RESPIRATION
Biological Oxidation:
All living organisms, without
any exception require free chemical energy for their multitude of physiological
functions. In most of the cases this
energy is supplied in the form of molecules.
However in many cases, GTP, CTP, phosphocreatine, NADH + H*, Arginine
phosphate, PEP etc., also supply the energy for specific reactions.
The ATP molecule is made up of
Adenosine and three phosphates. Three
phosphates are linked by high energy or anhydride bonds. These high energy bonds are called swigle
bonds. Break down of these terminal
bonds by specific enzymes yield 7.2 K.cals/mol –1 of free energy. This energy can be utilized for various
functions like transmission of nerve stimulus, muscular contraction,
osmoregulation, maintenance of body temperature, transpiration of materials
etc. At the same time the energy present
in these terminal bonds can be transferred to other molecules to build up
macromolecules like carbohydrates, proteins, nucleic acids, fats and other reserve
food substances.
Plants and animals use various
stored food materials as their energy source: starch, glycogen (it is animal
starch), fats and proteins act as the main energy sources. Organisms, during the course of evolution,
probably 3 or 3.5 billion years ago, with the onset of O2 liberation by
photosynthetic plants, have evolved a unique mechanism, by means of which
various food sources are systematically oxidized in a controlled degradative
process, in which the energy that is released is stored in the high energy
chemical bonds of ATPs and other molecules.
Thus they are made available for cellular metabolism. This process is often termed as “Biological
Oxidation”.
Biological oxidation per se
involves stepwise oxidation of reserve food materials in a controlled but
stepwise process. Depending upon the utilization of oxygen as an oxidant, this
process can be classified into anaerobic and Aerobic oxidation. Most of the organisms perform Aerobic
oxidation, but some bacteria, yeast and even some higher animals exhibit
Anaerobic oxidation, of which aerobic process is more complicated and yields
energy 8-10 times the anaerobic process.
Biological
Oxidation Process-Aerobic:
Irrespective of plants and
animals, aerobic process requires mitochondria as energy producing
organelles. These structures are found
in almost all organisms except, blue green algae, bacteria, Mycoplasma and
viruses. The number, position, shape and
size of these structures vary from organisms to organisms, and from
organ to organ and the
metabolic status has a significant say in the mitochondrial activity.
Biochemical
Pathway of aerobic respiration:
Reserve food materials like
Carbohydrates, Fats and Proteins are first subjected to hydrolysis by specific
enzymes. As a result simpler compounds
like Glucose, Keto acids and Acetyl Co-A are released into the Cytoplasm. From this pool various components are drawn
into the main oxidative pathway. For
example, carbohydrates, like starch or sugars are degraded into their
respective Glucose units by either starch phosphorylase or Amylases. Similarly proteins are degraded by various
proteinases into amino acids. Fats are
subjected to B-Oxidation to produce Acetyl-Co.A molecules.
The entire process of
biological oxidation takes places in 3 steps, at 3 different sites. The first step is Glycolysis. It takes place in cytoplasm. It does not require oxidation for its
function. The second step is Krebs or
TCA cycle. The glycolytic products are
drawn into mitochondrial matrix in which Krebs cycle operates with the
utilization of glycolytic products. The
final or the third step occurs in /on the inner mitochondrial membranes. Some of Krebs cycle products are used up by
the membranes and they are subjected to a process called terminal oxidation or
oxidative phosphorylation. This step requires
oxygen for its function.
Glycolysis an anaerobic pathway (EMP Pathway):
Glucose molecules are directly
utilized in the first step of this pathway.
There are many biochemical steps and require specific enzymes. In the first phase of this process 1 or 2
molecules of ATP are consumed as priming reactions. On the other hand in the latter part of these
reactions energy rich products are produced.
Glycolysis takes place exclusively in cytoplasm.
Glucose is primed by ATP with
the help of an enzyme-Hooknose. If the
glucose is already phosphorylated in the first position, as in the case of
starch-phosphorylation, Glucose-1, P is converted to Glucose-6-P by
phosphoglucomutase.
Glucose is isomerized to
Fructose-6-P by an enzyme phosphogluco-isomerase.
Fructose –6 – P is further
energized by another molecule of ATP by phosphofructo kinase.
Highly energized 6 carbon
Fructose diphosphate is acted upon by aldolase and it is split into 3 carbon
compounds like phospho-glyceraldehde and Dihydroxyacetone–P. These products are interconvertible by Triose
isomerase.
Phosphoglyceraldehyde is
oxidized to 1, 3 – diphosphoglyceric acid by Triose phosphate
dehydrogenase. One molecule of NADH + H*
is produced.
Energy rich 1, 3 –
diphospho-glycerate is converted to 3-Diphosphoglycerate by phosphoglyceryl
kinase. A molecule of ATP is synthesized
by substrate phosphorylation.
Phosphate in 3rd position is
shifted to 2nd position by phospho glycero-mutase.
A molecule of water is removed
from 2-PGA by Enolase.
Energy rich 2-PEP donates
energy rich phosphate bond to ADP to form ATP by pyruvate kinase.
This is another case of
substrate phosphorylation.
In the above glycolytic process
the two steps are of ATP energy utilizing reactions. There are three reactions in which high
energy bonds are transferred and conserved in NADH + H and ATP molecules. In this process one molecule of 62 carbon
glucose yields 2 molecules of 3 Carbon compounds like PGAKD and DiHAP which are
interconvertible, but PGALD is subjected to dehydrogenation reactions. Thus whatever happens of PGALD also happens
to DiHAP through the conversion of it into PGALD. Furthermore one PGALD at the end of
glycolysis yields 1 NADH + H and 2 ATP molecules. So one molecule of glucose on complete
glycolysis yields 2 NADH + H, 4 ATP and 2 Pyruvate molecules. This process is common for both aerobic and
anaerobic respiration.
Alcoholic Fermentation or Partial Biological Oxidation:
Anaerobic respiration:
Certain organisms like yeasts
(under anaerobic conditions), bacteria like Clostridium (an obligate anaerobic
bacterium), Lactic acid bacteria and muscular respiration in higher animals are
some of the examples where cells respire anaerobically. This process is a partial biological
oxidation in which a part of the glucose is oxidized intra-molecularly and 2
molecules of CO2 are released. And the
ultimate products are ethanol or lactate.
This is named after the discoverer of this process as EMP pathway. (Embden, Mayeroff, Parnas pathway). This process has been commercially exploited
very well in brewing and chemical industries.
In this pathway glucose is
subjected to series of oxidative processes and 2 molecules of pyruvate and
formed. The entire process is similar to
glycolytic pathway. But pyruvate
produced at the end of glycolysis is either converted to ethanol (in yeasts) or
to lactate (in the case of lactate bacteria or Muscular respiration).
Alcoholic
Fermentation: Yeast cells are capable of
oxidizing glucose and such substrates either in the presence of oxygen or
absence of oxygen. Under anaerobic conditions yeasts use glucose and convert it
into pyruvate by glycolytic reactions.
However, Pyruvate thus produced is subjected to decarboxylation first
and then reduction.
In this process, only two
carbon atoms of 6 carbon glucose are released by intra-molecular oxidation to 2
CO2. Two molecules of NADH + H* produced
in glycolytic process are used up in the reduction of acetaldehyde to
ethanol. Thus the net gain is just 2
ATPs per Glucose molecule.
Lactate
Formation: Lactate formation by anaerobic process is
found to operate in not only lactate bacteria (responsible for curdling of
milk) but also in muscular respiration.
As in the case of alcoholic fermentation, pyruvate generated by
glycolytic process is directly reduced by lactate dehydrogenase in which 2 NADH
+ H* molecules produced during glycolytic steps are used up. Again, the net gain is just 2 molecules of
ATP / glucose. It is interesting to note
that though human beings are highly evolved, they have retained evolutionarily
primitive biochemical process like anaerobic fermentation. This process is more predominant, when human
beings are subjected to violent exercises. The fatigue, the man experiences
during such activities is due to accumulation of Lactate in muscles which may
result in cramps.
This is due to the deficit
supply of oxygen. However, man recovers,
when he regains sufficient supply of oxygen by utilizing the stored lactate.
Kerbs
Cycle: Mitochondrial Process
A molecule of glucose on
glycolysis yields two molecules of 3 carbon compounds called pyruvate. Pyruvates thus synthesized in cytoplasm are
transported across the inner membrane into mitochondrial matrix. During this process pyruvate is subjected to
a series of complex steps,
in which decarboxylation and
dehydrogenation reactions take place and Acetyl-Co-A is liberated into the
matrix. The enzyme that is responsible
is a multiple enzyme complex called pyruvate dehydrogenase.
Acetyl Co-A, as it is
transported into mitochondrial fluid, it is drawn by a group of enzymes into
Krebs cycle (it is named after the discoverer of this cycle i.e. Hans Krebs).
The 2 carbon Acetyl Co.A
combines with a Ketonic 4 carbon oxaloacetate and undergoes a condensation
process by an enzyme called citrate synthetase to form 6 carbon citrate.
Citrate in turn undergoes a
successive dehydration and hydration steps to form Isocitrate. The enzyme responsible for this process is
Aconitase.
Then Isocitrate is subjected to
a sequential dehydrogenation and decarboxylation reactions by Isocitrate
dehydrogenase (the Co-enzyme is NAD or NADP).
alphaKetoglurate is further decarboxylated
and dehydrogenated to succinate by L-Ketoglutarate dehydrogenase. In this, a high energy bond is transferred to
GDP + Pi to form GTP which is another case of substrate phosphorylation.
Succinate is again converted to
Fumerate by a Flavoprotein enzyme called Succinate dehydrogenase where the
Co-enzyme is FAD.
Fumerate is then converted to
Malate with addition of one mol of water.
The enzyme responsible is Fumerase.
Malate is subjected to Malate
dehydrogenase reaction to form oxaloacetate.
The Oxaloacetate thus produced
again combines with Acetyl-Co.A to form citrate and Krebs cycle continues.
Krebs cycle which is also
called Tricarboxylic acid (TCA) cycle is very important for, it provides not
only energy rich compound like 4 NADH + H*, 1 FADH2 and 1 GTP but also it
provides some important intermediate compounds for other metabolic pathways.
In total, 2 pyruvates yield 8
molecules of NADH + H* 2 FADH2 and 2 GTP (2 GTP are converted to 2 ATP) and 6 mol.
of CO2.
When glycolysis and Kerbs cycle
are summed up, 1mol of glucose yields 10 NADH + H, 2 FADH2, 6 ATP and 6 CO2. It
is important to note that all 6 carbons of glucose molecule are oxidized, and
so it can be considered as complete oxidations.
Terminal
Oxidation
A
Phenomenon of Oxidative phosphorylation: Reduced products like NADH + H
and FADH2 that are synthesized during glycolytic and Krebs cycle reactions
contain lot of energy. In order to trap
and transfer this bond energy to ATP molecules, the reduced products are taken
up by the enzymes found in the inner membrane and the same are subjected to a
sequential Red-Ox process. The enzymes
that are involved in this process are Fe-S.FMN bond and Fe-S-FAD bond
reductase, NADP reductase Co enzyme Q (a labile molecule), Cyt. B. Oxidase, Cyt
C and Cyt a/a3 oxidase (copper containing enzyme) and these arranged
vectorially and sequentially.
Of these, Fe-FMN accepts
hydrogen from NADH + H, Fe- S-FAD accepts hydrogen from FADH2 and Co-Q accepts
hydrogen from both FMNH2 and FADH2.
However the Cytochrome containing Fe accepts only electrons and H are
released into the inner membrane. When
electrons reach the terminal enzyme Cyt a3, they are transferred to ˝ O3, simltaneoulsy
2H are also accepted by ˝ O2 to form a molecule of water. During this process of Terminal Oxidation,
simultaneous oxidation – reduction reactions occur. Depending upon the red-ox potential of
individual components, energy is released at 3 different sites. This energy is
used up by ATP synthetase enzyme to synthesize ATP from ADP + Pi. Hence, this phenomenon is also called
oxidative phosphorylation.
The most fascinating aspect of
terminal oxidation is the utilization of energy that is released at 3 sites
during the passage of electrons through the respiratory chain. Though the
mechanism of energy coupling had remained unsolved, many theories have been
proposed from time to time. Slater and
Chance proposed chemical coupling theory in 1939 and envisaged that during
electron transport, the energy that is released at the said sites are taken up
by an unknown intermediate compound with high energy bond, then this bond is
transferred to ADP and Pi to form ATP.
So far no one is able to detect or identify such high energy
intermediates. But Peter Mitchell (1961)
came out with an interesting hypothesis called Chemiosmotic hypothesis. He visualized, that the components of
electron transport chain are vectorially arranged (different components at
different sides of the membrane) various components accept NADH + H and FADH2
at different sites from inner surface and extrude proteins H on the outer
surface but the electrons are passed through the electron transport chain. This
extrusion of proteins to outside, builds up a proton gradient or proton motive
force, which is enough to bring about the formation of high energy phosphate
bond between ADP + Pi to form ATP. This theory became very popular and soon it
was widely accepted. Peter Michell was awarded
a Nobel Prize in chemistry in 1978.
However, the opponents of
Mitchell theory started getting new facts which were contrary to the
observation of Peter Mitchell’s supporters.
Boyer in 1974 proposed an alternative theory called conformational
coupling theory.
This was based on the
conformational changes observed during mitochondrial ATP synthesis. Certain Actin like components found in the
inner membrane of mitochondria is found to play a significant role in the
conformation coupling process. Currently
this theory is gaining more support.
Factors
As respiration involves very
many biochemical steps, many factors like water, substrate, oxygen, CO2
temperature etc., control the rate of respiration.
1.
Water: Water being the medium in which all the
cellular components are either suspended or dissolved, the rate of reaction
depend upon the concentration of the water in a given system. If a dry seed is taken for measuring its rate
of reactions depend upon the concentration of the water in a given system. If a dry seed is taken for measuring its rate
of respiration, it is seen that the respiration is at its minimum, this is
because the amount of water found in dry or resting seeds is just 2-8%. Hence movement of molecules is restricted to
a small space, so collisions between the reactants and enzymes become
infrequent and the rate of metabolism will be very low. As the dry seeds are allowed to imbibe water,
its concentration within the cells increase and rate of metabolism,
consequently the rate of respiration increases significantly.
2.
Substrate: Respiration or biological
oxidation virtually depends upon its substrates for the release of energy. Most of the plants / animals store starch/
glycogen as the foods material. Still
there are many organisms, which along with carbohydrates, store oil and
proteins (E.g. Castor bean, ground nut seed, cereal grain etc). When conditions are favorable for
respiration, plants utilize carbohydrates first, and then they utilize fats and
proteins in succession. Whether plants
are utilizing starch, fats or proteins as the substrate for respiration, it can
be determined by measuring Respiratory Quotient or RQ. Respiratory quotient
denotes the ratio between the CO2 released to the oxygen consumed.
CO2 released
RQ
=
-------------------
Q2 consumed
Different food sources exhibit
different R.Q. E.g. R.Q. of starch or carbohydrate is 1. R.Q. of fats or oil is
= 0.7 R.Q. of proteins ranges from 0.7 – 0.9 and organic acid shows 1.2.4.
The rate of respiration further
depends upon the availability of substrates. With the increase in the substrate
concentration the rate of respiration also increases; but beyond certain level
the rate remains constant because respiration depends upon the concentration of
enzymes which generally remain constant in a given set of conditions.
3,
Temperature: Heat is another form of energy
and it provides momentum for the molecules to move. Increase in temperature increases the energy
of the system and the rate of movement of molecules also increases. This increase in the rate of movement of
molecules increases the rate of collision between the respiratory enzymes and
the substrates. Thus the rate of
respiratory enzymes and the respiration is expressed as the function of temperature;
one finds the rate is minimum at lower temperature. With the increase of every 100C the rate doubles
and at a particular range of temperature the rate is maximum. This range of temperature is called the
optimum temperature. This optimum varies
from plant to plant for it is genetically determined. But higher temperature is always fatal
because, enzymes which are functional molecules, breakdown and become
nonfunctional.
4. Oxygen: Aerobes require oxygen for the
oxidation of substrates. The majority of
plants / animals are obligate aerobes.
However plants like yeasts can exist as aerobes as well as anaerobes and
their adaptability to these conditions is remarkable. But these are some bacteria which are
obligate anaerobes and the presence of oxygen is fatal for these organisms.
In aerobes, oxygen acts as one
of the substrate, because oxygen ultimately receives electrons and protons at
the terminal end of electron transport chain.
And this is extremely important.
Any inhibition of this step inhibits the production of ATP and it is
fatal.
In plants, respiration takes
place both at day time as well as at night time. But the respiration is very low at day times
particularly in tissues where cells are engaged in photosynthesis. This is because photosynthesis provides all
the energy that is required for the cellular metabolism.
Generally the increase in O2
concentration enhances the rate of respiration but beyond certain level the rate
remains constant. In facultative
anaerobes like yeasts, when they are respiring anaerobically, if oxygen is
supplied, alcoholic fermentation is completely stopped and the entire process
now switches over to aerobic metabolism.
This effect is called Pasteur’s effect.
During anaerobic process, the number of mitochondria per yeast cell is
as low as one or two. When only oxygen
is provided to such anaerobic cells, there is a sudden spurt in the
multiplication of mitochondria and in about 30 minutes or so the number of
mitochondria and in about 30 minutes or so the number of mitochondria increases
to 100-150 per cell. If such, cells,
which are fully geared up for aerobic respiration are subjected to anaerobic
conditions, one sees a dramatic change of sequence of events, where
mitochondria undergo breakdown and their number gets reduced to 1-2 / cell.
5.
CO2: The oxidation of substrates during respiration
yields CO2 as one of its products.
Normally CO2 diffuses out through transpiration system. However, if the concentration of CO2 builds
up to a greater extent CO2 acts as an inhibitor of respiration and it may have
a fatal effect on living system.
Generally plants do not suffer from the excess of CO2. On the other hand increased CO2 favors
photosynthesis and the yield increases.
6. Inhibitors: Many drugs like DNP, Rotenone, antimycin,
cyanide etc., inhibit respiratory oxidation.
This is because these drugs have specific inhibitory effect on specific
respiratory enzymes. For example,
cyanide binds to cytochrome oxidase and inhibits the transport of electrons to
O2 and stops oxidation which results in the complete inhibition of ATP
production. That is how cyanide causes
death. But some plants like Aeroids are
insensitive to cyanide poisoning, for they have a different mechanism for their
electron transport chain.
7. Internal Factors: The cellular factors like enzyme synthesis, the availability of CO enzymes, and cofactors and other nutrients are very essential for the normal metabolic processes. If any of these factors is either deficient or absent, it prevents the enzymes from doing their normal function and this causes the inhibition of respiration.