Oxidative phosphorylation

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Oxidative Phosphorylation
R. C. Gupta Professor and Head Dept. of
Biochemistry National Institute of Medical
Sciences Jaipur, India
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Energy
E M B - R C G
EMB-RCG
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• The ultimate source of energy is the food that we
  consume
• Carbohydrates, lipids and proteins present in
  food provide us energy
• These are present in food in the form of
  large complex molecules

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E M B - R C G
EMB-RCG
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High-energy phosphates
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There must be some way of storing energy so that
it may be readily available when needed
The energy released during catabolism is captured
in the form of high-energy phosphates
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Adenosine triphosphate
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Standard free energy (DGo) of hydrolysis of some
important organic phosphates (kcal/mol)
These are the values of DGo obtained in standard
laboratory conditions of 1M reactant
concentration at pH 7.0 at 25C values obtained
in living cells (DGo) are different as the
reactant concentrations, pH and temperature
are different
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EMB-RCG
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EMB-RCG
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Oxidation and reduction
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EMB-RCG
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Redox potential
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Measurement of redox potential
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Enzymes involved in biological oxidation
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Oxidases
Oxidases transfer hydrogen from a substrate to
oxygen forming water or hydrogen peroxide
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The enzymes forming water are metallo-enzymes
that usually contain copper e.g. cytochrome
oxidase and tyrosinase
The general reaction catalysed by oxidases is
AH2 ½ O2 ? A H2O
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Dehydrogenases
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Prosthetic group Examples
NAD Lactate dehydrogenase, malate dehydrogenase etc
NADP Glucose-6-phosphate dehydro-genase, 6-phosphogluconate dehydrogenase etc
Flavin nucleotides Succinate dehydrogenase, acyl CoA dehydrogenase etc
Iron- porphyrin Cytochrome a, cytochrome b etc
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Hydroperoxidases
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Mono-oxygenases
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EMB-RCG
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EMB-RCG
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Mitochondral hydroxylase system
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Adrenodoxin possesses an iron-sulphur centre
having catalytic activity
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Iron-sulphur centres
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NADPH? Adrenodoxin reductase
Cytochrome P?450 (mono-oxygenase)
FeS
FAD
Fe3
NADP
AH O2
A-OH H2O
NADPH H
Iron-sulphur protein
Mitochondral hydroxylase system
EMB-RCG
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EMB-RCG
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EMB-RCG
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EMB-RCG
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a-Ketoglutarate
dehydrogenase
CH2 COOH
CH2 COOH


NADHH
NAD
CH2 C COOH
CH2 C S CoA
CO2
CoA?SH
O
O
a-Ketoglutarate
Succinyl CoA
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Oxidative phosphorylation at the level of
respiratory chain
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Sequence of carriers in the respiratory chain
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Transport of reducing equivalents in
respiratory chain
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Reduced coenzyme Q transfers the reducing
equivalents to cytochrome b
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Cytochrome b is associated with iron-sulphur
protein

The subsequent carriers,
cytochromes c1, c and a, are typical
iron-porphyrin proteins
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Prosthetic group of cytochrome b
Prosthetic group of cytochrome c
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Components of respiratory chain do not function
as discrete carriers of reducing equivalents
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They are organized into four complexes
Each complex acts as a specific oxido-reductase
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Complex I, II, III and IV in respiratory chain
EMB-RCG
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EMB-RCG
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The ratio of ADP molecules phospho-rylated to
number of oxygen atoms reduced is known as PO
ratio
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PO ratio is three when the reducing equivalents
are accepted by NAD
PO ratio is two when the reducing equivalents
are accepted by FAD
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Mechanism of oxidative phosphorylation
Mechanism by which oxidation and phospho-rylation
are coupled remained unclear for long
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Several hypotheses were advanced to explain the
mechanism of this coupling
The chemiosmotic hypothesis is the most plausible
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Chemiosmotic hypothesis
Proposed by Mitchell
Inner mitochondrial membrane is impermeable to
protons (H)
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Energy released during transport of electrons
is used to actively eject H from the matrix of
mitochondria
This ejection establishes an electro-chemical
gradient across the membrane
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Re-entry of protons releases energy
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The energy released during influx of protons is
used to activate a membrane-bound enzyme
This enzyme, vectorial ATP synthetase, converts
ADP and Pi into ATP
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EMB-RCG
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The subunits of F0 and F1 components of
vectorial ATP synthetase
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When hydrogen ions flow back into the matrix

Two hydrogen ions
combine with two electrons and one oxygen
atom of inorganic phosphate
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Water is formed and inorganic phosphate is
converted into a highly reactive form
This form readily combines with ADP by a
high-energy bond
EMB-RCG
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There is strong experimental evidence in support
of chemiosmotic hypothesis
Building up of H gradient is the basic premise
of the hypothesis
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It is seen that on bathing mitochondria in a
fluid having a relatively high H concen-tration,
phosphorylation occurs in the absence of
oxidation
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The PH and HO ratios of various substrates
are also in agreement with the experimental
evidence
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Thus, the chemiosmotic hypothesis has now become
the accepted theory to explain oxidative
phosphorylation in the respiratory chain
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Inhibitors of oxidative phosphorylation
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Inhibitors of Oxidative Phosphorylation
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Uncouplers of oxidative phosphorylation
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An endogenous uncoupler
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Regulation of oxidative phosphorylation
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Oxidation of extra-mitochondrial NADH
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Two important mechanisms are
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Glycerophosphate shuttle
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Glycerophosphate shuttle
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Malate shuttle
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Malate shuttle
Cytosol
Mitochondrial Matrix
Oxalo-
Oxalo-
NADHH
NADHH
Aspartate
acetate
acetate
Aspartate
3
4
3
4
3
3
4
GOT
GOT
3
3
3
3
MDH
MDH
Glutamate
a
-Keto-
a-Keto-
Glutamate
3
5
glutarate
glutarate
3
4
Malate
Malate
NAD
NAD
4
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Transport across mitochondrial membrane
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Transport of pyruvate and H into mitochondria
is an example of a symport
Membrane
Mitochondrial matrix
Cytosol
Pyruvate
Pyruvate
H
H
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Some important antiports
Memb
Cytosol
Matrix
ADP
ADP
ATP
ATP
Malate
Malate
a-Ketoglutarate
a-Ketoglutarate
Glutamate
Glutamate
Aspartate
Aspartate
Malate
Malate
Citrate H
Citrate H


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Thank you