M.Prasad NaiduMSc Medical Biochemistry,

Ph.D,.

Theories of oxidative phosphorylation

1.Chemiosmotic theory

2.Boyer’s binding change mechanism

The Chemiosmotic Theory of oxidative phosphorylation, for which Peter Mitchell received the Nobel prize:

Coupling of ATP synthesis to respiration is indirect, via a H+ electrochemical gradient.

Matrix

H+ + NADH NAD+

+ 2H+ 2H+ + ½ O2 H2O

2 e – –

I Q III IV

+ +

4H+ 4H+ 2H+ Intermembrane Space

cyt c 3H+

F1

Fo

ADP + Pi ATP

Chemiosmotic theory proposed by Peter Mitchell

The transport of protons from matrix to intermembrane space is accompanied by

the generation of a proton gradient across the

membrane.

Protons (H+) accumulate intermembrane space creating an electrochemical potential difference, proton gradient or electrochemical gradient.

This proton motive force (PMF) drives the synthesis of ATP by ATP synthase complex.

H+

H+

4H+

H+

H+4H+

2H+

H+

4H+

2e- 2e-

IMS

ATP

ADP+Pi

4H+

4H+

2H+

4H+

IMM

OMM

H+

H+

H+H+

H+ H+

H+ H+

H+H+

H+

H+

H+

H+

H+

H+

H+

H+H+

H+H+

H+

H+

MATRIX

H+

H+

H+

H+

H+

H+

H+ H+H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+ H+

IIII

Iv

V

H+H+

H+

H+

IMM- Inner mitochondrial membrane IMS- Inter membrane space

OMM- outer mitochondrial membrane

Complex I, III and IV are proton pumps

CHEMIOSMOTIC THEORY Peter mitchel

Proton gradient / electrochemical gradient

Proton motive force

Proton dependant ATP synthese Uses proton gradient to make ATP Protons pumped through channel on enzyme

From intermembrane space into matrix ~4 H+ / ATP

Called chemiosmotic theory

NADH10 H+ X 1 ATP = 2.5 (3) ATP

4 H+

FADH2

6 H+ X 1 ATP = 1.5 (2) ATP 4 H+

Boyer ’s binding change mechanism:

ATP synthase is a protein assembly in the inner

mitochondrial membrane.

ATP synthase has two units

F1 - projects into matrix

-has 3 α , 3 β , gamma , delta, epsilon chains

-catalyses ATP synthesis

Peripheral catalytic sites are present on beta subunits.

Fo - embedded in membrane

- acts as channel for transport of H+

ADP + Pi ATP

F1

Fo

3 H+ matrix

intermembrane space

4

H+ H+ H+ H+H+ H+ H+ H+

Mechanism of ATP synthesis (Boyer’s Hypothesis)

Boyer’s binding change hypothesis

Synthesis of ATP occurs on the surface of F1.

Binding change mechanism states that 3 beta

subunits change CONFORMATIONS during catalysis with only one beta subunit acting as Catalytic site.

β subunits occur in 3 forms ‘O’ form (Open form). It has low affinity

for substrates ADP +Pi

‘L’ form (loose form). Can bind substrates ADP and Pi but catalytically it is inactive.

‘T’ form (Tight form). Binds substrates ADP + Pi tightly and catalyses ATP synthesis.

When protons pass through the disk of C subunits of F0 unit it causes rotation of γ sub unit.

The β subunits which are fixed to the membrane donot rotate.

ADP & Pi are taken up sequentially by the βsubunits which undergo conformational changes and form ATP.

Gamma subunit is in the form of axle . It rotates when protons flow.

ATP synthase is smallest known MOLECULAR MOTOR in the living cells.

ETC - inhibitors

Complex I : site I of ATP synthesis inhibitors

Rotenone, Peircidin, Amytal, Barbiturates

ComplexII:

Carboxin,Thenoyltrifluroacetone,malonate

Complex III : site II of ATP synthesis inhibitors

Antimycin, Myxothiazol , stigmatellin

Complex IV: site III of ATP synthesis inhibitors

Cyanide, azide , carbon monoxide

Complex – I inhibitors (Site I inhibitors)

Rotenone, insecticide, also used as fish poison. Binds to complex I and prevents the reduction of Ubiquinone.

Piercidin, Amytal (sedative), Barbiturates – inhibit by preventing the transfer of electrons from iron sulfur center of complex – I to Ubiquinone.

Complex – II inhibitors

Malonate acts as a competitive inhibitor with the

substrate succinate

Complex – III inhibitors (Site II Inhibitors)

Antimycin inhibit electron transfer from cytb to C1.

Myxothiazol and stigmatellin, antibiotics inhibit electron transfer from Cytb to C1.

Complex – IV (site III inhibitors)Cyanide and azide bind tightly to oxidized form

of heme a3 ( of complex iv ) preventing electron flow.

Cyanide is potent and rapidly acting poison.

Cyanide prevents binding of oxygen to

Cytochrome oxidase ( aa3 ).

Mitochondrial respiration and energy production stops cell death occurs rapidly.

Carbonmonoxide binds to the reduced form of

heme a3(Fe2+) competitively with oxygen and prevents

electron transfer to oxygen.

Uncouplers of oxidative phosphorylation :

Uncouplers will allow oxidation to proceed but

energy instead of being trapped as ATP is

dissipated as heat.

They are hydrophobic weak acids.

They are protonated in the intermembrane

space where a higher concentration of protons

exists.

These protonated uncouplers due to their

lipophilic nature rapidly diffuse across the

membrane into matrix where they are

deprotonated since matrix has a lower

concentration of protons.

Thus, the proton gradient is dissipated.

2-4 dinitrophenol a classical uncoupler – electrons

from NADH to oxygen proceeds normally but ATP not

formed as proton motive force across inner

mitochondrial membrane is dissipated .

2. Penta chloro phenol3. Dinitro cresol4.Bilirubin5.Thyroxine-Physiological uncoupler6.Valinomycin7.Nigericin

Note: They are Lipophilic

Intermembrane spacematrix

H+ H+ H+ H+

H+

H+

H+

H+

H+ H+

Physiological Uncouplers 1.Excessive thyroid hormones 2. Unconjugated hyper bilirubinaemia 3. In high doses aspirin uncouple oxidative phospharylation which explains fever that accompanies toxic over dosage of these drugs.

Uncoupling proteins

UCPs occur in the inner mitochondrial

membrane of mammals, including humans.

UCPs create a “proton leak”, that is they allow

protons to re-enter the mitochondrial matrix

without energy being captured as ATP.

Energy is released as heat, and the process is

called nonshivering thermogenesis.

UCP1, also called thermogenin, is responsible for the activation of fatty acid oxidation and heat production in the brown adipocytes of mammals.

Brown fat , unlike the more abundant white fat, uses almost 90% of its respiratory energy for thermogenesis in response to cold, at birth,etc.

Inhibitors of Oxidative phosphorylation :

Oligomycin – acts through one of the proteins present in F0 - F1 stalk .

Oligomycin blocks the synthesis of ATP by preventing the movement of protons through ATP synthase.

The regulation of the rate of oxidative phosphorylation by ADP level is called respiratory control.

The ADP level increases when ATP is consumed and so oxidation is coupled to the utilization of ATP.

Under physiological conditions, electron transport is tightly coupled to oxidative phosphorylation.

Electrons do not usually flow through the electron transport chain to O2 unless ADP is simultaneously phosphorylated to ATP.

In the presence of excess substrate and Oxygen, respiration continues until all ADP is converted to ATP.

After that the respiration rate or utilization of oxygen decreases

In the presence of adequate oxygen and substrate, ADP becomes rate limiting; it exerts a control over the entire oxidative phosphorylation process

The rate of respiration of mitochondria (Oxidative phosphorylation) can be controlled

by ADP. Oxidation cannot proceed via ETC without

simultaneous phosphorylation of ADP. Chance & Williams defined 5 conditions that

can control rate of respiration.

Generally most cells in the resting state are in state 4 , and respiration is controlled by the availability of ADP.

The availability of inorganic phosphate could also influence the respiration.

As respiration increases (Exercise) cell approaches state 3 ( ETC working to its full capacity ) or state 5 ( Availability of O2 is a limiting factor ).

ADP / ATP transporter may also be a rate limiting factor

P:O ratio (ADP : O ratio)

P:O ratio is defined as number of phosphates incorporated into ATP to 1 atom of oxygen utilized during the transfer of 2 electrons through ETC.

For NADH P:O ratio is 3 i.e 3 ATPs are produced (2.5)

For FADH2 P:O ratio is 2 i.e 2 ATPs are produced(1.5)