INVESTIGATION OF BIOLOGICAL
OXIDATION, OXIDATIVE PHOSPHORYLATION AND ATP SYNTHESIS. INHIBITORS AND UNCOUPLERS OF OXIDATIVE
PHOSPHORYLATION.
Bioenergetic
Bioenergetics is the part of biochemistry
concerned with the energy involved in making and breaking of chemical bonds in
the molecules
found in biological organisms.
Growth, development and metabolism
are some of the central phenomena in the study of biological organisms. The
role of energy is fundamental to such biological processes. The ability to harness
energy from a variety of metabolic pathways is a property of all living
organisms. Life
is dependent on energy transformations; living organisms
survive because of exchange of energy within and without.
In a living organism, chemical
bonds are broken and made as part of the exchange and transformation
of energy. Energy is available for work (such as mechanical work) or for other
processes (such as chemical synthesis and anabolic
processes in growth), when weak bonds are broken and stronger bonds are made.
The production of stronger bonds allows release of usable energy.
Living organisms obtain energy from
organic and inorganic materials. For example, lithotrophs
can oxidize minerals such as nitrates or forms of sulfur, such as
elemental sulfur, sulfites,
and hydrogen sulfide to produce ATP. In photosynthesis,
autotrophs
can produce ATP using light energy. Heterotrophs
must consume organic compounds. These are mostly carbohydrates,
fats, and proteins.
The amount of energy actually obtained by the organism is lower than the amount
present in the food; there are losses in digestion, metabolism, and thermogenesis.
The materials are generally combined with oxygen to
release energy, although some can also be oxidized anaerobically by various
organisms. The bonds holding the molecules of nutrients
together and the bonds holding molecules of free oxygen together are all
relatively weak compared with the chemical bonds holding carbon dioxide and
water together. The utilization of these materials is a form of slow combustion.
That is why the energy content of food can be estimated with a bomb calorimeter.
The materials are oxidized slowly enough that the organisms do not actually
produce fire. The oxidation releases energy because stronger bonds have been
formed. This net energy may evolve as heat, or some of which may be used by the
organism for other purposes, such as breaking other bonds to do chemistry.
Living organisms produce ATP from energy sources via oxidative phosphorylation. The terminal
phosphate bonds of ATP are relatively weak compared with the stronger bonds
formed when ATP is broken down to adenosine monophosphate and phosphate,
dissolved in water. Here it is the energy of hydration that results in energy
release. This hydrolysis of ATP is used as a battery to store energy in cells,
for intermediate metabolism. Utilization of chemical energy from such molecular
bond rearrangement powers biological processes in every biological organism.
Exergonic and
endergonic processes. Mechanism of energy releasing and storage in the
organism.
Exergonic refers to
chemical reactions that proceed spontaneously from reactants to products with
the release of energy. Endergonic reactions require energy input to proceed. Although
the terms are often used rather loosely, they are precisely defined
thermodynamic concepts based on changes in an entity called Gibbs free energy
(G) accompanying reactions. Reactions in which -G decreases are exergonic, and
those in which -G increases are endergonic. Exergonic reactions often involve
the breakdown of organic compounds found in food, whereas endergonic reactions
frequently entail synthesis of complicated molecules.
Biological metabolism contains many examples of both types, and living
organisms have developed elaborate techniques for coupling the two.
Exergonic reactions release free energy while endergonic
reactions consume free energy
Although a negative -G indicates that energy must be added to the system
before a reaction will occur, it tells us nothing about the rate at which it
will progress. As is often the case, it may go very slowly if substantial
activation energy is required to start the reaction. Living organisms have
found a way around this problem by forming protein catalysts, called enzymes,
that effectively reduce the amount of activation energy needed, and allow the
reaction to proceed at a satisfactory rate. Enzymes do not affect the free
energy of the reaction, and will not enable reactions to proceed that are not
energetically feasible.
By coupling exergonic and
endergonic reactions, organisms are able to use the available energy in food
they consume to construct complex proteins, lipids, nucleic acids and carbohydrates
needed for their growth and development. A well-known example involves coupling
the formation of energy-rich adenosine triphosphate (ATP) from adenosine
diphosphate (ADP) and phosphate (an endergonic reaction), with the transfer of
hydrogen, removed from organic food materials, to oxygen (an exergonic
reaction). The process is called oxidative phosphorylation. Energy stored in
ATP may be used subsequently when the exergonic conversion of ATP back to ADP
and phosphate is coupled with the endergonic synthesis of a needed cellular
component.
What is macroergic bond? Examples of high energy compounds.
A bond in
chemical compounds, which acts as an energy accumulator. Macroergic bond
is present in some
phosphorus-containing compounds in living organisms, e. g.* adenosinetriphosphate.
Macroergic bonds
are formed as a result of complex biochemical processes
and break
up when energy is released. The processes are reversible and can be repeated.
High-energy
phosphate can mean one of two things:
·
The
phosphate-phosphate bonds formed when compounds such as adenosine diphosphate
and adenosine triphosphate are created.
·
The compounds
that contain these bonds, which include the nucleoside
diphosphates and nucleoside triphosphates, and the high-energy storage
compounds of the muscle, the phosphagens. When people speak of a high-energy phosphate
pool, they speak of the total concentration of these compounds with these
high-energy bonds.
·
High-energy phosphate bonds are pyrophosphate
bonds, acid anhydride
linkages formed by taking phosphoric acid derivatives and dehydrating
them. As a consequence, the hydrolysis of these bonds is exergonic under physiological conditions,
releasing energy.
Energy released by high energy
phosphate reactions |
|
Reaction |
ΔG [kJ/mol] |
ATP + H2O → ADP +
Pi |
-30.5 |
ADP + H2O → AMP + Pi |
-30.5 |
ATP + H2O → AMP +
PPi |
-40.6 |
PPi + H2O →
2 Pi |
-31.8 |
AMP + H2O → A + Pi |
-12.6 |
Except for PPi → 2 Pi, these
reactions are, in general, not allowed to go uncontrolled in the human cell but
are instead coupled to other processes needing energy to drive them to
completion. Thus, high-energy phosphate reactions can:
·
provide energy to cellular processes,
allowing them to run;
·
couple processes to a particular
nucleoside, allowing for regulatory control of the process;
·
drive the reaction to the right,
by taking a reversible process and making it irreversible.
The one exception is of value because it allows a single
hydrolysis, ATP + 2H2O → AMP + PPi, to effectively
supply the energy of hydrolysis of two high-energy bonds, with the hydrolysis
of PPi being allowed to go to completion in a separate reaction. The
AMP is regenerated to ATP in two steps, with the equilibrium reaction ATP + AMP
↔ 2ADP, followed by regeneration of ATP by the usual means, oxidative phosphorylation or other
energy-producing pathways such as glycolysis.
Often, high-energy phosphate bonds are
denoted by the character '~'. In this "squiggle" notation, ATP
becomes A-P~P~P. The squiggle notation was invented by Fritz Albert Lipmann, who first
proposed ATP as the main energy transfer molecule of the cell, in 1941. It
emphasizes the special nature of these bonds.
Stryer states:
ATP is often called a high energy
compound and its phosphoanhydride bonds are referred to as high-energy bonds.
There is nothing special about the bonds themselves. They are high-energy
bonds in the sense that free energy is released when they are hydrolyzed,
for the reasons given above.
Lipmann’s term “high-energy bond” and his symbol ~P
(squiggle P) for a compound having a high phosphate group transfer potential
are vivid, concise, and useful notations. In fact Lipmann’s squiggle did much
to stimulate interest in bioenergetics.
The term 'high energy' with respect to
these bonds can be misleading because the negative free energy change is not
due directly to the breaking of the bonds themselves. The breaking of these
bonds, as with the breaking of most bonds, is an endergonic step (i.e., it
absorbs energy, not releases it). The negative free energy change comes instead
from the fact that the bonds formed after hydrolysis-or the phosphorylation of
a residue by ATP-are lower in energy than the bonds present before hydrolysis
(this includes all of the bonds involved in the reaction, not just the
phosphate bonds themselves). This effect is due to a number of factors
including increased resonance stabilization and solvation
of the products relative to the reactants.
Besides the adenosine nucleotide phosphates,
uracil, cytosine and guanine phosphates occur, too:
UMP, UDP, UTP, CMP, CDP, CTP, GMP, GDP, GTP.
The triphosphate nucleosides of these compounds and those of ATP are
components of RNA. They are integrated into the polymer by splitting off
pyrophosphate ( = PP). The corresponding desoxyribose derivatives (dATP, dGTP,
dCTP....) are necessary for DNA synthesis, where dTTP is used instead of dUTP.
The terminal phosphate residues of all nucleoside di- and triphosphates are
equally rich in energy. The energy set free by their hydrolysis is used for
biosyntheses. They share the work equally: UTP is needed for the synthesis of
polysaccharides, CTP for that of lipids and GTP for the synthesis of proteins
and other molecules. These specificities are the results of the different
selectivities of the enzymes, that control each of these metabolic pathways.
ATP formation
The Function of ATP
The ATP is used for many cell
functions including transport work moving substances across cell
membranes. It is also used for mechanical work, supplying the energy
needed for muscle contraction. It supplies energy not only to heart muscle (for
blood circulation) and skeletal muscle (such as for gross body movement), but
also to the chromosomes and flagella to enable them to carry out their many
functions. A major role of ATP is in chemical work, supplying the needed
energy to synthesize the multi-thousands of types of macromolecules that the
cell needs to exist.
ATP is also used as an on-off switch both to control
chemical reactions and to send messages. The shape of the protein chains that
produce the building blocks and other structures used in life is mostly
determined by weak chemical bonds that are easily broken and remade. These
chains can shorten, lengthen, and change shape in response to the input or
withdrawal of energy. The changes in the chains alter the shape of the protein
and can also alter its function or cause it to become either active or
inactive.
The ATP molecule can bond to
one part of a protein molecule, causing another part of the same molecule to
slide or move slightly which causes it to change its conformation, inactivating
the molecule. Subsequent removal of ATP causes the protein to return to its
original shape, and thus it is again functional. The cycle can be repeated
until the molecule is recycled, effectively serving as an on and off switch.
Both adding a phosphorus (phosphorylation) and removing a phosphorus from a
protein (dephosphorylation) can serve as either an on or an off switch.
How is ATP Produced?
ATP is manufactured as a result of
several cell processes including fermentation, respiration and photosynthesis.
Most commonly the cells use ADP as a precursor molecule and then add a
phosphorus to it. In eukaryotes this can occur either in the soluble portion of
the cytoplasm (cytosol) or in special energy-producing structures called
mitochondria. Charging ADP to form ATP in the mitochondria is called chemiosmotic
phosphorylation. This process occurs in specially constructed chambers
located in the mitochondrion’s inner membranes.
ATP-synthase
converts ADP into ATP, a process called charging.
Adenosine triphosphate (ATP) is an
organic molecule which stores energy used to carry out life processes. ATP
is made of an adenine nucleoside, ribose sugar, and three phosphate groups. The
high energy bonds between phosphate groups are broken when hydrolyzed, thus
releasing energy in the system. Either one or two phosphate groups can break
off, releasing Gibb's free energy,
which can then be used to drive other reactions.
The molecular structure of ATP which is formed from
a adenine nucleoside, ribose sugar, and three phosphate groups
ATP
can be formed from bonding either adenosine monophosphate (AMP) and two
inorganic phosphate groups (PPi) together or by bonding adenosine
diphosphate (ADP) and one inorganic phosphate group (Pi) together.
Energy is required to bond the adenosine to the phosphate groups, making it an
endergonic reaction. The energy used to bond the two molecules together is then
stored within covalent bonds between phosphate groups in ATP. ATP
can be formed through two different endergonic processes, either through
substrate-level phosphorylation or chemiosmosis.
ATP is needed
- as a source of energy for biochemical syntheses
- for transport processes
(active transport) and
- for mechanical work
like movements (ciliar movements, plasma currents etc.)
How
the Hydrolysis of ATP Performs Work
•The
bonds between the phosphate groups of ATP’s tail can be broken by hydrolysis
•Energy
is released from ATP when the terminal phosphate bond is broken
•This
release of energy comes from the chemical change to a state of lower free
energy, not from the phosphate bonds themselves
•The
three types of cellular work (mechanical, transport, and chemical) are powered
by the hydrolysis of ATP
•In
the cell, the energy from the exergonic reaction of ATP hydrolysis can be used
to drive an endergonic reaction
•Overall,
the coupled reactions are exergonic
ATP + H2O →
ADP + Pi
Releases -30.5
kJ/mol= ΔG˚ (when one
phosphate group breaks off)
ATP + H2O →
AMP + PPi
Releases -45.6 kJ/mol= ΔG˚ (when two phosphate groups break off)
Hydrolysis of ATP
The
Regeneration of ATP
•ATP
is a renewable resource that is regenerated by addition of a phosphate group to
adenosine diphosphate (ADP)
•The
energy to phosphorylate ADP comes from catabolic reactions in the cell
•The
ATP cycle is a revolving door through which energy passes during its transfer
from catabolic to anabolic pathways
http://www.youtube.com/watch?v=_PgjsfY71AM&feature=related
http://www.youtube.com/watch?v=YndC0gS3t6M&feature=related
Substrate-level and oxidative phosphorylation
Substrate-level phosphorylation
is a type of metabolic reaction that results in the formation of adenosine
triphosphate (ATP) or guanosine
triphosphate (GTP) by the direct transfer and
donation of a phosphoryl (PO3)
group to adenosine
diphosphate (ADP) or guanosine
diphosphate (GDP) from a phosphorylated reactive
intermediate. Note that the phosphate group does
not have to come directly from the substrate. By convention, the phosphoryl
group that is transferred is referred to as a phosphate group.
An alternative way to create ATP is
through oxidative
phosphorylation, which takes place during the
process of cellular
respiration, in addition to the substrate-level
phosphorylation that occurs during glycolysis and the Krebs cycle. During
oxidative phosphorylation, NADH is oxidized to NAD+, yielding 2.5
ATPs, and FADH2 yields 1.5 ATPs when it is oxidized. Oxidative
phosphorylation uses an electrochemical or chemiosmotic gradient of protons (H+)
across the inner mitochondrial membrane to generate ATP from ADP, which is a
key difference from substrate-level phosphorylation.
Unlike oxidative phosphorylation, oxidation
and phosphorylation
are not coupled in the process of substrate-level phosphorylation, although
both types of phosphorylation result in the formation of ATP and reactive
intermediates are most often gained in course of oxidation processes in catabolism.
However, usually most of the ATP is generated by oxidative phosphorylation in
aerobic or anaerobic respiration. Substrate-level phosphorylation serves as
fast source of ATP independent of external electron acceptors and respiration.
This is the case for example in human erythrocytes, which have no mitochondria, and in
the muscle during oxygen depression.
oxidative phosphorylation
The main part of substrate-level
phosphorylation occurs in the cytoplasm of cells as part of glycolysis and in mitochondria as part of the Krebs Cycle
under both aerobic
and anaerobic
conditions. In the pay-off phase of
glycolysis, two ATP are produced by
substrate-level phosphorylation: two and only two 1,3-bisphosphoglycerate
are converted to 3-phosphoglycerate by transferring a
phosphate group to ADP by a kinase; two phosphoenolpyruvate are converted to pyruvate
by the transfer of their phosphate groups to ADP by another kinase. The first
reaction occurs after the generation of 1,3-bisphosphoglycerate from 3-phosphoglyceraldehyde
and an organic phosphate via a dehydrogenase.
ATP is generated in a following separate step (key
difference from oxidative phosphorylation) by transfer of the high-energy
phosphate on 1,3-bisphosphoglycerate to ADP via the enzyme phosphoglycerate kinase,
generating 3-phosphoglycerate. As ATP is formed of a former inorganic phosphate
group, this step leads to the energy yield of glycolysis. The second
substrate-level phosphorylation occurs later by means of the reaction of
phosphenolpyruvate (PEP) to pyruvate via the pyruvate kinase. This reaction regenerates the ATP
that has been used in the preparatory phase of glycolysis to activate glucose
to glucose-6-phosphate and fructose-6-phosphate to fructose-1,6-bisphosphate,
respectively.
ATP
can be generated by substrate-level phosphorylation in the mitochondrial
matrix, a pathway that
is independent from the protonmotive force, pmf. In the mitochondrial matrix
there are two reactions capable of substrate-level phosphorylation: the
mitochondrial phosphoenolpyruvate carboxykinase (PEPCK), and the succinate-CoA
ligase (SUCL or succinate thiokinase or succinyl-CoA synthetase). Mitochondrial
PEPCK is thought to participate in the transfer of the phosphorylation
potential from the matrix to cytosol and vice versa. The enzyme is a
heterodimer, being composed of an invariant alpha subunit encoded by SUCLG1,
and a substrate-specific beta subunit, encoded by either SUCLA2 or SUCLG2. This
dimer combination results in either an ADP-forming succinate-CoA ligase
(A-SUCL, EC 6.2.1.5) or a GDP-forming succinate-CoA ligase (G-SUCL, EC
6.2.1.4). The ADP-forming succinate-CoA ligase is potentially the only matrix
enzyme generating ATP in the absence of a pmf, capable of maintaining matrix
ATP levels under energy-limited conditions, such as transient hypoxia.
Another
form of substrate-level phosphorylation is also seen in working skeletal
muscles and the brain. Phosphocreatine is stored as
a readily available high-energy phosphate supply, and the enzyme creatine
phosphokinase transfers a phosphate from
phosphocreatine to ADP to produce ATP. Then the ATP releases giving chemical
energy.
Apart
from this substrate-level phosphorylation can also be observed in fermentation,
for example, heterolactic
fermentation, butyric acid fermentation, and
propanoic acid fermentation.
The modern views on the biological
oxidation
Biological oxidation is that oxidation which occurs in
biological systems to produce energy.
Oxidation can occur by:
1-Addition of oxygen (less common)
2-Removal of hydrogen (common)
3-Removal of electrons (most common)
Electrons
are not stable in the free state, so their removal form a substance
(oxidation) must be accompanied by their acceptance by another substance
(reduction) hence the reaction is called oxidation-reduction reaction or redox
reaction and the involved enzymes are called oxido-reductases
Redoxpotential
It is the affinity of a substance to
accept electrons i.e. it is the potential for a substance to become reduced. Hydrogen has the lowest redoxpotential (-0.42
volt), while oxygen has the highest redoxpotential (+0.82 volt). The
redoxpotentials of all other substances lie between that of hydrogen and
oxygen.
Electrons are transferred from substances with low redoxpotential to
substances with higher redoxpotential.This transfer of electrons is an energy
yielding process and the amount of energy liberated depends on the
redoxpotential difference between the electron donor and acceptor.
Oxido-reductases
These enzymes
catalyze oxidation-reduction reactions.
They are classified into five groups:
1-oxidases. 2-aerobic dehydrogenises.
3-anaerobic dehydrogenises.
4-hydroperoxidasesand
5-oxygenases.
1. Oxidases
An oxidase
is any enzyme
that catalyzes
an oxidation-reduction
reaction involving molecular oxygen (O2) as the electron acceptor. In these
reactions, oxygen is reduced to water (H2O) or hydrogen
peroxide (H2O2). The oxidases are a subclass
of the oxidoreductases.
OXIDATION-REDUCTION OR REDOX REACTIONS
2. Aerobic Dehydrogenases(FlavoproteinLinked
Oxidases).
The coenzyme of aerobic
dehydrogenasesmay be:
•FMN (Flavinadenine mononucleotide) as
in L-amino acid oxidase.
•FAD (Flavinadenine dinucleotide) as in
D-amino acid oxidase, xanthineoxidase, aldehydedehydrogenaseand glucose
oxidase.
3. Anaerobic Dehydrogenases
Anaerobic dehydrogenasesare further
classified according to their coenzymes into:
•NAD+linked anaerobic dehydrogenasese.g.
a)Cytoplasmicglycerol-3-phosphate
dehydrogenase
b)Isocitratedehydrogenase.
c)Malatedehydrogenase.
d)β-HydroxyacylCoAdehydrogenase.
e)β-Hydroxybutyrate dehydrogenase.
•NADP linked anaerobic
dehydrogenasese.g.
a)Glucose-6-phosphate
dehydrogenase.
b)Malicenzyme.c)Cytoplasmicisocitratedehydrogenase
•FAD linked anaerobic
dehydrogenasese.g.
a)Succinatedehydrogenase.
b)Mitochondrial
glycerol-3-phosphate dehydrogenase.
c)Acy1 CoAdehydrogenase.
4. Hydroperoxidases
These enzymes use
hydrogen peroxide (H2O2) as substrate changing it into water to get rid of its
harmful effects.
They are further
classified into peroxidasesand catalases.
•Peroxidases: These enzymes need
a reduced substrate as hydrogen donorperoxidase
H2O2 + XH2 (reduced substrate)
----------→ X(oxidized substrate)+ 2H2O Example:
-Glutathione peroxidasegets rid of
H2O2from red cells to protect them from haemolysis
Glutathione Peroxidase
H2O2 + 2 G-S H -----------→2H2O +
G-S-S-G
•Catalases:These enzymes act on 2
molecules of hydrogen peroxide; one molecule is hydrogen donor & the other
molecule is hydrogenaccepetor.
2H2O2 +
catalase---------→ 2H2O + O2
Hydrogen peroxide is continuously
produced by the action of aerobic dehydrogenasesand some oxidases. It is also
produced by action of superoxide dismutase on superoxide (O•2). It is removed by
the action of peroxidasesand catalasesto protect cells against its harmful
effects.
5. Oxygenases
These enzymes
catalyze direct incorporation (addition) of oxygen into substrate.
They are further
classified into dioxygenasesand monooxygenases.
A. Dioxygenases(true oxgenases)
These enzymes catalyze direct
incorporation of two atoms of oxygen molecule into substrate e.g. tryptophan
pyrrolase, homnogentisicacid dioxygenase, carotenaseand
β-hydroxyanthranilicacid dioxygenase.
Dioxygenase
A + O2 → AO2
B. Mono-oxygenases(pseudo-oxygenases;
hydroxylases; mixed: function oxygenases)
AH + O2+ XH2 ------→A-OH
+ H2O + X
Fuctionsof cytochromeP450
Functions of microsomalcytochromeP450
1-It is important for detoxicationof
xenobioticsby hydroxylation. e.g. insecticides,carcinogens,mutagensand drugs.
2-It is also important for metabolism
of some drugs by hydroxylation e.g. morphine, aminopyrine, benzpyrineand
aniline.
drug-H + O2+ XH2 drug-OH + H2O + X→ drug-OH
+ H2O + X
Function of mitochondrial cytochromeP450
1-It has a role in biosynthesis of
steroid hormones from cholesterol in adrenal cortex, testis, ovary and placenta
by hydroxylation
2-It has a role in biosynthesis of bile
acids from cholesterol in the liver by hydroxylation at C26 by 26 hydroxylase.
3-It is important for activation of
vitamin D
CytochromeP450
It is a group of hydroxylaseswhich are
collectively referred to as cytochromeP450.
They are so called because their reduced
forms exhibit an intense absorption band at wavelength 450 nm when complexedto carbon
monoxide.
They are conjugated protein containing
haeme(haemoproteins).
According to their intracellular
localization they may be classified into:
•MicrosomalcytochromeP450.
It is present mainly in the microsomes of liver cells. It represents about 14% of
the microsomalfraction of liver cells.
•Mitochondrial cytochromeP450.
It is present in mitochondria of many
tissues but it is particularly abundant in liver and steroidogenictissues as
adrenal cortex, testis, ovary, placenta and kidney.
Tissue
Respiration.
Tissue respiration is the release of
energy, usually from glucose, in the tissues of all animals, green plants,
fungi and bacteria. All these living things require energy for other processes
such as growth, movement, sensitivity, and reproduction.
The most efficient form of
respiration is aerobic respiration: this requires oxygen. When oxygen is not
available, some organisms can respire anaerobically i.e. without air or oxygen.
Yeast can respire in both ways. Yeast gets more energy from aerobic
respiration, but when it runs out of oxygen it does not die. It can continue to
respire anaerobically, but it does not get so much energy from the sugar. Yeast
produces ethanol (alcohol) when it respires anaerobically and ultimately the
ethanol will kill the yeast.
We can respire in both ways too. Normally we use
oxygen, but when we are running in a race, we may not get enough oxygen into
our blood, so our muscles start to respire anaerobically. Unlike yeast we
produce lactic acid. Of course if we produced alcohol in our muscles it would
make us drunk! Fine thing if you are running away from a predator and you end
up drunk! Making lactic acid is not much better. Lactic acid causes cramp.
Glucose
+ Oxygen = Carbon Dioxide + Water + Energy
This word
equation means: “sugar and oxygen are turned into carbon dioxide and water
releasing energy”. You must memorise the word equation (and the balanced
chemical equation if you want a grade A, B or C). Get help memorising the
equations
Glucose
= Carbon Dioxide + Ethanol + Energy
This word
equation means: “glucose is turned into carbon dioxide and ethanol releasing energy”. You must
memorise this word equation.
Composition of respiratory chain
NADH
dehydrogenase (EC 1.6.5.3) (also
referred to as "NADH:quinone reductase" or "Complex I") is
an enzyme located in the inner mitochondrial membrane
that catalyzes the transfer of electrons from NADH to coenzyme Q (CoQ). It is
the "entry enzyme" of oxidative
phosphorylation in the mitochondria.
NADH
Dehydrogenase is the first enzyme (Complex I) of the mitochondrial electron
transport chain. There are three energy-transducing
enzymes in the electron transport chain - NADH dehydrogenase (Complex I), Coenzyme Q –
cytochrome c reductase (Complex III), and cytochrome c
oxidase (Complex IV). NADH dehydrogenase is the largest
and most complicated enzyme of the electron transport chain..
The reaction of NADH dehydrogenase is:
In
this process, the complex translocates four protons
across the inner membrane per molecule of oxidized NADH, helping to build the electrochemical
potential used to produce ATP.
The
reaction can be reversed - referred to as aerobic succinate-supported NAD+
reduction - in the presence of a high membrane potential, but the exact
catalytic mechanism remains unknown.
Complex
I may have a role in triggering apoptosis. [5] In fact,
there has been shown to be a correlation between mitochondrial activities and programmed
cell death (PCD) during somatic embryo
development.
All redox
reactions take place in the extramembranous portion of NADH dehydrogenase. NADH
initially binds to NADH dehydrogenase, and transfers two electrons to the flavin
mononucleotide (FMN) prosthetic group of complex I,
creating FMNH2. The electron acceptor - the isoalloxazine ring - of
FMN is identical to that of FAD. The electrons are then
transferred through the second prosthetic group of NADH dehydrogenase via a
series of iron-sulfur (Fe-S) clusters, and finally to coenzyme Q
(ubiquinone). This electron flow causes four hydrogen ions to be pumped out of
the mitochondrial matrix. Ubiquinone (CoQ)
accepts two electrons to be reduced to ubiquionol (CoQH2). [2]
FADH
dehydrogenase
In biochemistry, flavin
adenine dinucleotide (FAD) is a redox cofactor
involved in several important reactions in metabolism.
FAD can exist in two different redox states, which it converts between by
accepting or donating electrons. The molecule consists of a riboflavin moiety
(vitamin B2) bound to the phosphate group of an ADP molecule. The flavin group is bound to ribitol, a sugar
alcohol, by a carbon-nitrogen bond, not a glycosidic bond.
Thus, riboflavin is not technically a nucleotide; the name flavin adenine
dinucleotide is a misnomer.[1]
FAD can be reduced to FADH2, whereby it
accepts two hydrogen atoms (a net gain of two electrons):
FAD (fully
oxidized form, or quinone
form) accepts two electrons and two protons to become FADH2
(hydroquinone form). FADH2 can then be oxidized to the semireduced
form (semiquinone) FADH by donating one electron and one proton. The
semiquinone is then oxidized once more by losing an electron and a proton and
is returned to the initial quinone form (FAD).
FAD is an aromatic ring system,
whereas FADH2 is not. This means that FADH2 is
significantly higher in energy, without the stabilization that aromatic
structure provides. FADH2 is an energy-carrying molecule, because,
if it is oxidized, it will regain aromaticity and release all the energy represented
by this stabilization.
The
primary biochemical role of FADH2 in eukaryotes
is to carry high-energy electrons used for oxidative
phosphorylation. Its hydrogens remain in the
mitochondrial matrix, whilst FAD is tightly bound to a dehydrogenase enzyme
i.e. the second protein complex in the oxidative phosphorylation chain. FAD is
a prosthetic group in the
enzyme complex succinate
dehydrogenase (complex II) that oxidizes succinate to fumarate in the
eighth step of the citric acid cycle. The
high-energy electrons from this oxidation are stored momentarily by reducing
FAD to FADH2. FADH2 then reverts to FAD, sending its two
high-energy electrons through the electron
transport chain; the energy in FADH2 is
enough to produce 1.5 equivalents of ATP[2] by oxidative
phosphorylation. Another metabolic source of FADH2 is beta oxidation, where FAD
serves as a coenzyme to acyl CoA
dehydrogenase. A flavoprotein
is a protein that contains a flavin moiety, this may be in the form of FAD or
FMN (Flavin
mononucleotide) . There are many flavoproteins
besides components of the succinate dehydrogenase complex, including α-ketoglutarate dehydrogenase
and a component of the pyruvate
dehydrogenase complex.
Ubiquinones
These molecules are also known as coenzyme Q or mitoquinones. They are
involved in electron transport in mitochondrial preparations playing an
essential role in the oxidation of succinate or NADH via the cytochrome system.
They serves not only as a coenzyme but also, in their reduced forms, as
antioxidants. They are synthesized de novo in all animal tissues and cannot
thus be regarded as vitamins. Ubiquinones are present in all aerobic organisms,
plants, animals (the name ubiquinone was proposed with reference to their
ubiquitous occurrence) and bacteria, but are absent from Gram-positive
eubacteria and the archaebacteria. They
were discovered by the Morton's group in animal fat but their quinonoid
structure was revealed by Crane two years later in extracts from beef heart
mitochondria.
The
compound had a 2,3-dimethoxy-5-methylbenzoquinone nucleus and a side chain of
10 isoprenoid units and
was referred to as coenzyme Q 10 . Later, homologues with 6, 7, 8 and 9 units
were isolated from other organisms, bacteria or higher organisms. The main form
in man has 10 units but in rat has 9 units. Another system of nomenclature is
used: ubiquinone(x) in which x designates the total number of carbon atoms in
the side chain, it can be a multiple of 5.
Ubiquinones accept one electron and are transformed into semiquinone radicals
(UQH°) or two electrons to give ubiquinol (UQH2)
Coenzyme Q is
reducible by sodium dithionite or borohydride to its hydroquinone form, and can
in turn be reoxidized to the quinone by Ag2O or more slowly by oxygen. The
absorption spectra of the two forms are shown below. The quinone form has a
strong absorption band at 275 nm which disappears in the reduced form.
Cytochromes are, in
general, membrane-bound (i.e. inner mitochondrial membrane) hemeproteins
containing heme
groups and are primarily responsible for the generation of ATP
via electron transport.
They are found either as monomeric proteins
(e.g., cytochrome c)
or as subunits
of bigger enzymatic complexes that catalyze redox
reactions.
Cytochromes were
initially described in 1884 by MacMunn as respiratory pigments (myohematin or
histohematin).[1]
In the 1920s, Keilin
rediscovered these respiratory pigments and named them the cytochromes, or
“cellular pigments”, and classified these heme proteins, on the basis of the
position of their lowest energy absorption band in the reduced state, as
cytochromes a (605 nm), b (~565 nm), and c
(550 nm). The UV-visible spectroscopic signatures of hemes are still used
to identify heme type from the reduced bis-pyridine-ligated state, i.e., the
pyridine hemochrome method. Within each class, cytochrome a, b,
or c, early cytochromes are numbered consecutively, e.g. cyt c,
cyt c1, and cyt c2, with more recent
examples designated by their reduced state R-band maximum, e.g. cyt c559.[2]
The
heme
group is a highly-conjugated ring system (which allows its electrons
to be very mobile) surrounding a metal ion, which readily interconverts between
the oxidation states. For many cytochromes, the metal ion present is that of iron,
which interconverts between Fe2+ (reduced) and Fe3+ (oxidised)
states (electron-transfer
processes) or between Fe2+ (reduced) and Fe3+ (formal,
oxidized) states (oxidative processes). Cytochromes are, thus, capable of performing
oxidation and reduction.
Because the cytochromes (as well as other complexes) are held within membranes
in an organized way, the redox
reactions are carried out in the proper sequence for maximum efficiency.
In the
process of oxidative phosphorylation,
which is the principal energy-generating process undertaken by organisms, other
membrane-bound and -soluble complexes and cofactors are involved
in the chain of redox reactions, with the additional net effect that protons (H+)
are transported across the mitochondrial inner membrane. The resulting transmembrane proton gradient
([protonmotive force]) is used to generate ATP, which is
the universal chemical energy currency of life. ATP is consumed to drive cellular
processes that require energy (such as synthesis of macromolecules, active
transport of molecules across the membrane, and assembly of flagella).
Several kinds of cytochrome exist and can be
distinguished by spectroscopy, exact
structure of the heme group, inhibitor sensitivity, and reduction potential.
Three types of cytochrome are distinguished by their
prosthetic groups:
Type |
Prosthetic group |
The
definition of cytochrome c is not
defined in terms of the heme group. There is no "cytochrome e," but
there is a cytochrome f, which is
often considered a type of cytochrome c.
In mitochondria and chloroplasts, these
cytochromes are often combined in electron
transport and related metabolic pathways:
Cytochromes |
Combination |
a and a3 |
Cytochrome
c oxidase ("Complex IV") with
electrons delivered to complex by soluble cytochrome c
(hence the name) |
b and c1 |
Coenzyme Q - cytochrome c reductase
("Complex III") |
b6 and f |
A completely
distinct family of cytochromes is known as the cytochrome
P450 oxidases, so named for the characteristic Soret peak formed by
absorbance of light at wavelengths near 450 nm when the heme iron is
reduced (with sodium dithionite) and
complexed to carbon monoxide. These
enzymes are primarily involved in steroidogenesis
and detoxification.
Respiratory chain
The system of
mitochondrial enzymes and redox carrier molecules which ferry reducing
equivalents from substrates to oxygen are collectively known as the electron
transport system, or the respiratory chain. This system captures the free energy available
from substrate oxidation so that it may later be applied to the synthesis of
ATP. Many respiratory chain components were first identified in crude
homogenates through their spectral properties, which frequently change when a
carrier is oxidised or reduced. Fractionation of mitochondria in the presence
of mild detergents or chaotropic salts dissected the respiratory chain into
four large multi-subunit complexes containing the principal respiratory
carriers, named Complex 1 to Complex 4.
These
substantial protein "icebergs" float in the sheet of inner membrane lipids,
often presenting one face to the mitochondrial matrix
and another to the inter - membrane space.
Many of their components have now been isolated in a relatively pure form.
Other membrane bound enzymes such as the energy linked transhydrogenase (ELTH)
are also present which fulfil ancillary roles.
The main
components participate in the approximate order of their redox potentials, and the
bulky complexes are linked by low molecular weight mobile carriers which ferry
the reducing equivalents from one complex to the next. Except for succinate
dehydrogenase (complex 2) all these complexes pump protons from the matrix
space into the cytosol as they transfer reducing equivalents (either hydrogen
atoms or electrons) from one carrier to the next. The diagram above shows the
flow of reducing equivalents in purple, and movement of the positively charged
protons in red. Proton pumping is an arduous task which creates substantial pH
and electrical gradients across the mitochondrial inner membrane. These protons
eventually re-enter the matrix space via the F1 ATPase, driving the
synthesis of ATP as they return.
The number of
protons and the number of positive charges crossing the inner membrane need not
necessarily agree for each individual transmembrane protein, although the
accounts must balance for the whole ensemble. This discrepancy is illustrated
on the diagram above, and is explained in greater detail below.
Electrons
flow through the electron transport chain to molecular oxygen; during this flow,
protons are moved across the inner membrane from the matrix to the
intermembrane space. This model for ATP synthesis is called the chemiosmotic
mechanism, or Mitchell hypothesis. Peter Mitchell, a British
biochemist, essentially by himself and in the face of contrary opinion,
proposed that the mechanism for ATP synthesis involved the coupling between
chemical energy (ATP) and osmotic potential (a higher concentration of protons
in the intermembrane space than in the matrix). The inner membrane of the mitochondrion
is tightly packed with cytochromes and proteins capable of undergoing redox
changes. There are four major protein-membrane complexes.
Complex I and Complex II direct electrons to coenzyme Q. Complex I, also
called NADH-coenzyme Q reductase, accepts electrons from NADH. The NADH
releases a proton and two electrons. The electrons flow through a flavoprotein
containing FMN and an iron-sulfur protein. First, the flavin coenzyme (flavin
mononucleotide) and then the iron-sulfur center undergo cycles of reduction and
then oxidation, transferring their electrons to a quinone molecule, coenzyme
Q (see Figure ).
Complex
I is capable of transferring protons from the matrix to the intermembrane space
while undergoing these redox cycles. One possible source of the protons is the
release of a proton from NADH as it is oxidized to NAD, although this is not
the only explanation. Apparently, conformational changes in the proteins of
Complex I also are involved in the mechanism of proton translocation during
electron transport.
|
Complex
II, also known as succinate-coenzyme Q reductase, accepts electrons from succinate
formed during the TCA cycle. Electrons flow from succinate to FAD (the
flavin-adenine dinucleotide) coenzyme, through an iron-sulfur protein and a
cytochrome b550 protein (the number refers to the wavelength where
the protein absorbs), and to coenzyme Q. No protons are translocated by Complex
II. Because translocated protons are the source of the energy for ATP
synthesis, this means that the oxidation of a molecule of FADH2
inherently leads to less ATP synthesized than does the oxidation of a molecule
of NADH. This experimental observation also fits with the difference in the
standard reduction potentials of the two molecules. The reduction potential of
FAD is -0.22 V, as opposed to -0.32 V for NAD.
Coenzyme Q is
capable of accepting either one or two electrons to form
either a semiquinone or hydroquinone form. Coenzyme Q is not
bound to a protein; instead it is a mobile electron carrier and can float
within the inner membrane, where it can transfer electrons from Complex I and
Complex II to Complex III.
Complex
III is also known as coenzyme Q-cytochrome c reductase. It accepts
electrons from reduced coenzyme Q, moves them within the complex through two
cytochromes b, an iron-sulfur protein, and cytochrome c1. Electron
flow through Complex II transfers proton(s) through the membrane into the
intermembrane space. Again, this supplies energy for ATP synthesis. Complex III
transfers its electrons to the heme group of a small, mobile electron transport
protein, cytochrome c.
Cytochrome
c transfers its electrons to the final electron transport component, Complex
IV, or cytochrome oxidase. Cytochrome oxidase
transfers electrons through a copper-containing protein, cytochrome a, and
cytochrome a3, and finally to molecular oxygen. The
overall pathway for electron transport is therefore:
|
||
|
|
The
number n is a fudge factor to account for the fact that the exact
stoichiometry of proton transfer isn't really known. The important point is
that more proton transfer occurs from NADH oxidation than from FADH2
oxidation.
A theory
postulated by the biochemist Peter Mitchell in 1961 to describe ATP synthesis
by way of a proton electrochemical coupling is called chemiosmotic
hypothesis.
Accordingly, hydrogen ions
(protons) are pumped from the mitochondrial matrix to the intermembrane space
via the hydrogen carrier proteins while the electrons are transferred along the
electron transport chain in the mitochondrial inner membrane. As the hydrogen
ions accumulate in the intermembrane space, an energy-rich proton gradient is
established. As the proton gradient becomes sufficiently intense the hydrogen
ions tend to diffuse back to the matrix (where hydrogen ions are less) via the
ATP synthase (a transport protein). As the hydrogen ions diffuse (through the
ATP synthase) energy is released which is then used to drive the conversion of
ADP to ATP (by phosphorylation).
Chemiosmotic Hypothesis in a
simple form
In the 1960s,
ATP was known to be the energy currency of life, but the mechanism by
which ATP was created in the mitochondria
was assumed to be by substrate-level
phosphorylation. Mitchell's chemiosmotic hypothesis
was the basis for understanding the actual process of oxidative
phosphorylation. At the time, the biochemical
mechanism of ATP synthesis by oxidative phosphorylation was unknown.
Mitchell
realised that the movement of ions across an electrochemical
membrane potential could provide the energy needed to produce
ATP. His hypothesis was derived from information that was well known in the
1960s. He knew that living cells had a membrane potential;
interior negative to the environment. The movement of charged ions across a
membrane is thus affected by the electrical forces (the attraction of positive
to negative charges). Their movement is also affected by thermodynamic
forces, the tendency of substances to diffuse from regions
of higher concentration. He went on to show that ATP synthesis was coupled to
this electrochemical
gradient.
His
hypothesis was confirmed by the discovery of ATP synthase,
a membrane-bound protein that uses the potential energy of the electrochemical
gradient to make ATP.
The passage
back occurs via a specific proton channel. This passage is coupled to
ATP-synthesis, using the potential energy of the proton gradient for the
formation of the third phosphate bond of ATP.
We can now
calculate the end result of glucose degradation: the oxidation is coupled to a
decrease of the free energy; 686 kcal/mol (= 2881 kJ/mol) are obtained by the
complete oxidation of glucose. How much of this energy can the cell use?
1. Six
mol ATP per mol glucose are generated (substrate chain phosphorylation). This
is because all steps after the breaking down of fructose-1,6-phosphate have to
be counted twice (once for each of the two resulting C3 molecules),
so it is 3 x 2 ATPs. Of these six ATPs, two are needed to start glycolysis. That leaves four.
2.
During the course of glycolysis up to
acetyl-CoA, 2 x 2 NADH + H+ are generated. An additional 3 x 2 NADH
+ H+ and 1 x 2 FADH2 are produced in the citric acid
cycle. One NADH + H+ gives three, one FADH2 two ATPs when
fed into the respiratory chain. This sums up to 34 ATPs plus the 4 ATPs of
glycolysis. A total of 38 mol ATP are thus gained by the cell's degradation of
one mol glucose. Since each energy-rich bond of ATP contains 7.3 kcal/mol (=
-30.6 kJ/mol), the 38 ATP equal 277 kcal/mol (ca 1163 kJ/mol). This is 40.6% of
the theoretically possible gain. The other 59.4 percent are set free as heat.
This is a very high percentage compared to the gain of technical machines like
steam or petrol engines that is around or below 20 percent.
The ATP synthase
enzymes have been remarkably conserved through evolution. The bacterial enzymes
are essentially the same in structure and function as those from mitochondria
of animals, plants and fungi, and the chloroplasts of plants. The early
ancestory of the enzyme is seen in the fact that the Archaea have an enzyme
which is clearly closely related, but has significant differences from the
Eubacterial branch. The H+-ATP-ase found in vacuoles of the
eukaryote cell cytoplasm is similar to the archaeal enzyme, and is thought to
reflect the origin from an archaeal ancestor.
In most systems, the ATP synthase
sits in the membrane (the "coupling" membrane), and catalyses the
synthesis of ATP from ADP and phosphate driven by a flux of protons across the
membrane down the proton gradient generated by electron transfer. The flux goes
from the protochemically positive (P) side (high proton electrochemical
potential) to the protochemically negative (N) side. The reaction catalyzed by
ATP synthase is fully reversible, so ATP hydrolysis generates a proton gradient
by a reversal of this flux. In some bacteria, the main function is to operate
in the ATP hydrolysis direction, using ATP generated by fermentative metabolism
to provide a proton gradient to drive substrate accumulation, and maintain
ionic balance.
ADP + Pi
+ nH+P <=> ATP + nH+N
Because the structures seen in
EM, the subunit composition, and the sequences of the subunits appeared to be
so similar, it had been assumed that the mechanisms, and hence the
stoichiometries, would be the same. In this context, the evidence suggesting
that the stoichiometry of H+/ATP (n above) varied depending on
system was surprising. Values based on measure ATP/2e- ratios, and H+/2e-
ratios had suggested that n was 3 for mitochondria, and 4 for chloroplasts, but
these values were based on the assumption of integer stoichiometries. Although
all the F1F0-type ATP-synthases likely had a common
origin, both the assumption that the stoichiometries are the same, and that n
is integer, are called into question by emerging structural data (see below).
The structure of the soluble (F1)
portion of the ATP synthase from beef heart mitochondria has been solved by
X-ray crystallography. The pictures below are from Abrahams, J.P., Leslie,
A.G., Lutter, R. and Walker, J.E.
(1994) Structure at 2.8 Å resolution of F1-ATPase from bovine
heart mitochondria
The ATP synthase operates through
a mechanism in which the three active sites undergo a change in binding affinity
for the reactants of the ATP-ase reaction, ATP, ADP and phosphate, as
originally predicted by Paul Boyer. The change in affinity
accompanies a change in the position of the g-subunit relative to the a, b-ring, which involves a rotation of the one relative to the other. In the
direction of ATP synthesis, the rotation is driven by a flux of H+
down the proton gradient, through a coupling between the g-subunit, and the c-subunit of FO. This rotation has now been
demonstrated experimentally.
Respiratory control
The dependence of oxidative phosphorylation on ADP
reveals an important general feature of this process: Respiration is
tightly coupled to the synthesis of ATP. Not only is ATP synthesis
absolutely dependent on continued electron flow from substrates to oxygen, but
electron flow in normal mitochondria occurs only when ATP is being synthesized
as well. This regulatory phenomenon, called respiratory control, makes
biological sense, because it ensures that substrates will not be oxidized
wastefully. Instead, their utilization is controlled by the physiological need
for ATP.
In most
aerobic cells the level of ATP exceeds that of ADP by 4- to 10-fold. Respiration
depends on ADP as a substrate for phosphorylation. When ATP is consumed at high
rates, accumulation of ADP stimulates respiration, with concomitant activation
of ATP resynthesis. Conversely, in a relaxed and well-nourished cell, ATP
accumulates at the expense of ADP, and the depletion of ADP limits the rate of
both electron transport and its own phosphorylation to ATP. Thus, the
energy-generating capacity of the cell is closely attuned to its energy
demands.
Experimentally, respiratory control is
demonstrated by following oxygen utilization in isolated mitochondria. In the
absence of added substrate or ADP, oxygen uptake, caused by oxidation of
endogenous substrates, is slow. Addition of an oxidizable substrate, such as
glutamate or malate, has but a small effect on the respiration rate. If ADP is
then added, however, oxygen uptake proceeds at an enhanced rate until all of
the added ADP has been converted to ATP, and then oxygen uptake returns to the
basal rate. This stimulation of respiration is stoichiometric; that is,
addition of twice as much ADP causes twice the amount of oxygen uptake at the
enhanced rate. If excess ADP is present instead of oxidizable substrate, the
addition of substrate in limiting amounts will stimulate oxygen uptake until
the substrate is exhausted.
Two mechanisms of the control of respiration and ATP synthesis in
mitochondria according to the utilization of energy (ATP). The first mechanism of
respiratory control is based on the proton motive force Δp across the inner mitochondrial
membrane (grey). Activation of the ATP-synthase (blue) by ADP, taken up via the
ATP/ADP carrier (margenta), decreases Δp which in consequence stimulates the three proton pumps of the
respiratory chain (complexes I, III and IV). For simplicity, only complex IV
(cytochrome c oxidase) and its
substrate (cytochrome c) are
shown in green and red, respectively. The second mechanism of respiratory
control is based on the intramitochondrial ATP/ADP ratio. High ATP/ADP ratios
inhibit cytochrome c oxidase
activity allosterically. Uptake of ADP decreases the intramitochondrial ATP/ADP
ratio accompanied by exchange of bound ATP by ADP at the matrix domain of
subunit IV of cytochrome c
oxidase, with subsequent stimulation of respiration
Maintenance of respiratory
control depends on the structural integrity of the mitochondrion.
Disruption of the organelle causes electron transport to become uncoupled from
ATP synthesis. Under these conditions, oxygen uptake proceeds at high rates
even in the absence of added ADP. ATP synthesis is inhibited, even though
electrons are being passed along the respiratory chain and used to reduce O2 to
water.
Uncoupling
of respiration from phosphorylation can also be achieved chemically. Chemical
uncouplers such as DNP
or FCCP
act by dissipating the proton gradient. Addition of an uncoupler to
mitochondria stimulates oxygen utilization even in the absence of added ADP. No
phosphorylation occurs under these conditions because there is no ADP to be
phosphorylated.
The
phenomenon of respiratory control is the subject of today's studio
exercise. An oxygen electrode may be used to record [O2]
in a closed vessel (diagram p. 804). Electron transfer, e.g., from NADH to O2,
is monitored by recording the rate of disappearance of O2. At
right is an idealized representation of an oxygen electrode recording while
mitochondria respire in the presence of Pi, along with an electron
donor (e.g., succinate, or a substrate of a reaction that will generate NADH).
The dependence of respiration rate on availability of ADP, the substrate for
the ATP Synthase, is called respiratory control. The respiratory
control ratio is the ratio of slopes after and before ADP addition (b/a).
The P/O ratio is the moles of ADP added, divided by the moles of
O consumed (based on c) while phosphorylating the added ADP.
Chemiosmotic
explanation of respiratory control:
Electron transfer is obligatorily
coupled to H+ ejection from the matrix. Whether this coupled
reaction is spontaneous depends on the pH and electrical gradients.
Reaction |
Free energy change |
e- transfer (e.g., NADH to O2) |
a negative value* |
H+ ejection from
the matrix |
a positive value that varies
with the H+ gradient** |
e-
transfer coupled to H+ ejection |
algebraic sum of the above |
*DGo' = - nFDEo' = -218 kJ/mol, for
transfer of 2 e- from NADH to O2.
** For ejection of one H+
from the matrix:
DG = RT ln ([H+]cytosol/[H+]matrix)
+ F DY = 2.3 RT (pHmatrix - pHcytosol) + F DY
In the absence of ADP, H+
cannot flow back to the matrix through Fo. The pH and electrical
gradients (DpH & DY) are maximal. As respiration with outward H+ pumping proceeds,
the free energy change for H+ ejection (positive DG) increases and approaches the magnitude of that for electron transfer
(negative DG). When the
coupled reaction becomes non-spontaneous, respiration stops. This is
referred to as a static head. In fact there is usually a low rate
of respiration in the absence of ADP, attributed to H+ leaks. Protons
pumped out are carried by the uncoupler back into the mitochondrial matrix,
preventing development of a pH or electrical gradient.
Inhibitors of tissue respiration
Chemiosmosis can be disrupted by a variety of
chemicals. In oxidative phosphorylation, some of these inhibitors are quite
infamous:
The
electron transport chain was determined by studying the effects of particular
inhibitors.
Rotenone
is a common insecticide that strongly inhibits the electron transport of
complex I.
Rotenone
is a natural product obtained from the roots of several species of plants.
Tribes in certain parts of the world beat the roots of trees along riverbanks
to release rotenone into the water which paralyzes fish and makes them easy
prey.
Amytal
is a barbiturate that inhibits the electron transport of complex I. Demerol is
painkiller that also inhibits complex
Antibiotic.
Induces apoptosis, which is not prevented by the presence of Bcl-2. Inhibits
mitochondrial electron transport specifically between cytochromes b and c1. All
three of these complex I inhibitors block the oxidation of the Fe-S clusters of
complex I.
2-Thenoyltrifluoroacetone
and carboxin specifically block electron transport in Complex II
Antimycin
A is an antibiotic that inhibits electron transfer in complex III by blocking
the transfer of electrons between Cyt bH and coenzyme Q bound at the QN site.
Antibiotic. Induces apoptosis, which is not prevented by the presence of Bcl-2.
Inhibits mitochondrial electron transport specifically between cytochromes b
and c1.
Cyanide,
azide and carbon monoxide all inhibit electron transport in Complex IV. The all
inhibit electron transfer by binding tightly with the iron coordinated in Cyt a
This complex oxidizes cytochrome c and also reduces O2 to H2O.
Remember that cytochromes have heme cofactors -- this is important in our
discussion of cyanide and azide. Cytochrome c is a soluble
protein and also is a mobile carrier. Other inhibitors of cytochrome c
oxidase will not be discussed here, but are important biologically, such as
sulfide, formate, and nitric oxide.
Azide
and cyanide bind to the iron when the iron is in the ferric state. Carbon
Monoxide binds to the iron when it is in the ferrous state. Cyanide and azide
are potent inhibitors at this site which accounts for there acute toxicity.
Carbon monoxide is toxic due to its affinity for the heme iron of hemoglobin.
Animals carry many molecules of hemoglobin, therefore it takes a large quantity
of carbon monoxide to die from carbon monoxide poisoning.
CO
competes with oxygen for binding to the reduced form of cytochrome c
oxidase. Once bound to the cytochrome oxidase, oxygen cannot attach, and
electron transport is stopped. CO is a colorless, tasteless,
non-irritating toxic gas. When inhaled, the toxic gas enters the
bloodstream, depriving the heart and brain of the oxygen necessary to function
correctly. Sensing the body's need for more oxygen, the victim's heart rate increases
to pump more blood to the body's organs. If a person continues to inhale CO, he
or she faces the risk of breathing difficulty, cardiac trauma, brain damage,
coma and even death.
CN-,
like azide, binds to the iron atom of oxidized cytochrome, preventing binding
of oxygen. Again, since cytochrome oxidase is inhibited, oxygen
metabolism is prevented and thus so is energy generation. Cyanide has
long been known as a poison, sometimes used in warfare. As mentioned
above, lethal doses cause death in 15 minutes.
They
all cause similar toxic repercussions (with the exception of Carbon Monoxide,
which can bind to Hemoglobin causing your body to be unable to bind oxygen
properly and causing you to suffocate);
However, these three also cause chemical suffocation. They bind a protein in
our electron transport chain (cytochrome c oxidase). The electron transport
chain is our body's best way to create energy in the form of ATP. Without it,
we literally have next to no energy. The electron transport chain is the reason
we have pain in our muscles when we work out--if our body doesn't have oxygen
in those tissues and can't make energy, we make lactic acid, which causes our
muscles to hurt/burn. Anyway, cyanide, azide and carbon monoxide bind this
protein (cytochrome c oxidase) in the electron transport chain. Doing this
causes all of the electrons to stop transferring and no energy to be made. This
causes cell death. The first place to experience cell death from cyanide, azide
and carbon monoxide is your central nervous system--which is made up of your
brain and spinal cord.
So the first thing to die when someone dies of cyanide, azide or carbon
monoxide poisoning?
The
coupling between electron transport and oxidative phosphorylation depends on
the impermeability of the inner mitochondrial membrane to H+translocation. The
only way for protons to go from the intermembrane space to the matrix is
through ATP synthase. Uncouplers uncouple electron transport from oxidative
phosphorylation. They collapse the chemiosmotic gradient by dissipating protons
across the inner mitochondrial membrane. All of the uncouplers shown to the
left, collapse the pH gradient by binding a proton on the acidic side of the
membrane, diffusing through the inner mitochondrial membrane and releasing the
proton on the membranes alkaline side.
Uncouplers
of oxidative phosphorylation stimulate the rate of electron flow but not
ATP
synthesis.
(a)At
relatively low levels of an uncoupling agent, P/O ratios drop somewhat, but the
cell can compensate for this by increasing the rate of electron flow; ATP levels
can be kept relatively normal. At high levels of uncoupler, P/O ratios approach
zero and the cell cannot maintain ATP levels.
(b)
As amounts of an uncoupler increase, the P/O ratio decreases and the body
struggles to make sufficient ATP by oxidizing more fuel. The heat produced by
this increased rate of oxidation raises the body temperature. The P/O ratio is
affected as noted in (a).
(c)
Increased activity of the respiratory chain in the presence of an uncoupler
requires the degradation of additional energy stores (glycogen and fat). By
oxidizing more fuel in an attempt to produce the same amount of ATP, the
organism loses weight. If the P/O ratio nears zero, the lack of ATP will be
lethal
2,4-Dinitrophenol,
dicumarol and carbonyl cyanide-p-trifluorocarbonyl-cyanide methoxyphenyl
hydrazone (FCCP) all have hydrophobic character making them soluble in the
bilipid membrane. All of these decouplers also have dissociable protons
allowing them to carry protons from the intermembrane space to the matrix which
collapses the pH gradient. The potential energy of the proton gradient is lost
as heat DNP is a chemical uncoupler of electron transport and oxidative phosphorylation.
DNP
permeabilizes the inner mitochondrial membrane to protons, destroying the
proton gradient and, in doing so, uncouples the electron transport system from
the oxidative phosphorylation. In this situation, electrons continue to pass
through the electron transport system and reduce oxygen to water, but ATP is not
synthesized in the process. The compound, trifluorocarbonylcyanide
phenylhydrazone (FCCP), is also an
uncoupler.
The phenolic
group of DNP is usually dissociated at intracellular pH. However, a DNP
molecule that approaches the inner mitochondrial membrane from the outside
becomes protonated (because the pH is lower there). Protonation increases the
hydrophobicity of DNP, allowing it to diffuse into the membrane and, by
mass action, to pass through. Once inside, the higher pH of the matric
deprotonates the phenolic hydroxyl again. Thus, DNP has the effect of
transporting H+ back into the matrix, bypassing the F0 proton
channel and thereby preventing ATP synthesis.
The
link between electron transport and ATP synthesis is below. (a) In the presence
of excess phosphate and substrate and intact mitochondria, oxygen is consumed
only when ADP is added. When all of the added ADP has been converted into ATP,
electron transport stops and oxygen consumption ceases. (b) The addition of
2,4-dintrophenol uncouples electron transfer from ATP synthesis. The oxygen is
completely consumed in the absence of ADP. Endogenous Uncouplers Enable
Organisms to Generate Heat
.
The
uncoupling of oxidative phosphorylation from electron transport generates heat.
Hibernating animals and newborne animals (including human beings) contain brown
adipose tissue. The adipose tissue is brown due to the high mitochondria
content of the tissue. An endogenous protein called thermogenin uncouples ATP
synthesis from electron transport by opening up a passive proton channel
(UCP-1) through the inner mitochondrial membrane. The collapse of the pH
gradient generates heat. An uncoupling protein
(also called thermogenin) is produced in brown adipose tissue of newborn
mammals and hibernating mammals .
This protein
of the inner mitochondrial membrane functions as a H+
carrier. The uncoupling protein blocks development of a H+
electrochemical gradient, thereby stimulating respiration. The free energy
change associated with respiration is dissipated as heat. This
"non-shivering thermogenesis" is costly in terms of respiratory energy
unavailable for ATP synthesis, but it provides valuable warming of the
organism.
Valinomycin
combines with K ions to form a complex that passes through the inner
Valinomycin is a potent antibiotic which acts as a
potassium (K+) ionophore. Induces K+ conductivity in
cell membranes. Also active in vitro against Mycobacterium
Tuberculosis, and as an apoptosis inducer.
Valinomycin is obtained from the cells of several Streptomyces
strains, among which "S. tsusimaensis" and S. fulvissimus.
It is a member of the group of natural neutral ionophores because it does not
have a residual charge. It consists of enantiomers D- and L-valine (Val),
D-hydroxyvaleric acid and L-lactic acid. Structures are alternately bound via
amide and ester bridges. Valinomycin is highly selective for potassium ions
over sodium ions within the cell membrane. It functions as a potassium-specific
transporter and facilitates the movement of potassium ions through lipid
membranes "down" an electrochemical potential gradient. The stability
constant K for the potassium-valinomycin complex is 106 and for the
sodium-valinomycin complex only 10. This difference is important for
maintaining the selectivity of valinomycin for the transport of potassium ions
(and not sodium ions) in biological systems.
Oligomycin is a natural antibiotic isolated from Streptomyces
diastatochromogenes which inhibits mitochondrial H+-ATP
synthase. It is primarily found to act as an inhibitor of mitochondrial
respiration and swelling. This antibiotic is widely used as an
inhibitor of oxidative phosphorylation.1-2
Oligomycin inhibits the H+- ATP-synthase by binding to the
Oligomycin sensitivity-conferring protein (OSCP) at the F(o) subunits 6
and 9 which are found in the stalk of the F1F0-ATPase
complex. This binding blocks the proton conductance and inhibits the synthesis
of mitochondrial ATP.3-4 Because of its activity, it can also be
used to reduce the number of parameters (such as ER Ca2+
release, exocytotoxicity and apopotosis) which are affected by mitochondrial
depolarization.
Oligomycin, at high concentrations may also inhibit the plasma membrane
Na+-K+-ATPase. Interaction of Oligomycin with the Na+
occlusion site on the extracellular side of Na/K-ATPase, delays Na+
release to the extracellular side without inducing a conformational change.
Although Oligomycin stimulated Na+ binding to Na+/K+-ATPase,
it inhibited Na+/Na+ exchange, and did not affect
either Na+-dependent AD/-ATP exchange or K+-dependent
phosphatase activity.6-16
Proton
Motive Force Drives Transport The primary purpose of the proton gradient is togenerate
ATP by oxidative phosphorylation. The potential energy of the gradient can also
be used for active transport. The inner mitochondrial membrane is impermeable
to charged molecules.
ATP-ADP
Translocase There are two specific systems to transport ADP and Pi into the
mitochondrial matrix. A specific transport protein ATP-ADP translocase enables
ATP and ADP to transverse the inner mitochondrial membrane. The transport of
ADP in and ATP out are coupled. ADP only enters the matrix if ATP exits or vice
versa. ATP-ADP translocase has a single nucleotide binding site which binds ADP
and ATP with equal affinity. Due to the negative electrostatic charge of the
matrix, ATP-is bound on the N phase of the membrane because it has greater
negative charge than ADP.
Pentachlorophenol
(PCP) acts in a similar way to DNP. It was widely used
as a biocide, especially in pallet board manufacture as a fungicide, but is now
banned by the Biocidal Products Directive, because of its extreme toxicity and
environmental persistence.
DNP
PCP
Ways of energy usage in the organism.
o
1. From the smallest, single-celled
organism to the biggest and most complex mammals--including people--all living
things require energy for life. It's easy enough to understand that we and
other animals eat. Things get a little more puzzling when we think about fungi,
which absorb their food as organic molecules, from the surrounding environment.
Where do those molecules come from? Furthermore, where does the food come from
that we humans convert to energy? At the most basic level, all energy traces
back to plants. Plants are the basis of all the world's food systems, and their
unique ability to make organic materials from sunlight--called
photosynthesis--is what sustains nearly every other life form on the planet.
o
The powerhouse of energy production
in all plants is called a chloroplast. More than a million of these handy
devices occur in every quarter-inch of a leaf. They contain the pigment called
chlorophyll that makes most leaves green--and drives photosynthesis. The
reaction isn't all that complicated, as far as chemical reactions go. The
chloroplasts take in carbon dioxide, sunlight and water. They release oxygen
and a bit less water than they took in. The conversion of carbon dioxide to
oxygen is one life-sustaining function that plants perform for Earth and all of
its life. But plants do something equally as important when they keep a third
product behind: glucose, the sugar that sustains the plants---and anything, in
turn, that eats the plants.
o
In cellular respiration, glucose is
broken down by the removal of its hydrogen atoms. That process releases energy
in the form of electrons, negatively charged particles that fuel all of a
cell's other work in later reactions. So, plants make the glucose and
everything down the line---from plant-eaters to the carnivores that eat
them---break the glucose down again, and use its energy. That's the simple
story. Of course, life is rarely so simple, and there are exceptions to every
rule. Every so often, a new discovery comes along about living things that use
a non-living substance other than sunlight to make energy--like ammonia, or
even sulfur. These less-common organisms can harness electrons from chemical
sources instead of the sun. More amazing life forms have the potential to be
discovered at any time, anywhere on our planet---or beyond.
Cytochrome
P450
The
active site of cytochrome P450 contains a heme
iron center. The iron is tethered to the P450 protein via a thiolate
ligand derived from a cysteine
residue. This cysteine and several flanking residues are highly conserved in
known CYPs and have the formal PROSITE
signature consensus pattern [FW] - [SGNH] - x - [GD] - {F} - [RKHPT] - {P} - C
- [LIVMFAP] - [GAD].[7]
Because of the vast variety of reactions catalyzed by CYPs, the activities and
properties of the many CYPs differ in many aspects. In general, the P450 catalytic cycle
proceeds as follows:
1.
The substrate binds to the active
site of the enzyme, in close proximity to the heme group, on the side opposite
to the peptide chain. The bound substrate induces a change in the conformation
of the active site, often displacing a water molecule from the distal axial
coordination position of the heme iron,[8]
and sometimes changing the state of the heme iron from low-spin to high-spin.[9]
This gives rise to a change in the spectral properties of the enzyme, with an
increase in absorbance at 390 nm and a decrease at 420 nm. This can
be measured by difference spectrometry and is referred to as the
"type I" difference spectrum (see inset graph in figure). Some
substrates cause an opposite change in spectral properties, a "reverse type I"
spectrum, by processes that are as yet unclear. Inhibitors and certain
substrates that bind directly to the heme iron give rise to the type II
difference spectrum, with a maximum at 430 nm and a minimum at 390 nm
(see inset graph in figure). If no reducing equivalents are available, this
complex may remain stable, allowing the degree of binding to be determined from
absorbance measurements in vitro
2.
The change in the electronic state of
the active site favors the transfer of an electron from NAD(P)H via cytochrome P450 reductase
or another associated reductase[11]
This takes place by way of the electron transfer chain, as described above,
reducing the ferric heme iron to the ferrous state.
3.
Molecular oxygen binds covalently to
the distal axial coordination position of the heme iron. The cysteine ligand is
a better electron donor than histidine, which is normally found in
heme-containing proteins. As a consequence, the oxygen is activated to a
greater extent than in other heme proteins. However, this sometimes allows the
iron-oxygen bond to dissociate, causing the so-called "uncoupling
reaction", which releases a reactive superoxide radical and interrupts the
catalytic cycle.
4. A
second electron is transferred via the electron-transport system, from either cytochrome
P450 reductase, ferredoxins, or cytochrome b5, reducing the
dioxygen adduct to a negatively charged peroxo group. This is a short-lived
intermediate state.
5.
The peroxo group formed in step 4 is
rapidly protonated twice by local transfer from water or from surrounding
amino-acid side-chains, releasing one water molecule, and forming a highly
reactive species commonly referred to as P450. This highly reactive
intermediate was not "seen in action" until 2010,[12]
although it had been studied theoretically for many years. P450 Compound 1 is
most likely a iron(IV)oxo (or ferryl)
species with an additional oxidizing equivalent delocalized over the porphyrin
and thiolate ligands. Evidence for the alternative perferryl iron(V)-oxo [8]
is lacking.
6.
Depending on the substrate and enzyme
involved, P450 enzymes can catalyze any of a wide variety of reactions. A
hypothetical hydroxylation is shown in this illustration. After the product has
been released from the active site, the enzyme returns to its original state, with
a water molecule returning to occupy the distal coordination position of the
iron nucleus.
S: An
alternative route for mono-oxygenation is via the "peroxide shunt":
Interaction with single-oxygen donors such as peroxides and hypochlorites can
lead directly to the formation of the iron-oxo intermediate, allowing the
catalytic cycle to be completed without going through steps 2, 3, 4, and
C: If carbon
monoxide (CO) binds to reduced P450, the catalytic cycle is interrupted. This
reaction yields the classic CO difference spectrum with a maximum at
450 nm.
Free Radicals: Atoms contain a nucleus, and electrons move around the nucleus, usually
in pairs. A free radical is any atom or molecule that contains one or more
unpaired electrons. The unpaired electrons alter the chemical reactivity
of an atom or molecule, usually making it more reactive than the corresponding
non-radical. However, the actual chemical reactivity of radicals varies
enormously.
The hydrogen radical, the same as a hydrogen atom), which contains 1 proton and
1 electron (therefore unpaired), is the simplest free radical. Free-radical
chain reactions are often initiated by removal of from other molecules. A superscripted dot is
used to denote free radicals.
Free radicals and chain reaction:
Most molecules in the body are not
radicals. Hence any reactive free radical generated is likely to react with a non-radical.
When a free radical reacts with a non-radical, a free-radical chain reaction
results and new radicals are formed. Figure 1 shows two important reactions of
this type. Attack of reactive radicals on membranes or lipoproteins starts
lipid peroxidation, which is particularly implicated in the development of
atherosclerosis. If hydroxyl radicals are generated close to DNA, they can
attack the purine and pyrimidine bases and cause mutations. For example,
guanine is converted into 8-hydroxyguanine and other products.
Free radicals and diseases: Free radicals are
capable of damage biomolecules, provoke immune response, activate oncogens,
cause atherogenesis and enhance ageing process. However, in healthy conditions
nature has endowed human body with enormous antioxidant potential. Subtle
balance exists between free radical generation and antioxidant defence system
to cope with oxidative stress by various enzymes and vitamins at cellular level
which prevent the occurrence of disease. However, factors tilting the balance
in favour of excess free radicals generation lead to widespread oxidative
tissue damage and diseases. Therefore, trouble starts when there is an excess
of free radicals and the defence mechanism lags behind. Overwhelming production
of free radicals in response to exposure to toxic chemicals and ageing may
necessitate judicious antioxidant supplement to help alleviate free radical
mediated damage.
|
Free radicals
with pollutants: Highly
reactive molecules called free radicals can cause tissue damage by reacting
with polyunsaturated fatty acids in cellular membranes, nucleotides in DNA, and
critical sulfhydryl bonds in proteins. Free radicals can originate endogenously
from normal metabolic reactions or exogenously as components of tobacco smoke
and air pollutants and indirectly through the metabolism of certain solvents,
drugs, and pesticides as well as through exposure to radiation.
There is some evidence that
free radical damage contributes to the etiology of many chronic health problems
such as emphysema, cardiovascular and inflammatory diseases, cataracts, and
cancer. Defenses against free radical damage include tocopherol (vitamin E),
ascorbic acid (vitamin C), beta-carotene, glutathione, uric acid, bilirubin,
and several metalloenzymes including glutathione peroxidase (selenium),
catalase (iron), and superoxide dismutase (copper, zinc, manganese) and
proteins such as ceruloplasmin (copper). The extent of tissue damage is the
result of the balance between the free radicals generated and the antioxidant
protective defense system. Several dietary micronutrients contribute greatly to
the protective system. Based on the growing interest in free radical biology
and the lack of effective therapies for many of the chronic diseases, the
usefulness of essential, safe nutrients in protecting against the adverse
effects of oxidative injury warrants further st
WHAT
CAN STOP A FREE RADICAL? (ANTIOXIDANTS):
A free radical is stopped when the electron difference (gaining or losing an
electron) is corrected. Molecules that can correct the electron
difference are called Antioxidants. The process of damage by Free
Radicals is called oxidation (think rust on metal or the browning of
a cut apple), and the process that prevents it is anti-oxidation, and the
molecules which do the prevention are called Antioxidants. Antioxidants
are found in dark colored vegetables and fruit and in dietary supplements. The
life of a free radical has three stages: the initiation stage, propagation
stage, and finally the termination stage. Free radicals are terminated or
neutralized by nutrients (antioxidants), enzymatic mechanisms, or by
recombining with each other.