Plant Cell Energy Transductions 3

 

Biological Oxidations:

 

Respiration is a process of biological oxidation where various reserve food materials like starch, fats, proteins and their organic compounds are enzymatically converted to simpler products which are ultimately oxidized to CO2 and H2O.  During oxidative steps, the energy present in the chemical bonds in the above said components is transferred to high energy bonds of ATP and other energy rich molecules.  Majority of the organisms living on this planet use ATP as the main source of chemical energy. 

But compounds like GTP, UTP, PEP, CTP, DIPGA, creatine phosphate, etc., are also used by specific energy requiring metabolic pathways or signaling pathways.  Some of the compounds require the inputs of the following energy rich compounds.

 

Glucose is a simple six carbon monosaccharide which contains trapped solar energy in the form of chemical energy.  All living organisms require chemical energy for various biological activities and the same is supplied by the oxidation of glucose or similar carbon compounds including lipids.  Thus glucose is at the hub of energy supplying metabolic pathways.  The stepwise oxidation of glucose not only yields energy rich molecules but also provides many intermediary carbon skeletons for the synthesis of other components, like amino acids, nitrogen bases, nucleotides, lipids, etc. Based on the

 

 

utilization or non-utilization of oxygen, respiratory process is classified into aerobic type and anaerobic type.

 

The mechanism of biological oxidative process varies and it depends upon the organism involved, the metabolic status of the said organism and the availability of oxygen in the immediate environment.  In the presence of oxygen, most of the eukaryotic organisms respire aerobically, but in the absence of it, some like yeast cells resort to anaerobic type of respiration for their survival.  But some organisms are obligate anaerobes.  The anaerobic respiration results in the formation of ethanol and in some cases, lactate is produced.  However the glycolytic pathway of biological oxidation is common for both aerobic and anaerobic processes.  In aerobic reactions, the glycolytic products such as pyruvates are subjected to Kreb’s cycle events and terminal oxidation.  On the other hand, in anaerobic respiration, the glycolytic products are converted to either ethanol or lactate which depends upon the organism involved. A part of the oxidation process takes place in cytoplasm, and the other steps in Mitochondria.

 

 

 

 

 

 

AEROBIC RESPIRATION

 

 

 

 

 

The most important energy source for plants is starch and for animals glycogen or fat, again it depends upon the stage and status of the organism.  But in plants during germination of seeds it can be starch or fatty acid that is stored in them.  To start with, the complex compounds are hydrolyzed to simple forms like glucose or fructose, acetyl coA, or derivative of amino acids, which are drawn into glycolytic pathway that operates in cytosol or drawn into mitochondria.  All the required enzymes are found in cytosol.

 

 

If glucose is the starting material, it stores considerable amount of chemical energy (during photosynthesis) and the same is released in step wise manner and nearly 30 percent of it is captured and stored in ATP chemical bonds and the rest is released as heat.   The process is same in all organisms irrespective of their kinds and forms.

 
Glycolysis

 

This process operates in the cytoplasm but outside the mitochondria.  The enzymes needed for this process are found in cytosol and some of them are associated with the outer surface of the outer membrane of the mitochondria as granular particulates.

The glycolytic process takes place in two phases, i.e. first from glucose to phosphoglyceraldehyde and the second from PGALD to pyruvate.  These reactions are very well regulated by the end products or by the energy status of the cells.

 

To begin with, free glucose is raised to higher state of energy by a phosphorylation reaction with the help of an enzyme called hexose kinase, which utilizes a molecule of ATP.  This reaction is referred to as priming reaction.

 

 

Aldose Glucose (6C)

If starch is utilized, the breakdown product of starch by starch phosphorylase enzyme is glucose 1-P, which inturn is converted to glucose 6-P by phosphoglucomutase.  This enzyme is a regulatory enzyme.

 

Starch + P1 Phosphorylase Glucose 1-(P)

Glucose 1-(P) glucomutase Glucose 6-(P)

 

Other hexoses like galactose, mannose, etc., if available in the cells, are first converted to glucose 6-(P) before they are drawn into glycolytic pathway.

 

Mannose ->   Glucose 6–P

Galactose       ->   Glucose   6-P

Glucose 6-P is then converted to fructose 6-P by phosphoglucose-isomerase.  Immediately, the fructose 6-P is further primed up to fructose 1-6 diphosphate by an enzyme called phospho-fructokinase.  In this step another molecule of ATP is utilized.  This enzyme is allosteric in nature and the cellular ATP level regulates its activity.  If the cellular energy level is high i.e. high content of ATP, the enzyme activity is inhibited.  On the contrary, if the concentration of ATP is low (i.e. more of ADP) the enzyme is active and proceeds in the formation of fructose 1-6 diphosphate.

 

 

 

 

Fructose6P to fructorse1,6-diP is regulated, and it depends upon the availability of ATP.  And regulated by PFK   (phospho fructose kinase) is active and phosphorylates the F6P to F1,6 diP, otherwise PFK remains inactive, whose activity in turn is controlled by specific kinases.

 

 

 

The priming process is like expending the energy initially to push a big rock on the summit of a mountain to such a position, when it is dropped; it just rolls down all along the slop releasing its energy at every step of its movements.  Similarly, further glycolytic reactions of fructose 1-6 diphosphate is a downhill process.  Then fructose 1-6 diphosphate is cleaved into two 3 carbon compounds, called glyceraldehyde 3-phosphate (PGALD) and di hydroxy acetone phosphate (DHAP).  The enzyme that performs this reaction is fructose diphosphate aldolase which can reverse this reaction by aldol condensation. 

Glyceraldehyde 3 phosphate and di hydroxy acetone phosphates are isomers and they can be easily inter-converted by an enzyme named triose phosphate isomerase.

 

Further reactions depends upon the energy status of the cell.  If cells require more energy.  DHAP is first converted to PGALD which is then subjected to the second phase of glycolytic reactions.  On the contrary, if the cellular ATP molecules are found in sufficient quantities, DHAPs are channeled towards glycerol and lipid production.  This part of the reaction is regulated by the ATP and ADP levels of the cells or ATP/ADP ratios.

 

If more energy is required, the second phase of glycolytic process proceeds where glyceraldehyde 3 phosphate is oxidized to 1.3 di phosphoglycerate.  The enzyme glyceraldehyde phosphate dehydrogenase which brings about this reaction utilizes inorganic phosphates and at the same time, its coenzyme NAD gets reduced to energy rich NADH+H.  So a part of energy is stored in NADH+H (the energy held in NADH+H is substantial).

PGALD + Pi + NAD ->1-3 PGA (DiPGA) +NADH2

DiPGA + ADP-> ATP + 3’PGA,

3”PGA -> 2’PGA,

2’PGA-> H2O + PEP

PEP + ADP -> Pyruvate + ATP

 

 

The DIPGA is an energy rich compound and the same is then subjected to substrate phosphorylation by an enzyme called phosphoglycerate kinase.  In this process, the energy rich anhydride phosphate bound is transferred to ADP to produce energy rich ATP molecule.  Note the ATP generated is by substrate phosphorylation.  The resultant product of this molecule is 3’phosphoglyceric acid, which is then subjected to transfer of phosphate group from 3’ position to 2’ position by phosphoglyceromutase.  The product is 2’ phosphoglycerate which is then acted upon by enolase enzyme, where a molecule of water is removed from 2’ carbon position to yield another energy rich compound called phosphoenol pyruvate.

The high energy phosphate bond of phosphoenol pyruvate is then transferred to ADP to produce an ATP by an enzyme called pyruvate kinase.  This reaction also produces the ultimate product of glycolytic reaction called pyruvate.

At the end of glycolytic pathway, one mole of glucose yields 2 moles of pyruvates; 2 moles of NADH+H and 4 moles of ATP.  Initially 2 moles of ATPs are used for priming reactions of the first phase of glycolytic reactions; the net gain of ATP in these glycolytic reactions is 2 moles of ATP for every mole of glucose.

Glucose + 2 ATP + 2 NAD + 4 ADP + 2PI -> 2 Pyruvate

2 NADH + H + 4 ATP + 2 ADP

The fate of the pyruvate depends upon the availability of O2.  In aerobic process the pyruvate is drawn to Kreb’s cycle. Instead, if oxygen is not available, certain organisms like yeasts, and lactate bacteria use pyruvate and produce ethanol and lactate respectively by intramolecular biological oxidation.

Citric acid cycle or Kreb’s Cycle:

Pyruvate a 3 carbon kenotic acid produced at the end of glycolytic pathway is transported from cytosol into mitochondrial matrix, across the mitochondrial membrane. 

 

While in transit pyruvate is oxidized to Acetyl-coA by an very important enzyme complex called pyruvate dehydrogenase. Then, 2C acetyl-coA is drawn into a cyclic biochemical pathway called citric acid cycle.  As the cyclic reactions were elucidated by Han’s Kreb’s, this pathway is also called Kreb’s cycle.

 

The enzyme requires some important vitamins in the form of coenzymes such as NAD, FAD, TPP and Pentothinate.

 

This is a complex of 12 subunits which has been isolated and electro density micrograph is taken.  The same is presented below.

                                     

 

 

 

 

The mitochondrial matrix which fills the internal mitochondrial chambers possesses all the enzymes needed for Kreb’s cycle.  The matrix also contains other enzymes required for β-oxidation of fatty acids, amino acid metabolism, protein metabolism and nucleic acid metabolism.  The mitochondrial chamber is incompletely compartmentalized by the folds developed from the inner membrane called Cristae.

 

 

Pyruvate, a 3 carbon compound is oxidized to two carbon acetyl Co.A.  This is a multistep reaction executed by a multiple enzyme complex called pyruvate dehydrogenase.

 

 

The Pyruvate dehydrogenase complex is regulated by specific kinases and phosphotases.

There are five steps in which 3 enzymes containing 5 coenzymes take part by dehydrogenation and decarboxylation reactions.  Once the substrate, i.e. pyruvate is bound to this multiple enzyme complex, the reaction goes on till the last products produced is  released from the surface of the enzyme and some of the intermediate products remain bound to the enzymes.

 

In the first step, the pyruvate dehydrogenase enzyme removes CO2 from the pyruvate through thiamine pyrophosphate to produce hydroxy ethyl derivate of the thiozole ring of TPP.

 

E1-TPP+ CH3COCOOH  ->    E1-TPP-CH3-CHOH+CO2

 

In the second step, the hydroxy ethyl group is dehydrogenated resulting in the transfer of acetyl group to the sulphur moiety of lipoic acid, a prosthetic group of enzyme called lipoyl acetyl transferase.

 

The third step involves enzymatic transfer of acetyl group from the lipoate to thiol group of co-enzyme A.  The acetyl Co.A formed in this reaction is released from the enzyme surfaces.  During this reaction, lipoate reduces dihydrolipoamide.

 

In the fourth step, lipoamide dehydrogenase removes the hydrogen from dihydrolipoamide, where the co-enzyme FAD gets reduced to FADH2 and lipoid acid is regenerated.

 

In the fifth step, the hydrogen from the E3 FADH2 (still bound to the enzyme) is transferred to NAD to form NADH+H.  Thus, one molecule of pyruvate on dehydrogenation decarboxylation reactions produces one molecule of acetyl Co.A, one molecule of Co2 and one molecule of NADH2.  The enzyme pyruvate dehydrogenase, brings about this multistep reaction is a multiple enzyme complex and it is regulated by Ca2+ and ATP.  These reactions take place while the pyruvate is transferred across the inner mitochondrial membrane.  The carrier for pyruvate is now known to be carnitine phosphate.

 

The acetyl Co.A condenses with another ketonic acid called oxoloacetate (OAA) with the help of citrate synthase.  The product is citric acid, a six carbon dicarboxylic acid.  Citrate synthase is a regulatory enzyme and its activity is regulated by the levels of succinyl Co.A and ATP.  In fact, this acts as a pacemaker.

 

 

 

 

 

 

Then the enzyme aconitase brings about a reversible catalysis of citrate to isocitrate through an intermediate compound called aconitate by adding and removing a molecule of water

 

The isocitrate is then subjected to dehydrogenation and decarboxylation by an enzyme complex called isocitrate dehydrogenase to produce alfa ketogluterate.

 

The coenzyme involved in this reaction is mostly NAD.  Once again, the activity of this enzyme is modulated by the levels of ADP/ADP ratio.

 

Alfa ketogluterate is a key intermediate compound in the cellular metabolism, because it is also used in the synthesis of glutamate and other amino acids.  However, in citric acid cycle.  Alfa ketogluterate is converted to succinate in two steps reactions by an enzyme called alfa ketogluterate dehydrogenase.  The catalytic action of this multiple enzyme complex is similar to that of pyruvate dehydrogenase.  But the interesting reaction involved in this is the formation of high energy bond in succinyl Co.A.  This high energy bond in succinyl Co.A. is used in the substrate phosphorylation of GDP to GTP.

Alpha Ketogluterate -> Succinyl Co A (energy rich bond)

Suucinyl CoA + GTP +P -> Succinate + GTP

The succinate is a 4 carbon dicarboxylic acid.  It is further converted to Fumerate by the action of succinate dehydrogenase, whose coenzyme FAD gets reduced to FADH2.  Malonate acts as the competitive inhibitor for this enzyme.  Then the fumerate is converted to malate by the addition of H2O by an enzyme called Fumerase.

Succinate +FAD-> Fumarate + FADH2

Fumerate + H2O -> Malate

 

Malate is then subjected to dehydrogenation by malate dehydrogenase to produce oxoloacetate.

Malate + NAD -> NADHH + OAA

The OAA thus produced, then combines with another molecule of acetyl Co. A and the citric acid cycle is repeated.

OAA + Acetyl CoA-> Citrate

 

At the end of Kreb’s cycle. 1 mole of glucose yields 6 002, 10 NADH2 and 6 ATPs of which 2 ATPs are used in the first two priming steps of glycolysis.

 

TERMINAL OXIDATION (Oxidative phophorylation)

 

Energy rich compounds like NADH+H and FADH2 that are produced during Kreb’s cycle within mitochondria are drawn into inner cristae membrane, where they are subjected to a series of oxido reduction reactions during which energy is released.  This process is referred to as terminal oxidation. As in this process ADP is phosphorylated to ATP it is also called oxidative phosphorylation.  In this process the chemical energy released during electron transport is coupled for energy rich phosphate unhydride bond formation in ATP.

 

The process of terminal oxidation takes place in series of steps.  The oxido reductases  that are responsible for this process are found in the cristae membranes as specific groups they have different red-ox potential, because of this difference, certain amount of energy is released at different oxido reduction steps.  The energy released at these steps is used in the formation of high energy anhydride bonds in ATP.  Such a process is termed as oxidative phosphorylation.  Oxidation of one mole of NADH2 yields 3 moles of ATPs; and 1 mole of FADH2 yields 2 moles of ATPs.  The unused energy is released in the form of heat.

 

Organization of Electron Transport Chain:

 

The oxido-reductive enzymes are found in the inner membrane of the mitochondria and the same are organized into 5 complexes.  Complex I to complex IV are found buried within the membranes and the complex V is found on the outer surface of the oristae membrane slightly buried, but its head projects out into the mitochondrial matrix and in line with the electron transport chain.

 

Complex I consist of NADH2 dehydrogenase with FMN as the prosthetic group and 16-24 Fe-S protein. 

Complex II is made up of FADH2 dehydrogenase with FAD as the coenzyme and 6-8 FE-3 containing proteins. 

Complex III has heme containing cytochrome b and Cytochrome-C proteins.  Cyt.C is mobile, it is also found perimitochondrial space.  The release of this in to cytoplasm can lead to apoptosis.  In addition, it also possesses 2 Fe-S containing proteins. 

Complex IV consists of Cyt.c & Cyt.a3 oxidase proteins with 2 copper and 2 heme moieties. 

The complex V is made up of F1 protein called ATP synthetase and a protein to secrete Protons.  In addition, it also possesses an oligomycin sensitive conformational protein (OSCP).

 

 

The first four complexes from complex I to complex IV are vectorially disposed within the core of inner mitochondrial membrane,  but the complex V is located on the surface of the inner membrane in such a way, the head i.e. F1 is projected out into the mitochondrial matrix and the basal part is buried in the lipid core of the membrane.  But the OSCP acts as a stalk between Fo and F1 part of the ATP synthase enzyme complex. 

 

 

Such particles are uniformly distributed over the surface of the inner membrane.  However, this complex when it is free from the membrane (probably only) with the head portion, it acts as the hydrolysing ATPase enzyme.  On the contrary, if it is associated with the membrane (with fo & OSCP) it functions as ATP synthetase.  Along with the above said complex they are some other components in the membrane which are free and labile.  In fact, they act as intermediary components between complexes.  They are coenzyme Q and cyt. C.  the coenzyme Q acts as a shuttle between complex I and complex III and complex II and complex II, but not between complex I and II, similarly, cyt. C acts as a shuttle between complex III and complex IV.  During terminal oxidation process, NADH2 reacts with complex I but not with any other complex.  Similarly, FADH2 reacts with complex II only. 

 

MECHANISM OF TERMINAL OXIDATIONS

 

When NADH2 reacts with complex I, NADH2 gets oxidized to NAD and the FMN of complex I gets reduced to FMNH2.  Because of the difference in oxido reduction potential between the oxidant and the reductant.  12.2 K.cals/mole of energy is liberated and the same is used in the formation of ATP by oxidative phosphorylation process.  On the other hand, FADH2 reacts with complex II where the prosthetic group of complex II enzyme i.e. FAD gets reduced to FADH2.  The redox potential between these two is almost equal and the free energy released is nil.

 

 

 

The coenzyme Q which acts as the shuttle between complex I and complex III and complex II and complex III gets reduced to QH2.  The coenzyme Q accepts hydrogen either from FMNH2 or FADH2.  The energy released in this redox reaction is negligible to produce any energy rich bond.  The most interesting step is the reaction between QH2 and complex III, where the reaction results in the separation of H protons and electrons.  Only the electrons are channeled to cyt. B of the complex III and H ions is released into the membrane.  As cyt. B Fe3 + accepts the electron and it gets reduced to Cyt.B fe2+.  In this redox reaction, some amount of energy is released but not sufficient to produce an ATP.  The reduced cyt. B Fe 2+ reacts with oxidized cyt.c1 FE3+.  Where cyt.bfe2 gets oxidized and cyt.c Fe3 gets reduced to cyt.Gfe2.  The energy released in this redox couple is about 9.9 K.cals/mole and the same is used up n the formation of another energy rich ATP molecule.

Then cyt.c1fe2+ reacts with cyt.C fe3+, where cyt.cfe3 gets reduced to cyt.C fe2+ and cyt.c1fe2 gets oxidized to cyt.c1fe3+.  The free energy released in this reaction is not sufficient to produce ATPs.  From cyt.cfe2+ the electrons are transported through cyt.a and cyt.a3.  It is during the redox reactions between cyt. A and cyt.a3, hereby 23.8 K.cals/mole of energy is released and the same is utilized in the generation of ATP.  Finally, the electrons from cyt.a3fe2+ are transferred over to o2, which simultaneously receives 2H+ to produce a water molecule. 

 

 

 

 

It is very important to remember that during electron transportation, only one electron is shuttled between the redox couples at a time, but the flow of electrons is very fast.  However, the synthesis of ATPs is calculated for a pair of electrons flow at any given step.  Thus the oxidation of NADH+H yields 3 ATPS and FADH2 yields 2 ATP.

 

The presence of 3 sites in the electron transport chain at which ATPs generate has been identified by using drugs such as rotenone, antimycin and cyanide.  Based on these studies, it has been identified that rotenone binds to complex I, antimycin binds to complex III and cyanide binds to cytochrome oxidase complex and prevents the electron flow and also prevents ATP formation.  So the sites of ATP formation are between NADH2 and FMN of complex I, Cyt.b and Cyt.c1 of complex III and Cyt.a and Cyt.a3 of the complex IV.

 

 

 

 

 

All in all, one mole of glucose on complete glycolysis, Kreb’s cycle and terminal oxidation yields a total of 40 ATPs of which 2 ATPs are used in the early glycolytic reactions.  So the net gain in terms of ATP production is 38 per mole of glucose.  In actuality 1 mole of glucose possesses 686 K.cals energy out of which 38x 7.2 K.cals of energy is trapped is in the formation of 38 ATPS.  The rest of the energy is released as heat.  Thus the efficiency of aerobic type of biological oxidation in terms of energy conserved is hardly 39.3%, it is not 100%, but energy released as heat is required to maintain the body temperature.  Animals and plant that live in Arctic and Antarctic regions do store lot of fat, during hibernation period they use that fat in oxidative process, where the released energy in electron transport is used. It also uses electron transport process only to release heat rather ATPs

 

Mechanism of ATP Synthesis:

 

Though the energy is released at different sites during the electron transport the mechanism by which the energy is utilized is in the formation of energy rich phosphate bond in ATP is not completely elucidated.  In this text book, three important theories have been discussed.

 

HIGH ENERGY CHEMICAL INTERMEDIATE THEORY

 

The protagonists of this theory namely Slater, Britten chance and others believe that the energy released at specific sites in the electron transport chain is trapped as an high energy bond in an unknown compound called X.  Then the same energy rich bond is transferred to inorganic phosphate,

 

 

 

which is then transferred to Pi and than to ADP to generate ATP.  This concept was invoked, because the formation of high energy bond can be inhibited by the addition of an uncoupler of oxidative phosphorylation called dinitrophenol (DNP), where all the energy released during electron transport is lost in the form heat, interestingly in this process the flow of elctron is not inhibited but ATP synthesis is completely prevented.  This indicates the presence of some intermediate compound or component which acts as the receiver of energy for coupling reactions in generating ATPs.  Unfortunately, the presence of such energy rich substance has not been detected so far.  Hence, this theory is not favored by many scholars in this field.

CHEMIOSMOTIC THEORY

 

Peter Mitchell proposed this theory and it was supported by many.  But there are others who vehemently argue against this theory.  In spite of it, he was awarded a Nobel Prize in 1978, for his significant contribution in the field of bioenergetics; no other field has generated so much controversy and heated debates as in the case of mitochondrial bioenergetics.

 

 

 

 

 

Mitchell’s chemiosmotic hypothesis is based on the assumption that NADH2 and FADH2 are drawn into inner mitochondrial membrane by the electron transport system and then the protons (H+) are released at the outer

surface of the membrane into periplasmic space and the electrons are transported along the electron transport chain.  Though the electrons that are transported along the chain end up in oxygen, the extrusion of positively charged H+ at the outer surface of the inner mitochondrial membrane, builds up greater positive charge outside and negative charge inside the membrane.  The membrane potential thus generated provides a strong proton motive force for the generation of high energy bonds between ADP and Pi, which results in the formation of ATP.  The differential charge is believed to be neutralized by the synthesis of ATP, during which the OH ad H produced are released to outside and inside of the time of anhydride bond formation between ADP and Pi .

 

One of the best evidence that has been presented in favor of this hypothesis is from Jogendorf’s experiments on chloroplasts.  After soaking the isolated chloroplasts to saturation in a buffered medium with pH 4 (acid) in dark, when the said chloroplasts are transferred to another buffered medium with alkaline pH 8.5 containing ADP and Pi, resulted in a burst of ATP synthesis.  This explains how membrane potential with high negative charges and positive charges operating on the opposite surfaces of the membranes can generate energy rich bond between ADP and Pi to synthesize ATPs.

 

 

In spite of overwhelming circumstantial evidences, research workers who were contradicting the chemiosmotic hypothesis developed new and sophisticated microelectrodes to measure the smallest change in the pH in the shortest possible time.  With such new tools and techniques they measured the time, required for the generation of proton motive forces across the mitochondrial membranes and the time required for the synthesis of ATP in mitochondria.  Such studies demonstrated that the time required for the building up of proton motive force to generate ATB molecules runs to a few milliseconds, but in actuality ATPs are synthesized within a few nano seconds after the elctron transport. T he time lag between the generation of sufficient proton motive force across the mitochondrial membranes to generate ATP synthesis , the actual time required for ATP synthesis due to electron transport within the membrane, is found to be a strong evidence against chemiosmotic hypothesis.  In similar experiments people have shown that blocking proton transfer does not prevent the synthesis of ATP into mitochondrial inner membranes. Furthermore, in plant cells, where auxin causes  secretion of H ions outside the plasma membrane to such an extent  the pH at the outer surface of the membrane falls from 6.8 to 4.0.  Though ADP and Pi are available at the site ATPs are not synthesized some of there objections are solid and chemiosmots hypothesis has no answer to them.

 

CONFORMATIONAL CHEMICAL COUPLING THEORY

 

In actuality, the conformational hypothesis was initiated by David Green et al. But later Boyer and others developed a new hypothesis and gave new dimensions to the above said theory.  In Mitochondria, in its resting state when internal membranes are in a relaxed condition, but when mitochondria are fully charged with ATP, the internal cristae membranes show maximum contraction where even ATP synthetase complex with its head and stalk show drastic changes in their morphological conformations. The analysis of internal membrane protein components of mitochondria indicates the presence of some contractile proteins similar to actions.  Furthermore, such proteins are believed to act as semiconductors of electronic energy .

This hypothesis envisages that during the electron flow from high energy state to lower levels, because of the difference in redox potential between the oxidants and reductants of different complex, certain amount of energy is released at different sites.  The same energy is low drawn into some contractile proteins associated with ATP syntheses.  By virtue of energitization of these proteins, the ATP synthetase undergoes conformational change.  As ADP and Pi are already bound to specific sites on the surface of ATP synthetase, the conformational change brings the ADP and Pi together and the energy found in contractile proteins is used up in the formation of energy found in contractile proteins is used up in the formation of energy rich bonds between ADP and Pi.  Once the energy is used up the contractile proteins and ATP syntheses enzymes relax to their almost simultaneously original state, ATP is released from the enzyme surface.  And this process is repeated again and again to produce more ATP molecules.  This process takes place very rapidly.  Hence this hypothesis appears to explain all the observed facts about the mechanism of ATP synthesis.  The explanation is very simplistic and seminal.  Recently the mitochondrial people consider the proton motive force is very important in producing ATP molecules.  Accordingly they consider that the proton binding to ATP synthase, particularly binding and  release of them determines the rate of ATP synthesis.

 

 

 

ANAEROBIC RESPIRATION

 

Respiration without utilizing oxygen or in the absence of oxygen is called anaerobic respiration.  Yeasts and some bacteria respire even in the absence of oxygen and ethanol or lactate is produced as the end products.  Surprisingly, even higher animals exhibit anaerobic respiration under certain conditions where oxygen supply is inadequate.

Yeast cells are capable of performing respiratory processes both aerobically and anaerobically.  Under aerobic conditions, yeast cells possess 150-200 active mitochondria per cell.  On the other hand, if such are subjected to anaerobic conditions, the number of mitochondria reduces dramatically to 1 to 2 per cell.  But with the restoration of aerobic conditions, the original number of mitochondria will be restored.  Thus yeast cells show remarkable adaptations.

 

 

 

During anaerobic respiration, glucose is subjected to same biochemical reaction as in the case of glycolytic steps.  For that matter there is no difference between anaerobic and aerobic glycolytic pathways.  The pyruvates produced the end products of glycolytic reactions, are first decarboxylated and then reduced to ethanol by alcohol dehydrogenase enzymes.  In this process, I mole of glucose yields 2 moles of CO2, 2 moles of ethanol and the net gain in ATP is 2 moles.  This process is also called partial biological oxidation because a part of the glucose is lost as CO2 (Fig. 12.14.).

 

In the case of lactate bacteria, the pyruvate produced at the end of glycolysis is converted to lactate by the action of lactate dehydrogenase activity.  Here, even CO2 is not released.  Hence this kind of respiration is often referred to as intramolecular respiration.  The net gain of ATP in this process is also 2.

 

Even in higher mammals, muscular respiration under inadequate supply of oxygen during intense muscular work, pyruvate is converted to lactate.  The accumulation of lactate in the muscles causes fatigue and pain.

 

 

 

 

 

MEASUREMENT OF RESPIRATION

 

Respiration can be measured quantitatively or qualitatively in terms of either the uptake of oxygen or the liberation of C2 with reference to the substrate used in a given time.  Such studies can be made by using whole cells or parts of the plant body like leaves, floral buds, embryos, etc.  It is also possible to isolate mitochondria from any part or parts of the plant body and the uptake of O2, release of CO2 or the P/O2 ratio can be determined.

 

Instruments like oxygen polarograph, Warburg’s manometers can be used to determine the oxygen uptake, O2 output or P/o2 ratio under various parameters.

 

RESPIRATORY QUOTIENT RQ

 

The ratio between the amount of CO2 evolved and the amount of oxygen consumed by a given weight of tissue in a given time at standard temperature is called respiratory quotient (RQ)

RQ CO2 released / O2 consumed

 

Determination of RQ of a tissue gives valuable information about the rate of respiration and the substrate used.  By just measuring RQ it is possible to find out what substrate is consumed during respiration.  Generally, carbohydrate gives RQ value to be 1, fats 0.7, proteins 0.8 to 0.99 and organic acids like malate, tartarate oxalate, etc., more than 1.

 

Carbohydrates:  Glucose: C6H12O6 + 602 -> 6C02 + 6HO2

 

 

 

FACTORS THAT CONTROL RESPIRATION:

 

Temperature:  most of the biochemical processes mediated by enzymes or not are dependent on the temperature of the media.  This is because temperature provides energy for the movement of molecules within the medium which in turn has an important effect on the frequency of collision between reactants and the enzyme.  Thus the rate of the reaction is controlled by the prevailing temperature.  Majority of the plants exhibit 25-30 degree as the optimal temperature but some plants living near hot springs show 40C-60C degree as the optimal temperature.  Higher temperature affects the biochemical process because enzymes get denatured and loose their functional abilities.

 

OXYGEN

 

The availability of oxygen ultimately determines the respiratory process which may be aerobic or anaerobic type.  The concentration of oxygen in the surrounding medium has a profound effect on the rate of respiration because oxygen acts as the oxidant, where it receives electrons during terminal oxidation.  Increase and decrease in the concentration results in the increase and decrease I the rate of respiration.  However, some cells respire both under aerobic and anaerobic conditions.  But in certain cases like yeast cells, if the cells that are respiring under aerobic conditions, transferred to anaerobic media, the process of respiration immediately stops.  Interestingly, the number of mitochondria in the yeast cell go down from 200 to 1 or 2 per cell.   But when such cells are transferred to aerobic condition, within the next 30 minutes or so, mitochondria regenerate and multiply and restore the normal process of respiration.  On the contrary, if the cells that are growing under strict anaerobic condition are suddenly transferred to aerobic condition the rate of respiration goes down instead of increase in the rate.  This effect is called Pasteur’s effect.  This peculiar behavior has been explained as due to the effect of oxygen on glycolytic pathway.  This is a transitory effect, with time the normal rate of respiration is restored.  However, in leaves, increase in oxygen initiates photo respiration.

 

GLUCOSE REPRESSION

 

Similar to oxygen’s effect, in yeast cells, higher concentration of glucose brings about the repression of respiratory process; such a process is called glucose repression.  During glucose repression, the mitochondria are degraded by the lysosomal activities.  Glucose repression persists for a period of 60-90 minutes, and then mitochondria regenerate and restore the normal process of respiration.  This effect is virtually similar to that of catabolitic repression fund in bacteria. 

 

CARBON DIOXIDE

 

In animals higher concentration of CO2 has deleterious effect, because it brings about a change in the pH in the body fluid.  In plants, however, CO2 has no direct effects.  In daytimes, most of the CO2 available is used in photosynthesis, but at nights the concentration of CO2 increases.  As a result of it, stomata close, thereby it affects the availability of oxygen to plants but it does not impose any ill effects on the plants.  Nonetheless, CO2 brings about an inhibitory effect on ethylene induced cyanide insensitive respiration.

 

PHYTOHORMONES:

 

Plant hormones such as IAA and GA generally enhance the rate of respiration due to certain changes in membrane permeability and also due to the activation of some respiratory enzymes.  At later stage, as phytohormones activate and enhance transcription and translation, the rate of respiration also increases because of the grater demand for the energy.  As most of the growth promoting plant hormones accelerate cellular metabolism leading to growth, the rate of respiration increases.  On the other hand, hormones like ethylene, which induces fruit ripening, enhances the cyanide insensitive respiration to climactric state.  This brings about many metabolic changes leading to softening and sweetening of the fruits.  Even wounding of plant parts induces and enhances the rate of respiration because of the activity of traumatic acid.  Due to the action of traumatic acid, the surface cells where the wound is inflicted, become meristematic and start cell divisions.  So the cells require more energy for cellular metabolism and multiplication.  That is why the rate of respiration increases at region of wound.  However, growth inhibiting hormones like abscisic acid (ABA) lowers respiratory activity is plant cells.

 

PLANT ORGANS

 

The whole plant cannot be taken as a unit for measuring or determining respiratory activity.  Unicellular plants can be used for such measurements.  But higher plants with complex organizations exhibit different rates of respiration activity at different stages of growth and development.  While germinating seeds, young leaves, apical buds, root tips, floral buds, developing inflorescence and such others exhibit higher rate of respiration, dormant seeds, dormant buds, mature fruits and seeds show minimum respiratory activity.

 

OXIDATIVE PENTOSE PHOSPHATE PATHWAY

 

This pathway is called by other names like phosphogluconate pathway or hexose monophosphate shunt.  B.L. Horeker and E. Racker have made extensive studied on this pathway.  Both plant and animal cells exhibit this pathway in cytoplasm outside the mitochondria.  However its activity depends upon the plants requirements.  In cellular metabolism, pentose sugars are required for the synthesis of nucleotides; NADPH2s are required for various reductive processes like fatty acid synthesis, reductive animation, nitrate or nitrite reduction etc.  Under such demanding conditions, hexose monophosphate shunt is activated and it provides the required intermediary compounds.  Thus this pathway is found to be very useful in providing many intermediary products and reducing power required for other metabolic processes.

 

The overall reactions of this pathway are as follows:-

6 Glucose 6-P+12 NADP+7H2) – 5 glucose 6-P+6CO2+12 NADPH2+Pi

 

For every 6 moles of glucose phosphates used, 6 moles of CO2, 12 moles of NADPH2 and one mole of phosphates are released, thereby 1 mole of glucose is completely oxidized to 6 moles of CO2  but 5 moles of glucose are recovered.  Hence, equation can be rewritten as

Glucose 6-P + 12 NADP + 7H2O –> 6 CO2 + 12 NADPH2+Pi

 

To begin with, glucose 6-P is converted to phosphogluconlactone by the activity of glucose 6, P dehydrogenase enzyme which is called as Zwischenferment or Horecker Racker enzyme.  In this process, one NADHP2 is produced.

 

The phosphogluconate, thus produced, is immediately hydrated to produce 6phosphogluconate.  Then this is subjected to successive dehydrogenation and decarboxylation steps to produce ribulose 5,Ps by the enzyme activity of phosphogluconate dehydrogenase enzymes.  Then ribulose 5P is converted to ribose 5, P by isomerase enzymes.  Once ribose and ribulose are produced they are subjected to cyclic reactions.

The following are the diagrams presented by various authors for you appreciation or comments.