chemiosmotic theory
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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)
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