Lec 21. Proton motive force

PMF (Proton-motive force)

ATP synthesis is powered by the movement of

protons across the membrane

PMF is what type of gradient

Electrochemical gradient = pH and charge gradient

Which is the greater contributor to the protonmotive

force across the inner mitochondrial membrane?

The charge gradient generated by protons

The charge gradient contributes what percent of free energy in PMF

85%

The chemiosmotic hypothesis (PMF) was confirmed by

Racker & Stoeckenius who created vesicles containing the

ATP synthase and bacteriorhodopsin…

Bacteriorhodopsin

proton pump, activated by light

Racker & Stoeckenius-ATP synthase and bacteriorhodopsin

light‑activated proton pumping alone could drive ATP synthesis in artificial vesicles, the experiment proved that ATP synthase requires only a proton gradient

PMF comes from the

electron transport chain

ATP synthase is composed of

a proton-conducting unit (Fo) and a catalytic unit (F1)

Each ATP synthase has

three active sites located on the three β subunits.

β subunits of ATP synthase exist in

3 possible conformations

The rotation of the γ subunit of ATP synthase

interconverts the three β subunit conformations, allowing ATP synthesis.

Binding change mechanism of ATP synthase

conformational (“mechanical”) change that “forces” ATP formation

If F₁ is separated from F₀, what reaction does it perform?

It hydrolyzes ATP (acts as an ATPase).

What does the proton do once it enters the F0 channel

bind to a glutamate or aspartate residue on one of

the subunits of the c ring

rotation of the c ring is powered by

The force of the proton gradient

The rotation of the c ring powers the

movement of the γ subunit

the movement of the γ subunit

alters the conformation of the β subunits

The c ring subunits can only rotate into the interior of the membrane if the

negative charge on the glutamate (or aspartate) residue is neutralized by binding a proton
NOT ROTATION UNTIL PROTON IS BOUND

One complete rotation of C ring requires

8 protons, and makes 3 ATP molecules

Glycerol 3-phosphate Shuttle

transfers the electrons from NADH to FAD to QH2

Glycerol 3-phosphate Shuttle is especially important for

insect flight muscles

Malate-Aspartate Shuttle (most common)

transfers the electrons from NADH…to

another NADH…on the matrix side

Difference between malate and oxaloacetate

Malate crosses the mitochondrial membrane,

while oxaloacetate cannot

ATP-ADP translocase

exchanges an ATP

(out) for an ADP (in)

What does it mean that ~25% of oxidative phosphorylation energy is used for transport?

one‑quarter of the proton‑motive force is consumed to move ATP, ADP, phosphate, pyruvate, and other metabolites across the inner mitochondrial membrane rather than to make ATP

Of the ~ 30 -32 molecules of ATP formed by the complete

combustion of glucose, how many are from oxidative phosphorylation

26 -28 ATP

Of the ~ 30 -32 molecules of ATP formed by the complete

combustion of glucose, how many are from substrate level phosphorylation

4 ATP

Normally, electrons do not flow through the

electron-transport chain unless

ADP is available to be converted into ATP

ATP synthase stops turning when

ADP is depleted

Uncouplers

allow electron transport without any ATP production

Example of uncoupler

DNP and UCP

DNP

drug that artificially uncouples oxidative phosphorylation

Why does 2,4-dinitrophenol uncouple

oxygen consumption from the need

for ADP substrate (i.e. ATP formation)?

it collapses the proton gradient, removing the back‑pressure that normally stops the ETC when ATP synthase has no ADP to work with. With no gradient to oppose it, the ETC runs at full speed and consumes oxygen even though ATP cannot be made

Why does oxygen consumption fall when ADP is depleted?

ATP synthase stops, the proton gradient becomes too steep, and the ETC cannot pump more protons, so electron flow stalls.

Why does DNP allow oxygen consumption to continue even without ADP?

dissipates the proton gradient, so the ETC never encounters back‑pressure and keeps running at maximum speed

Most mammals (but not pigs)

can uncouple electron

transport from ATP synthesis

in order to

regulate body

temperature

UCP1

natural proton channel

that can uncouple

Electron transport inhibitors

poisons that stop ATP synthesis

Examples of Electron transport inhibitors

Rotenone
MPTP
Cyanide
Azide
CO

Cyanide, azide, and CO

all inhibit Complex IV by binding to the heme iron in cytochrome c oxidase, preventing oxygen from accepting electrons and completely halting the ETC.
NO PEROXIDE BRIDGE

Rotenone

a piscicide that blocks Complex I

MPTP

neurotoxin whose mode of action

has been attributed to its ability to block electron transport by inhibiting Complex 1

MPTP role in parkinson’s

Preferentially kills

dopaminergic

neurons

in the substantia

nigra

Conditions potentially cause by free radical injury

Parkinsons
Alcoholic liver disease
Diabetes