chapter 6 biological oxidation
DESCRIPTION
Chapter 6 Biological Oxidation. The biochemistry and molecular biology department of CMU. Biological oxidation is the process in which substances (carbohydrate, Lipid, AAs) are oxidized in living organism. Oxidation types: Dehydrogenation Electron lost Oxygenation. - PowerPoint PPT PresentationTRANSCRIPT
Chapter 6
Biological Oxidation
The biochemistry and molecular biology department of CMU
Biological oxidation is the proc
ess in which substances (carb
ohydrate, Lipid, AAs) are oxidiz
ed in living organism.
Oxidation types:
Dehydrogenation
Electron lost
Oxygenation
Nature of biological oxidation :1. 37 , pH 7.4, enzymatic reaction.℃2. Energy released gradually.
3. Formation of H2O.
4. Formation of CO2 by decarboxylation.
CO2
ATP ADP + Pi1/2 O2
H2O
acetyl CoA CoASH
G glycerol, FAs AAs
glycogen lipids proteinsfirst
phase
secondphase
thirdphaseoxidative
phosphorylation
Pyr
TCAC
cytosol
Mit.
2H
2H
General situation of biological oxidation
§1 Respiratory Chain
§2 Oxidative Phosphorylation
§3 ATP
§4 Shuttle Systems
§ 1 Respiratory Chain
A chain in the mitochondria con
sists of a number of redox carriers f
or transferring hydrogens removed f
rom the substrate to oxygen to form
water. The chain is termed a respirat
ory chain, also called electron trans
port chain (ETC).
Composition of Respiratory Chain Complexes
Complexes NameProsthetic Groups
Complex I NADH-CoQ Reductase
FMN, Fe-S
Complex II Succinate-CoQ Reductase
FAD, Fe-S
Complex III CoQ-Cyt c Reductase
iron-porphyrine
Fe-S
Complex IV Cytochrome Oxidase
iron-porphyrine Cu
Position of Respiratory Chain Complexes
Order of Respiratory Chain Complexes
NADH
succinate
FMN£¨Fe-S£©
FAD£¨Fe-S£©
Cyt b, £¨Fe-S£©CoQ Cyt aa3 O2Cyt ccompex I
compex II
compex III compex IV
c1
Composition of ETC
Hydrogen carrier : NAD +
FMN
FAD
CoQ
electron carrier : Fe-S Cyt
• NAD+/NADH (Nicotinamide Adenine Dinucleotide, Co )Ⅰ
• NADP+/NADPH (Nicotinamide Adenine Dinucleotide Phosphate, Co
)Ⅱ
The nicotinamide is the vitamin PP.
1. Nicotinamide coenzymes
NAD+
O
H H
OH OH
H H
CH2 O O
OHOH
HHH
CH2
H
OP
OH
O
N
N
N
N
NH2
N
CONH2
O P
OH
O
NADP+
O
H H
OH OH
H H
CH2 O O
OOH
HHH
CH2
H
OP
OH
O
N
N
N
N
NH2
N
CONH2
O P
OH
O
PO
OH
OH
N
H
CONH2
R
+ H + H + + e
N
H
CONH2
R
H
+ H +
NAD +/NADP + NADH/NADPH
FMN: Flavin Mononucleotide
FAD: Flavin Adenine Dinucleotide
They contain the riboflavin (Vit B2).
2. Flavin prosthetic groups
FMN
CH2 OHO P
OH
OC
C
H OH
C
H OH
H OH
CH H
NH
N
N
N O
O
H3C
H3C1
45
8 9
10Isoalloxazine
ribitol
FAD
CH2 O O
OHOH
HHH
CH2
H
OP
OH
O
N
N
N
N
NH2
O P
OH
OC
C
H OH
C
H OH
H OH
CH H
NH
N
N
N O
O
H3C
H3C1
45
8 9
10
R
NH
N
N
N O
O
H3C
H3C
FMN/FAD
1
45
8 9
10
R
NH
NH
HN
N O
O
H3C
H3C1
45
8 9
10+ 2H
FMNH2/FADH2
NAD+ is a coenzyme, that
reversibly binds to enzymes.
FAD is a prosthetic group, that
remains tightly bound at the active
site of an enzyme.
Iron-sulfur centers (Iron-sulfur
protein, Fe-S) are prosthetic groups
containing 2, 3, 4 or 8 nonheme iron
atoms complexed to elemental and
cysteine S.
3. Fe-S
Fe
Fe
S
S
S
Fe
Fe
S
S
S
SS
Cys
Cys
Cys
Cys
S
Fe
S
Fe
S
S
S
S
Cys
CysCys
Cys
Iron-sulfur centers
2 Fe iron-sulfur center of ferredoxin.
2 Fe colored orange; elemental & Cys S yellow.
Different types of iron-sulfur centers
Iron-sulfur centers transfer only
one electron.
Fe3+ + e- Fe2+
Coenzyme Q (CoQ, ubiquinone) is very hydrophobic. It dissolves in the membrane.
Coenzyme Q functions as a mobile e- carrier within the mitochondrial inner membrane.
4. CoQ
O
O
H3CO
H3CO
CH3
(CH2 CH
C
CH3
CH2)nH
CoQ
isoprene
H2C C C CH2
CH3
H
O
O
CH3O
CH3CH3O
R
OH
OH
CH3O
CH3CH3O
R
+ 2H
ubiquinone ubiquinol
Cytochromes (Cyt) are proteins with heme prosthetic groups. They absorb light at characteristic wavelengths.
Hemes in the 3 classes of cytochrome (a, b, c) differ slightly in substituents on the porphyrin ring system.
5. Cytochromes
Hemes a & a3 are often referred to as cytochromes aa3.
Cytochrome c is a small, water-soluble protein with a single heme group.
N
N
N
N
CH3 CH
CH2
CH3
CH CH2
CH2
CH2
COO
CH3
HC
CH2CH2 OOC
Fe
O
Heme b
N
N
N
N
CH3 HC
CH3
S CH2
CH3
CH S CH2
CH3
CH2
CH2
COO
CH3
H3C
CH2CH2 OOC
protein
protein
Fe
Heme c
Heme c
Cytochrome c
Lys13 Lys 72
heme
complex IV
cyt. c
N
N
N
N
CH3 HC
CH2
CH3
CH CH2
CH2
CH2
COO
CH3
HC
CH2CH2 OOC
Fe
OH
CH2 CH C CH2
CH3
3 H
O
Heme a
The heme iron can undergo
an electron transition between
ferric and ferrous states:
Fe3+ + e- Fe2+
The sequence of the components in the respiratory chain has been deduced in several ways.
1. Inhibitor.
2. O2 is suddenly introduced into the system.
3. Standard redox potential.
Inhibitor
Standard reduction potentials for respiratory chain and related electron carriers
1. NADH respiratory chain :
2. Succinate respiratory chain : c1 c aa3 O2
FAD£¨ Fe-S£©
succinate CoQ Cyt b
NADH FMN£¨Fe-S£©
c1 c aa3 O2
FAD£¨Fe-S£©
succinate¦Á-glycerophosphate
CoQ Cyt b
malate¦Â-hydroxy fatty acyl CoA¦Â-hydroxybutyrateisocitriteGlu
FAD
lipoic acid
pyruvate¦Á-ketoglutarate
FAD
fatty acyl CoA
§2
Oxidative Phosphorylation
Oxidative phosphorylation:
The phosphorylation of ADP t
o ATP coupled to electron transfer
from a substrate to molecular oxy
gen.
Substrate level phosphorylation :
Phosphorylation of ADP or GD
P to ATP or GTP coupled to the deh
ydrogenation of an organic substra
te.
Coupled site:
1. P/O ratio is the number of
inorganic phosphates
incorporated into ATP per
oxygen atom consumed.
(number of ATP / 2H)
P/O ratio of some substrate
substratescomponent
sP/O ratio
Number of ATP
-hydroxybutyrate NAD+→O2 2.4~2.8 3
succinate FAD →O2 1.7 2
Vit C Cyt c→O2 0.88 1
Cyt c Cyt aa3→O20.61~0.6
81
∆E0'E0'
NADH FMN£¨ Fe-S£©
CoQ Cyt b Cyt c1 Cyt c Cyt aa3 O2
FAD£¨ Fe-S£©
succinate
energyADP + Pi ATP
energyADP + Pi ATP
energyADP + Pi ATP
-0.32 -0.22 +0.04 +0.08 +0.23 +0.25 +0.29 +0.82
0.36V 0.21V 0.53V
69.5kJ/mol 40.5kJ/mol 102.3kJ/mol∆G0'
-hydroxybutyrate
succinate
Vit C
Three Coupled sites
① NADH → CoQ
② CoQ → Cyt c
③ Cyt aa3 → O2
• NADH ETC has 3 coupled site , P/O ratio =3,
3mol ATP / 2H.
• Succinate ETC has 2 coupled site , P/O ratio =2 , 2mol ATP / 2H.
Coupled mechanism:
1. Chemiosmotic hypothesis
The chemiosmotic hypothesis is the concept that a proton concentration gradient serves as the energy reservoir for driving ATP formation and was originally formulated by Peter Mitchell in the early 1960s .
Respiratory chain is in cristae of the inner membrane.
inner membranecristae
outer membrane
Intermembrane space
matrix
Spontaneous electron flow
through each of complexes I, III, &
IV is coupled to H+ ejection from the
matrix.
A total of 10 H+ are ejected from the mitochondrial matrix per 2 e- transferred from NADH to oxygen via the respiratory chain.
Matrix
NADH + H+ NAD+
2H+ + ½ O2 H2O
2 e – – I Q III IV
+ +
4H+ 4H+ 2H+ Intermembrane Space
cyt c
2. ATP synthase (complex V )
ATP synthase, embedded in cristae of the inner mitochondrial membrane, includes:
F1 catalytic subunit, made of 5 kinds of polypeptides α3β3γδε.
F0 complex of integral membrane proteins mediates proton transport.
Looking down at the membrane, they are arranged as a ring of alternating α & β subunits.
F1 in cross
section
ADP + Pi ATP
ATP
ATP ADP
+ Pi ATP ADP
+ Pi
ATP open tight
binding
loose binding
The mechanism for ATP synthase
Matrix
NADH + H+ NAD+ 2H+
+ ½ O2 H2O
2 e – – I Q III IV
+ +
4H+ 4H+ 2H+ Intermembrane Space
cyt c 3H+
F1
Fo
ADP + Pi ATP
3H+ 3H+
Transport of ATP, ADP, & Pi
summary
2.5 ~P bonds synthesized during oxidation of NADH produced in the matrix. (10 H+ pumped; 4 H+ used up per ATP).
1.5 ~P bonds synthesized during oxidation of FADH2 produced in the
matrix.
Regulation of Oxidative
Phosphorylation
1. The factors affecting ETC
A. Inhibitors of ETC
NADHFMN
£¨Fe-S£© CoQ Cyt b Cyt c1 Cyt c Cyt aa3 O2
FAD£¨Fe-S£©
succinate
Rotenone, Amytal, piericidin A
dimercaptopropanol Antimycin A
CN-, N-3,
CO, H2S
B. Uncoupling agent
A compound that disrupts the
usual tight coupling between elect
ron transport and phosphorylation
of ADP.
Uncoupling reagents dissolve in the membrane and function as carriers for H+.
OH
NO2
NO2
2,4-dinitrophenol
Uncouplers block oxidative phosphorylation by
dissipating the H+ electrochemical gradient.
Matrix
NADH + H+ NAD+ 2H+
+ ½ O2 H2O
2 e I Q III IV
4H+ 4H+ 2H+ H+ Intermembrane Space
cyt c
uncoupler
Endogenous
Uncouplers E
nable Organi
sms To Gene
rate Heat.
C. Inhibiter of oxidative phosphoryl
ation
A compound that Inhibits electron
transport and phosphorylation of A
DP.
Oligomycin
2. ADP
[ADP]↑→ Oxidative phosphorylation↑
[ADP]↓→ Oxidative phosphorylation↓
pyruvate
ADP
succinate
Mt.
rotenone
oligomycin
antimycin A ( CN-)
time
DNP
Influence of different substrates and inhibitors on quantity consuming oxygen
3. Thyroxine
Na+,K+-ATPase →ATP catabolism → ADP↑ → Oxidative phosphorylation↑ → formation of ATP↑. Formation and catabolism of ATP↑ , consuming of O2↑, Generation of Heat↑.
4. mtDNA mutation
Free radical → mutation of DNA → inhibition of Oxidative phosphorylation → energy↓
§3 ATP
N
N
N
N9
NH2
O
OHOH
HH H
CH2
H1'
2'
glycosidicbondOP
O-
O
O
PO
O-
O
P
O-
-O
O¦Á¦Â¦Ã
ester bond
Adenosine triphosphate, ATP
α bond G△ 0′ = - 14.3KJ
β bond G△ 0′ = - 32.2KJ
γ bond G△ 0′ = - 30.5KJ
>30KJ ( 7kcal ) -high energy bond
s
Compound Go' of phosphate hydrolysis, kJ/mol
Phosphoenolpyruvate (PEP)
Phosphocreatine
Pyrophosphate
ATP (to ADP)
Glucose-6-phosphate
Glycerol-3-phosphate
ΔG0' of phosphate hydrolysis of some compound
Examples of high-energy compounds
Type of bond General formula Example
ΔG0'kJ/mol
Phospho-guanidines
Creatine phosphate 43.9
Enol phosphates PEP 61.9
Phosphoric-carboxylic
1,3-bisphospho-
glycerate41.8
Phosphoric acid
anhydrides
ATP, GTP, UTP, CTP 30 .5
Thiol estersAcetyl CoA,
Acyl CoA31.4
R C O
CH2
~ PO3H2
R C N
NH
H
~ PO3H2
R C O
O~ PO3H2
P O
O
~P OHOH
O
OH
R C
O
~ SCoA
Potentially, 2 ~P bonds can be cleaved, as 2 phosphates are released by hydrolysis from ATP.
AMP~P~P AMP~P + Pi
(ATP ADP + Pi)
AMP~P~P AMP + P~P
(ATP AMP + PPi)
P~P 2 Pi
(PPi 2Pi)
Adenylate Kinase:
2 ADP ATP + AMP
Nucleoside Diphosphate Kinase catalyzes
reversible reactions such as:
ATP + GDP ADP + GTP,
ATP + UDP ADP + UTP, etc.
Phosphocreatine is used in nerve and muscle for storage of ~P bonds.
creatinephosphate
+ ADP
creatine
+ ATP
creatinekinase
COOH
CH2
N
C
H3C
NH2
NH
COOH
CH2
N
C
H3C
N
NH
H
P
Production and application of ATP
creatinephophate
creatine
ATP
~P
ADP
~P
oxidativephosphorylation
Substrate level phosphorylation
mechanism energyosmotic energychemical energyelectric energyheat energy
§4 Shuttle Systems
Permeable to :
Pyr, succinate, α-ketoglutarate, malate, citrite, Glu etc.
Impermeable to :
H+, NADH, NADPH, OAA etc.
The inner membrane of mitochondria is quite impermeable to some molecules and ions.
• Oxidation of extramitochondrial NA
DH is mediated by shuttle systems.
- Glycerophosphate shuttle system
Brain, skeletal muscle
Malate-asparate shuttle ( heart , liver )
NAD+
NADH+H+
Malate
OAA
Malate
OAAGluGlu
Asp Asp¦Á-KG ¦Á-KG
NAD+
NADH+H+
ETC
3ATP
MDH
transaminasetransaminase
MDH
cytosol matrix