ATP Synthase

What drives ATP synthesis?

Proton transport through the ATP synthase

ATP Synthase aka

F1F0-ATPase

ATP synthase structure

consists of two parts F1 and F0

Five polypeptides of F1

alpha, beta, gamma, delta, epsilon

Alpha-Beta arrangement

Pseudo-threefold symmetry

F1 is

catalytic subunit, only beta does catalysis though

Conformations of F1

O state, L State, T state

Gamma rod of F1

goes through center of alpha beta complex

F0 includes

three hydrophobic subunits denoted a, b, and c

F0 forms

the transmembrane pore or channel through which protons move to drive ATP synthesis

A and B subunits

compromise part of the stator

C-subunits

form a ring that is part of the rotor

What drives the F1 conformation changes?

the rotations of F0 that are transduced by gamma

Loose Conformation

bind ADP, medium affinity for ATP/ADP

Tight Conformation

forms ATP, high affinity for ATP/ADP

Open conformation

releases ATP, low affinity for ATP/ ADP

How are protons carried across the membrane?

Through combined action of a subunit and c subunit

Where do H+ enter?

A subunit

What allows the rotor to turn?

H+ neutralize a negative charged Asp in the middle of TM helix of c subunit- allows rotor to turn

The entry point and exit point of H+ on the subunit

Are dislocated from each other

Ions and metabolites enter mitochondria via _________

transporters

Malate Aspartate shuttles

Oxaloacetate is reduced by NADH to malate, which then passes through the membrane and is oxidized by NAD+ (which reforms NADH)

glycerophosphate shuttle

DHAP from glycolysis is reduced to 3-phosphoglycerol by NADH and 3-phosphoglycerol dehydrogenase where electrons are then transferred to FADH2

The malate-aspartate shuttle

Is reversible

ADP-ATP translocator

moves ATP from matrix to intermembrane space and ADP into the matrix

Conformations of ATP-ADP translocase

binding site facing the matrix (When ATP binds) or binding site facing the inter membrane space (where ADP binds)

What helps characterize the transporter?

poisoning the transporter by natural products atractyloside and bongkrekic acid

Atracyloside

blocks outer surface of inner membrane

Bongkrekic acid

blocks inner surface of inner membrane

Cost of transporting 1 ATP out

-1 per ATP

ATP movement out

one -1 change is moved to the cytosol and one H+ is moved into the matrix

Every ATP transported out

costs one H+

One ATP synthesis costs about

3 H+

Making and exported an ATP costs

4 H+

How many H+ are yielded form electron transport chain

10 H+ per electron pair from NADH

P/O ratio

number of ATP produced per oxygen reduced

For electrons entering as succinate (FADH2)

about 6 H+ pumped per electron pair to oxygen

The amount of ATP produced depends on

the shuttle used to move NADH equivalents into the matrix

malate-aspartate shuttle

use malate to carry electrons across the membrane

glycerophosphate shuttle stores

electrons in glycerol-3-P which transfers electrons to FAD

NADH produced in TCA

3 per cycle or a total of 6

FADH2 produced in TCA

1 per cycle or a total of 2

NADH produced in glycolysis

2 NADH

NADH produced in bridging reaction

2 NADH

ATP produced in glycolysis

2 ATP

ATP produced in TCA

2 ATP

How many ATP per glucose are produced if glycerol-3-P shuttle?

30 ATP

How many ATP per glucose are produced if malate-aspartate is used to shuttle ATP?

32 ATP

Uncouplers

disrupt the coupling between electron transport and oxidative phosphorylation by passively dissipating the proton gradient

Uncouplers have

the net effect of generating heat

Uncouplers are often

hydrophobic molecules with a dissociable proton

Example of an uncoupler

2,4-dinitrophenol (DNP)

2,4-dinitrophenol

shuttle back and forth across the membrane carrying protons

used as a diet drug in the 30's

caused hyperthermia

UCP1

membrane channel in mitochondria that is a natural uncoupler

UCP1 opens or closes its channel

in response to cAMP signaling

Most people use

> 200 mol of ATP per day

total ATP present at one time

< 0.1 mol

ATP consumption change between rest and vigorous activity

~100 fold change

Complex I-III operate

near equilibrium

Complex IV is

the irreversible regulated step

Complex IV regulated by

-Cyt C (2+)

What determines Cyt C (2+) concentration

concentrations of NADH/ NAD+ and ADP, Pi/ATP

ATP Synthase is regulated by

protein called IF1

When matrix pH is high

IF1 forms tetramer

When pH drops (such as when O2 availability is low)

tetramer dissociates and binds to La state of alpha/ beta dimer which prevents the conformational change

Mitochondria play a significant role

in apoptosis

Apoptosis

the programmed death of cell

How does apoptosis occur

mitochondria do this in part by partitioning some of the apoptotic active molecules (cytochrome C)

What releases cytochrome c from the inner membrane

oxidation of bound cardiolipins

Releases cytochrome c from the mitochondria

opening of pores in the outer membrane

Apaf-1

apoptotic protease activating factor, which is a multidomain protein

Where do Cyt C bind

Apaf-1 in the cytosol

Apoptosomes

assembled from several Apaf-1 and looks like an earth-orbiting space station

Where does Cyt C anchor

At the inner mitochondrial membrane by association with cardiolipin

Cardiolipin is required for:

oxidative phosphorylation, stabilization of respiratory supercomplexes, association of respiratory supercomplexes with ATP-ADP translocase, biogensis of proteins destined for the mitochondrial matrix and/ or the inner membrane, fission and fusion of mitochondria

Cardiolipin is most commonly associated with

inner mitochondrial membrane

Parts of Apaf-1

N-terminal caspase-recruitment domain (CARD), a nucleotide-bind and oligomerization domain (NOD) and several WD40 domains

Cyt C

is an apoptosis activator molecule

Cardiolipin

lipid in the intermitchodrial membrane

Cyt C oxidizes

Cardiolipin and in then released into the cytosol

As Cyt C binds to Apaf1

ATP hydrolysis takes place

ATP hydrolysis causes

a change in Apaf1 structure to the semi open conformation

Nucleotide change occurs which leads to

the formation apoptosome