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Biological Oxidation
WANG Zhao
Department of Biochemistry and Molecular Biology
Jiamusi University
The oxidation processes of substances in organisms are known as
biologic oxidation. When carbohydrate, fat, and proteins are degraded to
form CO2 and water, some chemical energy is released and captured by
ADP to form ATP for living processes, and part chemical energy liberated
as heat to maintain body temperature.ⅤⅥ
Chemically, oxidation is defined as the removal of electrons and
reduction as the gain of electrons, as illustrated by the oxidation of
ferrous to ferric ion. This principle of oxidation-reduction applies equally
to biochemical systems, and is the important concept concerning with the
nature of biologic oxidation. In addition to this, dehydrogenation and
oxygenation are also named oxidation and opposite direction is reduction.
Biologic oxidation conforms to the general laws of thermodynamics.
One mole substance can be oxidized completely in vivo to get the same
quantity of energy as that in vitro. But the reaction condition, reaction
model, and the type of energy librated are difference if we compare
biologic oxidation with combustion in vitro (see Table 7-1).
Section I Oxidative system for ATP production
1. Respiratory chain
The respiratory chain in mitochondria consists of a number of redox
carriers that proceed from the NAD-linked dehydrogenase systems,
through flavoproteins and cytochromes, to molecular oxygen. Not all
substrates are linked to the respiratory chain through NAD-specific
dehydrogenases; some substances have more positive redox potentials,
they are linked directly to flavoprotein dehydrogenases, which in turn are
linked to the cytochromes of the respiratory chain.
The differences between biologic oxidation and combustion in vitro
Biologic oxidation
combustion
Reaction conditions
Reaction occurs at 37C, neutral High temperature and dry
pH, water involved
conditions
Reaction models
Reaction si under the catalysis of Reaction occurs break out
enzymes, O2 accepts 2 electrons suddenly
without
and then combines with proton catalysis, hydrogen and
to form water, CO2 is formed by oxygen combine directly
decarboxylation of substances.
to form water and CO2.
Type
of
energy During the reaction, energy is All energy bursts out as
liberated
liberated stepwise, part energy is the form of heat and light.
accumulated as chemical energy
for living processes, part energy
is released as heat to keep body
temperature.
Respiratory chain is present in the inner mitochondrial membrane as
four protein-lipid respiratory chain complexes, which span the membrane.
Cytochrome c and CoQ are soluble and more mobile.
NAD+
FpH2
2Fe3+
substrate
NAD-dependent
dehydrogenase
Flavoprotein
Cytochromes
A
NADH
Fp
2Fe2+
AH2
H+
H+
H2O
1/2 O2
2H+
2H+
respiratory chain
Choline
Succinate
Proline
3-Hydroxyacyl-CoA
3-Hydroxybutyrate
Glutamate
Malate
Isocitrate
pyruvate
Fp
(FAD)
FeS
II
I
lipoate
Fp
(FAD)
Fp
(FMN)
FeS
NAD
Fp
(FAD)
FeS
a-ketoglutarate
IV
III
Cyt b
FeS
CoQ
Cyt c1
Cyt c
Cyt aa3
Cu
O2
FeS
ETF
(FAD)
Fp
(FAD)
Glycerol 3-phosphate
Acyl-CoA
Sarcosine
Dimethylglycine
respiratory chain for some important substrates
The positions of the components in respiratory chain depend on the
standard redox potential of the component. Electrons always flow from
the negative potential to positive potential. From Table 7-2 the direction
of flow of the electrons can be predicted from relative negative redox
couple to relative positive one.
Table 7-2 standard redox potentials of redox couples in respiratory chain
Redox couples
H+/H
2
NAD+/NADH
FMN/FMNH2
FAD/FADH2
Cyt b Fe3+/Fe2+
Q10/Q10H2
Cyt c1 Fe3+/Fe2+
Cyt c Fe3+/Fe2+
Cyt a Fe3+/Fe2+
Cyt a3 Fe3+/Fe2+
1/2O2/H2O
E0’ volts
-0.42
-0.32
-0.30
-0.06
+0.04 or 0.01
+0.07
+0.22
+0.25
+0.29
+0.55
+0.82
E0’ volts: standard electrode potentials(detected at pH 7.0, 25C , in 1mol/L of
reactants)
1-1
Complex I
Complex I is NADH-ubiquinone reductase responsible for transporting
electron from NADH to ubiquinone. Complex I is composed of
flavoprotein and iron-sulfur protein, flavin mononucleotide (FMN) and
iron-sulfur cluster are their prosthetic group resectively. FMN receives 2
protons and 2 electrons from NADH(+H+), and transfers electrons to FeS.
The function of FeS is to transport electrons from FMN to ubiquinone.
Iron-sulfur protein (FeS) is another component found in respiratory
chain with the flavoproteins and with cytochrome b, and transports a
single electron by the oxidoreduction between Fe2+ and Fe3+.
NH 2
CONH 2
+
N
adenine
nicotinamide
O
H2C
O
H
P
O
_
P
H
_
CH 2
O
O
H
OH
H
N
ribose
H
OH
H
OH
AMP
H
2H
H
H
CONH 2
+
O
H
H
OH
nicotinamide mononucleotide
H
N
N
O
O
O
H
H
N
N
CONH 2
+
H
H
N
H
2H
R
R
The structure of NAD+
NH 2
adenine
ribitol
H2C
H
H
H
C
C
C
H2
C
OH OH OH
N
N
H3C
H3C
N
C
O
C
P
_
O
O
P
N
N
O
O
O
N
N
_
O
CH2
O
O
H
H
OH
OH
H
H
O
NH
ribose
flavin
FMN
FAD
R
R
2H
H3C
H3C
N
N
N
C
C
O
H3C
NH
H3C
2H
N
H
N
N
H
C
O
The structure of FAD and FMN
O
C
NH
O
Pr
Cys
S
Fe
S
Cys
Pr
S
Fe
S
Fe
S
S
Cys
S
Fe
Pr
S
Cys
Pr
The structure of iron-sulfur protein
1-2
Ubiquinone
An additional carrier is present in the respiratory chain liking the
flavoproteins to cytochrome b. this substance is named ubiquinone or
coenzyme Q. CoQ is a constituent of the mitochondrial lipids
characterized by the possession of a polyisoprenoid side chain.
O.
O
H3CO
CH 3
H3CO
CH 3
(CH 2 CH C CH 2)nH
+
H +e
H3CO
O
OH
CH 3
H3CO
+
H +e
H3CO
R
semiquinone form
(free radical)
R
OH
OH
ubiquinone
(full oxidized or
quinone form )
CH 3
H3CO
dihydroubiquinone
(reduced or quinol form)
The structure of ubiquinone
n = number of isoprenoid units, which is 10 in higher
animals, ie, Q10
1-3
Complex III
Complex III is ubiquinone-cytochrome c reductase responsible for
transferring electron from ubiquinone to cytochrome c. complex III
consists of two types of cytochrome b (Cyt b 562 and b566), cytochrome c1,
and iron-sulfur protein.
Cytochromes are a group of enzymes containing iron prophyrins (hemes)
as their prosthetic groups responsible for transferring electrons.
According to their specific absorption spectrum, cytochromes contained
in respiratory chain can be divided into cytochrome a, b, c.
Lys
Cys
Ala
Gln
CH 3
S CH
CH 3
Ile
Cys
CH 3
H3C
CH
N
Fe3+
Met
N
S
N
His
N
CH 3
H3C
Lys
Thr
CH 2
CH 2
CH 2
CH 2
COO -
COO -
The structure of cytochrome c
1-4
Cytochrome c
Cytochrome c is soluble in water, and easy to be separated from inner
membrane of mitochondria. Its function is to transfer electron from
complex III to complex IV.
1-5
Complex IV
Complex IV is cytochrome c oxidase responsible for transferring
electron from cytochrome c to oxygen to form water. Complex IV
contains cytochrome a and cytochrome a3. more recent studies show that
two cytochromes are combined with a single protein, and it is difficult to
separate. The complex is known as cytochrome aa3.it contains two
molecules of heme, each having one Fe atom that oscillates between Fe3+
and Fe2+ during oxidation and reduction. Furthermore, two atoms of Cu
are present, each associated with a heme unit.
1-6
Complex II
Some compounds have more positive redox potentials, they link with
complex II directively. Complex II is succinate-ubiquinone reductase
containing flavoprotein with flavin adenine dinucleotide (FAD, see Fig
7-4) as its prosthetic group, iron-sulfur protein, and cytochrome b. the
function of complex II is to transfer electron from succinate to
ubiquinone.
2. Oxidative phosphorylation
We have studied one model of ATP production, i.e. ATP production
at the substrate level. Most ATPs form in mitochondria at respiratory level
known as oxidative phosphorylation. Mitochondria are powerhouse of
cells and ATPs are energy currency. Most enzymes involved for ATP
production locate in mitochondria (Table 7-3).
Talbe 7-3 the distribution of some enzymes and
coenzymes in mitochondria
Intermembrane space
Outer membrane
Inner membrane
Monoamine oxidase
AcylCoA synthetase
Cytochrome b,c1,c,aa3
CoQ
FMN
NADHdehydrogenase
Fatty acid elongation enzymes
Succinate dehydrogenase
Aconitase
Fumarase
Malate dehydrogenase
Phospholipase A
-keto acid dehydrogenase
Fatty acid -oxidation enzymes
Phosphocholine transferase
Carnitine acyltransferase
Glutamate dehydrogenase
Kynurenine hydroxylase
Cytochrome c reductase
Adenylyl kinase
Creatine kinase
NDP kinase
matrix
Citrate synthase
Isocitrate dehydrogenase
-phosphoglycerl dehydrogenase
Glycerophosphate acyltransferase
-hyroxybutyrate dehydrogenase
Ornithine carbamoyl transferase
Cytochrome b5
Fatty acid elongation enzymes
ATPase
Adenylyl translocase
endergonic
exergonic
mechanical energy
(muscular contraction)
oxidative
phosphorylation
ATP
osmotic energy
active transport of
substances
creatine
~
P
creatine
phosphate
phosphorylation
at substrate level
ADP
ATP production and utilization
~
P
chemical energy
(syntheses)
electric energy
(biologic electricity)
heat energy
(keep body
temperature)
2-1 Coupling phosphorylation to oxidation
Common reactions can be divided into exergonic and endergonic.
Exergonic reactions are accompanied by loss of free energy (G is
negative); endergonic reactions are accompanied by gain of free energy
(G is positive). The free energy liberated from exergonic reactions can
be captured by a common high-energy compound such as ATP for
endergonic reactions. Respiratory chain are exergonic, phosphorylation of
ADP to
ATP is endergonic, the couple of two processes is named
oxidative phosphorylation.
Free energy change (Gibbs change) can be expressed as G0’ (under
conditions of pH 7.0, 25C,1mol/L reactants). When ATP terminal
phosphate is hydrolyzed, G0’ is –30.5kj/mol. The positions of
respiratory chain in which G0’ more than –30.5kj/mol. Or E0’ is more
than 0.2 volt conformably can be considered as the coupling position of
oxidative phosphorylation.
Examination of intact respiring mitochondria reveals that when
substrates are oxidized via an NAD-linked dehydrogenase and the
respiratory chain, approximately 3 mol of inorganic phosphate are
incorporated into 3 mol of ADP to form 3 mol of ATP per 1/2 mol of O 2
consumed; i.e., P:O ratio = 3. On the other hand, when a substrate is
oxidized via a flavoprotein-linked dehydrogenase, only 2 mol of ATP are
formed; i.e., P:O = 2.
Dehydrogenations in the pathway of catabolism of glucose in both
glycolysis and the citric acid cycle, plus phosphorylations at the substrate
level, can now account for 68% of the free energy resulting from the
combustion of glucose , captured in the form of high-energy phosphate. It
is evident that the respiratory chain is responsible for a large proportion
of total ATP formation.
Succinate
Fp
(FAD)
FeS
II
III
I
Fp
(FMN)
FeS
NAD
E0'
-0.32
CoQ
+0.04
-0.22
Cyt b
FeS
+0.08
0.36V
ADP+Pi
IV
Cyt c1
+0.23
Cyt c
+0.25
Cyt aa3
Cu
+0.29
+0.55
ADP+Pi
+0.82
0.53V
0.21V
ATP
O2
ATP
ADP+Pi
ATP
2-2 The coupling mechanism of oxidative phosphorylation
The powerful explain for the coupling mechanism of oxidative
phsophorylation is chemiosmotic theory. Mitchell’s chemiosmotic theory
postulates that the energy from oxidation of components in the respiratory
chain is coupled to the translocation of hydrogen ions (protons, H+) from
the inside to the outside of the inner mitochondrial membrane. The
electrochemical potential difference resulting from the asymmetric
distribution of the hydrogen ions is used to drive the mechanism
responsible for the formation of ATP (Fig. 7-10).
From Fig. 7-10 the oxidation in the respiratory chain couples with
proton translocation from the inside to the outside of the membrane. Each
of the respiratory chain complexes I, III, and IV acts as a proton pump.
The energy from the proton gradient is utilized to promote
phosphorylation of ADP to ATP under the catalysis of ATP synthase.
ATP synthase is so called
complex V, consists of several
protein
subunits
(33)
collectively known as F1, which
project into the matrix and
which contain the ATP synthase.
These subunits are attached by
a stalk to a membrane protein
complex known as F0. the stalk
also contains several subunits,
one
of
these
is
called
oligomycin-sensitivity-conferri
ng protein (OSCP), onec it is
combined by oligomycin, ATP
synthase will be inhibited. F0
extends through the membrane
and also consists of several
protein subunits (see Fig. 7-10). Protons pass through the F0-F1 complex,
leading to the formation of ATP from ADP and Pi.
3. Some factors affecting oxidation phosphorylation
Much information about the respiratory chain has been obtained by the
use of inhibitors, and conversely, this has provided knowledge about the
mechanism of action of several poisons (see Fig. 7-12).
3-1 Inhibitors
For descriptive purposes, inhibitors may be divided into inhibitors of
the respiratory chain proper, inhibitors of oxidative phosphorylation, and
uncouplers of oxidative phosphorylation.
(1) Inhibitors of respiratory chain
Inhibitors that arrest respiration by blocking the respiratory chain act at
three sites. 1) Inhibitors, such as amobarbital, piericidin A, and fish
poison rotenone, block the transfer from FeS to CoQ in the respiratory
chain via an NAD-linked dehydrogenase. 2) Inhibitors, such as
dimercaprol (BAL) and antimycin A, inhibit the respiratory chain
between cytochrome b and cytochrome c. 3) The classic poisons H 2S,
carbon monoxide, and cyanide inhibit cytochrome oxidase and can
therefore totally arrest respiration. In addition, carboxin specifically
inhibit transfer of reducing equivalents from succinate dehydrogenase to
CoQ, whereas malonate is a competitive inhibitor of succinate
dehydrogenase.
(2) Uncouplers
The action of uncouplers is to dissociate oxidation in the respiratory
chain from phosphorylation. 2,4-denitrophenol is most frequently used
uncoupler. Uncouplers are amphipathic and increase the permeability
of the lipoid inner mitochondial membrane to protons, thus reducing
the electrochemical potential and short-circuiting the ATP synthase. In
this way, oxidation can proceed without phosphorylation. The action of
uncouplers results in respiration becoming uncontrolled, since the
concentration of ADP or Pi no longer limits the rate of respiration. The
free energy liberated from the respiration is not captured as
high0energy phosphate, and released as heat. We could not get ATP.
pyruvate dehydrogenase complex
isocitrate dehydrogenase
a-ketoglutarate dehydrogenase complex
matate dehydrogenase
3-phosphoglycerol dehydrogenase
b-hydroxyacyl CoA dehydrogenase
b-hydroxybutyrate dehydrogenase
glutamate dehydrogenase etc
Succinate
Malonate
Rotenone
Amobarbital
Piericidin A
Fp
(FAD)
FeS
TTFA
Antimycin A
a Fe chelating
agent
III
I
NAD
Fp
(FMN)
FeS
H+m
H2S
CO
CN-
II
Cyt b
FeS
CoQ
H+m
H+c
IV
Cyt aa3
Cu
Cyt c
Cyt c1
H+c
H+m
O2
H+c
Uncouplers
e.g. 2,4-dinitrophenol
(DNP)
H+c
H+m
V
ADP+Pi
Oligomycin
F1,F0,OSCP
ATP
ATP
ADP+Pi
ATP
ADP+Pi
Oxicative phosphorylation and its inhibitors
Inhibitors of oxidative phosphorylation
Antibiotic
oligomycin
completely
blocks
oxidation
and
phosphorylation in intact mitochondria. Oligomycin combines with
oligomycin-sensitivity-conferring protein (OSCP) at the stalk of complex
V and inhibits ATP synthase activity.
3-2 The regulatory effect of ATP on respiration]
the rate of respiration of mitochondria can be controlled by the
concentration of ADP. This is because oxidation and phosphorylation are
tightly coupled; i.e., oxidation cannot proceed via the respiratory chain
without concomitant phosphorylation of ADP. When we do heaby
physical exercises with ATP consumed, at that time increased ADP is
transported into mitochondria and stimulates oxidative phosphorylation to
meet the use of ATP.
3-3 Thyroid hormone
thyroid hormone can induces biosynthesis of both Na +-K+-ATPase and
uncoupling protein. The results are both increase of oxidative
phosphorylation and uncoupling of oxidation and phosphorylation, and
increase of oxygen consumption and liberation of heat.
4. The oxidation of cytosolic NADH
The inner bilipoid mitochondrial membrane is freely permeable to
uncharged small molecules, such as oxygen, water, CO2, and NH3, and to
monocarboxylic acids, such as 3-hydroxybutyric, acetoacetic and acetic.
Long-chain fatty acids ae transported into mitochondria via the carnitine
system, and there is also a special carrier for pyruvate. However,
dicarboxylate and tricarboxylate anions and amino acids require specific
transporter or carrier systems to facilitate their transport across the
membraone.
NADH can not penetrate the mitochondrial membrane, but it is
produced continuously in the cytosol by 3-phosphoglyceraldehydr
dehydrogenase, an enzyme in the glycolysis sequence. However, under
aerobic conditions, extramitochondrial NADH does not accumulate and is
presumed to be oxidized by the respiratory chain in mitochondria. The
mechanism of transfer is known as glycerophosphate shuttle (see Fig.
7-13) and malate shuttle (see Fig. 7-14).
Out
er
2
Inne
me
A
rmbr
T
ane
mem
bran
e
H
2
Ps
O
In the glycerophosphate shuttle, mitochondrial enzyme is linked to the
respiratory chain via a flavoprotein rather than NAD, only 2 rather than 3
mol of ATP are formed per atom of oxygen consumed. The malate shuttle
system is linked to the NAD –linked respiratory chain, 3 mol of ATP are
formed per atom of oxygen consumed.
Section 2 Other oxidation systems
1. Aerobic dehydrogenase and oxidase
Oxidases, with prosthetic group as Cu, catalyze the removal of
hydrogen from a substrate using oxygen as a hydrogen acceptor. They
form water as a reaction product. Aerobic dehydrogenase are
flavoproteins, with prosthetic group as FMN or FAD, catalyze the
removal of hydrogen from a substrate, and via the transfer of FMN or
FAD, using oxygen as a final hydrogen acceptor. They form hydrogen
peroxide as a reaction product. In some textbook, aerobic dehydrogenase
and oxidase are all called oxidases.
2Cu2+
SH2
O2-
H2O
oxidase
2Cu+
S
1/2O2
2H+
SH2
S
FMN
aerobic
dehydrogenase
FMNH2
H2O2
O2
2. Oxidases in peroxisomes
The term “oxidase” is sometimes used collectively to denote all
enzymes that catalyze reactions involving molecular oxygen. In
peroxisome there are two kinds of hydroperoxidases: peroxidase and
catalase. Hydroperoxidases protect the body against harmful peroxides.
Accumulation of peroxides can lead to generate free radicals, which in
turn can disrupt membranes and perhaps cause cancer and atherosclerosis.
2-1 Peroxidases
Peroxidases are found in milk and in leukocytes, platelets, and other
tissues involved in eicosanoid metabolism. The prosthetic group is
protoheme, which unlike the situation in most hemoproteins, is only
loosely bound to the apoprotein.
peroxidase
H2O2 + AH2
2H2O + A
2-2 Catalase
Catalase is a hemoprotein containing four heme groups, in addition to
possessing peroxidase activity, it is able to use one molecule of H 2O2 as a
substrate electron donor and another molecule of H2O2 as oxidant or
electron accepter.
H2O2
catalase
H2O+ O2
3. Superoxide dismutase
oxygen can be reduced in tissues to the superoxide anion free radical
( O2 ), it is suggested that the toxicity of oxygen is due to its conversion
to superoxide.
Superoxide is formed when reduced flavins -------present, for example,
in xanthine oxidase ------are reoxidized univalently by molecular oxygen.
Superoxide dismutase catalyzes the formation of H2O2 from O2 .
O2
+ O2 + 2H+  H2O2 + O2
H2O2 + O2
 H2O + OH + OH
H2O2 + Fe2+  Fe3+ + OH + OH
Several powerful oxidants, named reactive oxygen, are produced
during the course of metabolism, in both blood cells and most other cells
of the body. These include superoxide, hydrogen peroxide (H 2O2),
peroxyl radicals (ROO), and hydroxyl radicals (OH). The last is a
particularly reactive molecule and react with proteins, nucleic acids,
lipids, and other molecules to alter their structure and produce tissue
damage.
Peroxidation of lipids exposed to oxygen is responsible not only for
stinking foods (rancidity) but also for damage to tissues in vivo, where it
may be a cause of cancer, inflammatory diseases, atherosclerosis, aging,
etc. Lipids peroxidation is a chain reaction providing a continuous supply
of free radicals that initiate further peroxidation. Peroxyl lipids combine
with proteins to form lipofuscin, it is concerned with aging.
Chemical compounds and reactions capable of generating potential
toxic oxygen species can be referred to as pro-oxidants. On the other
hand, compounds and reactions disposing of these species, scavenging
them, suppressing their formation, or opposing their actions are
antioxidants and include compounds such as NADPH, GSH, ascorbic
acid, and vitamin E.
4. Oxidases in microsomes
4-1 Monooxygenases
Monooxygenases are also termed as mixed-function oxidases or
hydroxylases incorporating only one atom of molecular oxygen into the
substrate. The other oxygen atom is reduced to water, an additional
electron donor or cosubstrate being necessary for this purpose.
RH + NADPH + H+ + O2  ROH + NADP+ + H2O
This monooxygenases are found in the microsomes of the liver
together with cytochrome P450 (there is a maximum absorption peak at
450 nm). NADPH donates reducing equivalents for the reduction of these
cytochromes. This reaction is catalyzed by NADPH-cytochrome
p450
reductase. Electron from NADPH is transferred to flavoprotein of this
enzyme, then it further is transported to iron-sulfur protein (iron-redox
protein). The latter gives 1 electron to substrate-contained P450, and
further combines with a molecule oxygen to form RH-P450-Fe3+-O2-.
Then this oxidized compound receives the second electron to form
reduced compound. At that time, one oxygen atom oxidized substrate to
form hydroxylated substrate, another oxygen atom combines with 2H +
(from NADPH) to form water (see Fig. 7-15).
P450
su b stra te A -H
A-H
Fe 3+
P450
NADP
NADPH+H
P450
e_
Fe 3+
+
FADH
+
2 Fe 2 S 2
2
e
FAD
O2
_
CO
2 Fe 2 S 2 2 +
2H
+
H 2O
A-OH
A-H
Fe 2+
3+
P450
A-H
Fe 2+
O2
P450
A-H
Fe 2+
O2
4-2 Dioxygenases
Dioxygenases incorporate both atoms of molecular oxygen into the
substrate.
R + O2  RO2
PROBLEMS FOR CHAPTER 7 BIOLOGIC OXIDATION
I. Explain the following contents:
1. Biologic oxidation
2. Oxidative phosphorylation
3. ATP synthase
4. Respiratory chains
5. P/O ratio
6. Uncouplers
7. High energy phosphate bond
III. Answer the following questions
1. What is the differences between biologic oxidation and combustion in vitro?
2. How many models are there for ATP production?
3. Indicate the components and arrangements of respiratory chains.
4. How does the cytosolic NADH incorporate oxidative phosphorylation? Describe
the processes.
5. What will happen when DNP is injected into the rat?