human saliva.It grows only on succinate, fumarate, aspartate, malate, oxaloacetate, andpyruvate.

InOut

Organic acid Organic acid

methylmalonyl-CoA

2Na+Acetate

ATP

methylmalonyl-CoA

CO2

Propionyl-CoApropionate

2Na+

Symport

Acidaminococcus fermentans

InOut

glutamate glutamate

glutaconyl-CoA

COyNa+

yNa+

CO2

crotonyl-CoA

butyrateacetate

yNa+

Symport

Acetatebutyrate

K. pneumoniae

In

Out

citrate

citrate

Na+

Na+

Na+

Na+ NAD NADH CO2

UQUQH2

Na+

citrateNa+

Na+

NAD NADH

acetate

OAAPyruvate

CO2

formate

Acetyl-CoAAcetyl-PATP

CoASH

CO2

Oxalobacter formigenesAnaerobic bacterium that is part of the normal flora in mammalianintestines.

In

OutOxalic acid

formate

2 negative charges come inside and 1 goes outIn addition one proton is consumed inside

Creating a ∆Ψ3 mole of oxalic acid to 1 mole of ATP formed

In

Oxalic acid FormateCO2

H+