RESPIRATORY CHAIN

& OXIDATIVE
PHOSPHORYLATION
 Respiratory chain
• Enzyme complexes in mitochondria 
collects and transports reducing equivalents
 directing them to final reaction with
oxygen  form water and ATP
• Reducing equivalents flow through from
redox potential negative to positive
• There are 4 enzyme complexes:
- NADH-Q dehydrogenase / I
- Succinate-Q dehydrogenase / II
- Cytochromes dehydrogenase / III
- Cytochrome oxidase / IV
 AH2 NAD+ FpH2 2Fe3+ H2O

Substrate Flavoprotein Cytochrome

s
A NADH Fp 2Fe2+ 1
/2O

H+ H+ 2H
+ 2H+ 2

Transport of reducing equivalents through the respiratory

chain
 Mitochondrial
• Powerhouses of the cell  most of energy
captured takes place inside it
• Outer membrane  permeable to most
metabolites, contain various enzym (acyl Co-
A synthetase, glycerolphosphate
acyltransferase )
• Inner membrane  selectively permeable
• Matrix  contain phospholipid cardiolipin
together with enzymes of resp chain
• Intermembrane space has similar
composition with cytoplasmic and contain
adenylyl kinase and creatine kinase
 A B MATRIX
F1 subunits
OUTER
Phosphorylating F0 subunits
MEMBRANE
B complexes
INNER
MEMBRANE

MATRIX

Sonication
Cristae

INNER
MEMBRANE
OUTER
MEMBRANE
Submitochondrial particel
Formed from fragments of
the inner membrance
 Respiratory chain

• Not all substrates are linked to resp chain

through NAD-D-ase
• Co-Q (ubiquinone)  mobile component,
collects reducing equivalents from
flavoprotein complexes and passes them on
to cytochrome b (the lowest redox pot)
• Cytochrome oxidase has a very high
affinity for oxygen  resp chain to
function at maximum rate until tissue
depleted of O2 irreversible reaction
 Proline Succin
3-Hydroxyacyl- ate
CoA Cholin
3-Hydroxybutyrate e
Pyruvat Glutamate Fp
e Malate (FAD)
Isocitrate FeS

Fp Fp Cyt aa3
Lipoate Cyt b Cyt c1 Cyt O2
(FAD) NAD (FMN) Q
FeS c Cu
FeS
FeS
- ETF
Fp FeS : Iron-sulfur
Ketoglutarate (FAD)
(FAD) protein
FeS ETF : Electron-
Fp transferring
(FAD) flavoprotein
Fp : Flavoprotein
Acyl-CoA Q : Ubiquinone
Sarcosine Cyt : Cytochrome
Glycerol 3-phosphate Dimethylglycin
 Oxidative phosphorylation
• Oxidative reaction Coupled by
phosphorylation to the generation of high
energy intermediate (ATP or other high
phosphagen)
• Oxidative phosphorylation at resp chain
level  via NAD D-ases form 3 mol ATP
and via flavoprotein D-ases form 2 mol ATP
• Phosphorylations at the substrate level 
captured smaller energy  eg:a) High
energy phosphates are captured in kreb’s
cycle during the conversion of succinyl Co-
A to succinate. And b) in glycolytic
reactions on cytoplasmic.
 Mechanism of oxidative
phosphorylation
• Mitchell’s chemiosmotic theory:
- energy from oxidation in resp chain 
translocation of H+ (protons) 
electrochemical potential difference in
matrix and intermembrane space 
drive the mechanism of responsible for
the formation of ATP (ATP synthase)
 Mechanism of oxidative
phosphorylation
• Complexes I, III and IV of resp chain is a
proton pump
• Pi + ADP  ATP, by ATP synthase
• ATP synthase is a complex enzyme 
consist of several protein subunits (F1),
which attached to membrane protein
complex (F0)
• F1  project into matrix and contain the
phosphorylation mechanism
F0  spans the membrane and forms
the proton channel
 Exchange metabolites at inner
mitochondrial membrane
- Exchange of anions against OH- ions and
cations against H+ ions  for transport of
ionized metabolites
- Freely permeable to uncharged small
molecules  O2 , H2O , CO2 , NH3
monocarboxylic acids (3 hydroxy butyric,
acetoacetic, acetic)
- Long chain fatty acids need carnitine system
- Symport pyruvate - H+
 Exchange metabolites at inner
mitochondrial membrane
• Dicarboxylate and tricarboxylate anions
require specific carrier  linked to
inorganic phosphate (H2PO4- )
• Exchange ATP / ADP by adenine nucleotide
transporter
• Transport of oxaloacetate need
transamination process
Oxidation of extramitochondrial NADH

- NADH cannot penetrate mitochondrial

membrane  produced continuously in
cytosol by 3 phosphoglyceraldehyde D-ase
- Aerobic conditions: not accumulated  be
oxidized by resp chain
- Transfer of reducing equivalents from
cytosol to mitochondrial require substrate
pairs, linked by suitable D-ase
Oxidation of extramitochondrial NADH
- The mechanism:
1. Glycerophosphate shuttle  only 2 mol
ATP are formed per atom oxygen
consumed  present in brain, muscle,
adipose, liver but deficient in heart muscle
2. Malate shuttle  more universal utility
 more complex, due to the
impermeability of mitochondrial membrane
to oxaloacetate
 OUTER INNER
MEMBRANE MEMBRANE

MITOCHONDRION
CYTOSOL

NAD+
Glycerol 3- Glycerol 3-
phosphate phosphate FAD
GLYCEROL-3- GLYCEROL-3-
PHOSPHATE PHOSPHATE
DEHYDROGENASE DEHYDROGENASE
(CYTOSOLIC) (MITHOCONDRIAL)
FDH2
Dehydroxyacetone Dehydroxyacetone
NADH + phosphate phosphate
H+
Respiratory Chain

Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol

into the mitochondrion
 INNER
MAMBRAN
CYTOSOL E MITHOCOND
RION
1
NAD+ Malat Malate NAD+
e

MALATE MALATE
DEHYDROGENASE DEHYDROGENASE
NADH NADH
+H+ Oxaloacetat -KG -KG Oxaloacetat +H+
e e
TRANSAMI TRANSAMI
NASE NASE
Glutamate Asp Asp Glutamate

H+ H+

Malate shuttle for transfer of reducing equivalents from the cytosol into the
mitocondrion. 1. Ketoglutarate transporter, 2. glutamate-aspartate transporter (note
the proton symport with glutamate)
 Creatine phosphate shuttle
• Facilitating transport of high energy
phosphat from mitochondria in active
tissues
• Isoenzyme of creatine kinase (CKM), in
intermembrane space  catalyzing
transfer ~ P (ATP) to creatine:
~ P(ATP) + creatine  creatine-P ,
transported into cytosol via protein pores
 available for generation of
extramitochondrial ATP
 H

P
N CREATINE H2N
KINASE

C NH C NH

N H3C N

ΔGO’ = 12.6
COO- kJ/mol COO-

Creatine
Creatine
phosphate
 Resp chain & oxd phos inhibitors
• Inhibitors of resp chain
1. Blocking electrons transfer from Fe-S to
co-Q , ie: barbiturates , pierisidin-A ,
rotenon , carboxine ,
succinate D’ase competitive inhibitor:
malonate
2. Blocking electrons transfer from cty b to
cyt c, ie: dimercaprol , antimycin A
3. Inhibitors of cytochrome oxidase: H2S , CO
and CN
 Resp chain & oxd phos inhibitors

• Inhibitors of oxidative phosphorylation, ie:

oligomycine, atractyloside
• Un-couplers (dissociate oxidation in resp
chain from phosphorylation)  respiration
to become uncontrolled, ie: dinitrophenol,
dinitrochressol, pentachlorophenol, chloro
carbonyl cyanide phenilhydrazon (cccp)
 Uncoupling Protein
• In mitochondrial
membrane protein
regulate proton
channel or
transporter.
• Uncoupling
Protein :
thermogenesis
• Hibernat animal
• Producing : heat
• Salicylic acid : Uncoupling agent if
taken excess
 FAD
Succinate
FeS
H2S
BAL CO
Antimycin A CN -

Complex I Complex III Complex IV

NADH FMN, FeS Cyt b, FeS, Cyt c Cyt a Cyt a3 O2

Q
Cyt C1 Cu Cu

Piericidin A
Amobarbital
Rotenone Uncouplers
Uncouplers

Oligomycin
Oligomycin

ADP + P1 ATP ADP + P1 ATP ADP + P1 ATP

 Clinical aspects

• Fatal infantile mitochondrial myopathy and

renal dysfunction  due to severe
diminution / absence of most
oxidoreductase
• MELAS (mitochondrial encephalopathy,
lactic acidosis and stroke)  due to
complex I or complex IV deficiency 
mutation in mt DNA