Krebs Cycle and ETS

 

           

(1) Oxidative decarboxylation / Formation of acetyl CoA.

(2) Kreb's cycle / TCA cycle / Citric acid cycle.

(3) Electron Transport System

 

(1) Oxidative decarboxylation of pyruvic acid : If sufficient O₂ is available, each 3-carbon pyruvate molecule \[\left( C{{H}_{3}}COCOOH \right)\] enters the mitochondrial matrix  where its oxidation is completed by aerobic means. It is called gateway step or link reaction between glycolysis and Kreb's cycle. The pyruvate molecule gives off a molecule of CO₂ and releases a pair of hydrogen atoms from its carboxyl group (-COOH), leaving the 2 carbon acetyl group \[(C{{H}_{3}}CO)\]. The reaction is called oxidative decarboxylation, and is catalyzed by the enzyme pyruvate dehydrogenase complex (decarboxylase, TPP, lipolic acid, transacetylase, Mg²⁺) . During this reaction, the acetyl group combines with the coenzyme A (CoA) to form acetyl coenzyme A with a high energy bond\[\left( C{{H}_{3}}CO-CoA \right)\]. Most of the free energy released by the oxidation of pyruvate is captured as chemical energy in high energy bond of acetyl coenzyme A. From a pair of hydrogen atoms released in the reaction, to electrons and one H⁺ pass to\[NA{{D}^{+}}\], forming, \[NAD{{H}^{+}}{{H}^{+}}\]. The NADH forms 3 ATP molecules by transferring its electron over ETS  described ahead.

Decarboxylation and dehydration :

        

Image pending

 

**TPP=Thiamine pyrophosphate

 **LAA=Lipoic acid amide

Acetyl CoA is a common intermediate of carbohydrate and fat metabolism. Latter this acetyl CoA from both the sources enters Kreb's cycle. This reaction is not a part of Kreb's cycle.

(2) Kreb's cycle / TCA cycle / Citric acid cycle

(i) Discovery : This cycle has been named after the German biochemist in England Sir Hans Krebs  who discovered it in 1937. He won Noble Prize for this work in 1953. Krebs cycle is also called the citric acid cycle after one of the participating compounds.

(ii) Site of occurrence : It takes place in the mitochondrial matrix.

(iii) Kreb’s cycle

 

 

 

 

          

(iv) Enzymes of Kreb's cycle

 

Step	Enzyme	(Location in mitochondria)	Coenzyme(s) and cofactor (s)	Inhibitor(s)	Type of reaction catalyzed
(a)	Citrate synthetase	Matrix space	CoA	Monofluoro-acetyl- CoA	Condensation
(b)	Aconitase	Inner membrane	\[F{{e}^{2+}}\]	Fluoroacetate	Isomerization
(c)	Isocitrate dehydrogenase	Matrix space	\[NA{{D}^{+}},\text{ }NAD{{P}^{+}},\text{ }M{{g}^{2+}},\text{ }M{{n}^{2+}}\]	ATP	Oxidative decarboxylation
(d)	\[\alpha \]-ketoglutarate dehydrogenase complex	Matrix space	TPP,LA,FAD,CoA, NAD+	Arsenite,Succinyl-CoA, NADH	Oxidative decarboxylation
(e)	Succinyl-CoA synthetase	Matrix space	CoA	-	Substrate level phosphorylation
(f)	Succinate dehydrogenase	Inner membrane	FAD	Melonate, Oxaloacetate	Oxidation
(g)	Fumarase	Matrix space	None	-	Hydration
(h)	Malate dehydrogenase	Matrix space	NAD+	NADH	Oxidation

 

(v) Steps in Kreb's cycle :  Kreb's cycle consists of 8 cyclic steps, producing an equal number of organic acids. Each step is catalyzed by a specific enzyme. In Kreb's cycle, the entrant molecule  is 2-carbon acetyl CoA and the  receptor molecule is 4- carbon oxaloacetate.

(a) condensation :  Acetyl coenzyme A reacts in the presence of water with the oxaloacetate normally present in a cell, forming 6-carbon citrate and freeing coenzyme A for reuse in pyruvate oxidation. The high-energy bond of acetyl CoA provides the energy for this reaction. The reaction is catalyzed by the citrate synthetase enzyme. The citrate has 3-carboxyl group. Hence, Krebs cycle is also called tricarboxylic acid cycle, or TCA cycle after its first product.

\[\text{Oxaloacetate}+\text{Acetyl}\,\text{CoA }+{{\text{H}}_{\text{2}}}O\underset{\text{Synthetase}}{\mathop{\xrightarrow{\text{Citrate}}}}\,\text{Citrate}+CoA\,(\text{Reused})\]

(b) Reorganisation (Dehydration) : Citrate undergoes reorganisation in the presence of an enzyme,  aconitase , forming 6-carbon cisaconitate and releasing water.

\[\text{Citrate}\xrightarrow{\text{Aconitase}}\text{Cisaconitate}+{{H}_{2}}O\]

(c) Reorganisation (Hydration) :  Cisaconitate is further reorganised into 6-carbon isocitrate by the enzyme, aconitase, with the addition of water.

\[\text{Cisaconitate}+{{H}_{2}}O\xrightarrow{\text{Aconitase}}\text{Isocitrate}\]

(d) Oxidative decarboxylation I :  This is a two stage process :

Stage I : Hydrogen atoms from isocitric acid react with NAD to form NAD.  2H forming  oxalosuccinic acid. The pair of hydrogen  atoms give two electrons and one H⁺ to NAD⁺ forming NADH +H⁺.  The enzyme isocitrate dehydrogenase catalyses the reaction in the presence of Mn²⁺. NADH generates ATP by transferring its electron over the ETS.

\[\text{Isocitric}\,\text{acid}+NAD\underset{M{{n}^{2+}}}{\mathop{\xrightarrow{\text{Isocitric}\,\text{dehydrogenase}}}}\,\text{Oxalosuccinic}\,\text{acid}+NAD.2H(or\,NADPH.2H)\]

Stage II : Decarboxylation of oxalosuccinic acid occurs forming \[\alpha \]-ketoglutaric acid, which is a first 5-C carbon molecule of Kreb's cycle.

\[\text{Oxalosuccinic}\,\text{acid}\underset{{}}{\mathop{\underset{M{{n}^{2+}}}{\mathop{\xrightarrow{\text{Carboxylase}}}}\,\alpha -\text{Ketoglutaric}\,\text{acid}+C{{O}_{2}}}}\,\]

(e) Oxidative decarboxylation II : This is also a 2 stage process :

Stage I :  Coenzyme A reacts with  \[\alpha \]-ketoglutarate, forming 4-carbon succinyl-coenzyme A and releasing CO₂ and a pair of hydrogen atoms. The reaction is catalysed by \[\alpha \]-ketoglutarate dehydrogenase complex enzyme. the pair of hydrogen atoms pass two electrons and one H⁺ to NAD⁺, forming NADH + H⁺

\[\alpha -\text{ketoglutarate}+CoA+NA{{D}^{+}}\underset{\text{dehydrogenase}}{\mathop{\xrightarrow{\alpha -\text{ketoglutarate}}}}\,\text{Succinyl}-CoA+C{{O}_{2}}+NADH+{{H}^{+}}\]

Stage II :  Succinyl -coenzyme A splits into 4-carbon succinate and coenzyme A with the addition of water. The coenzyme A transfers its high energy to a phosphate group that joins GDP (Guanosine diphosphate), forming GTP (Guanosine triphosphate). The latter is an energy carrier like ATP. This is the only high-energy phosphate produced in the Krebs cycle. The stage 2 reaction is catalysed by succinyl-CoA synthetase enzyme. The formation of GTP is called substrate level phosphorylation.\[\]

\[\text{Succinyl}-CoA+{{H}_{2}}O+GDP/ADP\underset{\text{Synthetase}}{\mathop{\xrightarrow{\text{Succinyl}-CoA}}}\,\text{Succinate}+CoA+GTP/ATP\]

In a plant cell, this reaction produce ATP from ADP and GTP from GDP or ITP (Inosine triphosphate) in animals.

Oxygen to oxidize a carbon atom to CO₂ is taken in steps 4 and 5 from a water molecule.

(f) Dehydrogenation : This process converts succinate into 4-carbon fumarate with the aid of an enzyme, succinate dehydrogenase,  and liberates a pair of hydrogen atoms. The latter pass to FAD⁺ (Flavin adenine dinucleotide), forming FADH₂. Hydrogen is carried by FAD in the form of whole atoms.

Succinate+FAD+Fumarate +FADH2

(g) Hydration : This process changes fumarate into 4-carbon maltate in the presence of water and an enzyme, fumarase.

Fumarate +H2O   Maltate

(h) Dehydrogenation : This process restores oxaloacetate by removing a pair of hydrogen atoms from maltate with the help of an enzyme maltate dehydrogenase. The pair of hydrogen atoms pass two electrons and one H+ to NAD+ , forming NADH+H+.

Maltate +NAD+Oxaloacetate \[+NADH\text{ }+{{H}^{+}}\]

                                         

          

Oxaloacetate combines with acetyl coenzyme A to form citrate, and so the cycle continues.

(vi) Summary of Kreb's cycle

(a) All the enzymes, reactants, intermediates and products of TCA cycle also are found in aqueous solution in the matrix, except the enzyme a-ketoglutarate dehydrogenase and succinate dehydrogenase which are located in the inner mitochondrial membrane. Both are called mitochondrial marker enzyme.

(b) Oxidation of one mole of acetyl CoA uses 4 molecules of water and releases one molecule of water.

(c) Liberates 2 molecules of carbon dioxide.

(d) Gives off 4 pairs of hydrogen atoms.

(e) Produces one GTP/ ATP molecule during the formation of succinate.

(f) One mole of acetyl CoA gives 12 ATP during oxidation in Krebs cycle.

(g) Regenerates oxaloacetate used in last cycle for reuse.

 

The above summary is for one molecule of acetyl coenzyme A. There are two acetyl coenzyme A molecules formed from one molecule of glucose by glycolysis and oxidative decarboxylation of pyruvate. The entire Krebs cycle may be represented by the following equation -

\[2\text{Acetyl}\,\text{coenzyme}\,A+8{{H}_{2}}O+6NA{{D}^{+}}+2FA{{D}^{+}}+2GDP/ADP+2Pi\]                                                                                   \[\to 4C{{O}_{2}}+2{{H}_{2}}O+6\,NADH+2FAD{{H}_{2}}+2GTP/ATP+6{{H}^{+}}\]

 

(vii) Difference between Glycolysis and Kreb's cycle

 

Glycolysis	Kreb?s cycle
It takes place in the cytoplasm.	It takes place in the matrix of mitochondria.
It occurs in aerobic as well as anaerobic respiration.	It occurs in aerobic respiration only.
It consists of 9 steps.	It consists of 8 steps.
It is a linear pathway.	It is a cyclic pathway.
It oxidised glucose partly, producing pyruvate.	It oxidises acetyl coenzyme A fully.
It consumes 2 ATP molecules.	It does not consume ATP
It generates 2 ATP molecules net from 1 glucose molecules.	It generates 2 GTP/ATP molecules from 2 succinyl coenzyme A molecules.
It yields 2 NADH per glucose molecule.	It yields 6 NADH molecules and 2 FADH2 molecules from 2 acetyl coenzyme A molecules.
It does not produce CO2.	It produces CO2.
All enzyme catalysing glycolytic reactions are dissolved in cytosol.	Two enzymes of Krebs cycle reactions are located in the inner mitochondrial membrane, all others are dissolved in matrix.

 

(viii) Product form during aerobic respiration by Glycolysis and Kreb’s cycle.

 

(a) Total formation of ATP    

 

ATP formation in Glycolysis
	Steps		Product of reactions		In terms of ATP
ATP formation by substrate phosphorylation	1, 3-diphosphoglyceric acid (2 moles) ®  3 phosphoglyceric acid (2 moles) Phosphoenolpyruvic acid (2 moles) ® Pyruvic acid (2 moles)		2 ATP 2 ATP		2 ATP 2 ATP
			Total		4 ATP
ATP formation by oxidative phosphorylation or ETC	1, 3 - disphosphoglyceraldehyde (2 moles) 1, 3 - diphosphoglyceric acid (2 moles)		2 NADH2		6 ATP
	Total ATP formed		4 + 6 ATP =		10 ATP
ATP consumed in Glycolysis	Glucose (1 mole) ® Glucose 6 phosphate (1 mole) Fructose 6 phosphate (1 mole) ® Fructose 1, 6-diphosphate (1 mole)		- 1 ATP - 1 ATP		- 1 ATP   - 1 ATP
			Total		2 ATP
	Net gain of ATP = total ATP formed - Total ATP consumed		10 ATP - 2ATP		8 ATP
ATP formation in Kreb?s cycle
ATP formation by substrate phosphorylation	Succinyl CoA (2 mols) ® Succinic acid (2 mols)	2 GTP		2 ATP
		Total		2 ATP
ATP formation by oxidative phosphorylation or ETC	Pyruvic acid (2 mols) ® Acetyl CoA (2 mols) Isocitric acid (2 mols) ®  Oxalosuccinic acid (2 mols) a-Ketoglutaric acid (2 mols) ®  Succinyl CoA (2 mols) Succinic acid (2 mols) ®  Fumaric acid (2 mols) Malic acid (2 mols) ®  Oxaloacetic acid (2 mols)	2 NADH2   2 NADH2   2 NADH2   2 FADH2   2 NADH2		6 ATP   6 ATP   6 ATP   4 ATP   6 ATP
		Total		28 ATP
	Net gain in Kreb?s cycle (substrate phosphorylation + oxidative phosphorylation)	2ATP + 28 ATP		30 ATP
Net gain of ATP in glycolysis and Kreb?s cycle	Net gain of ATP in glycolysis + Net gain of ATP in Kreb?s cycle	8 ATP + 30 ATP		38 ATP
Over all ATP production by oxidative phosphorylation or ETC	ATP formed by oxidative phosphorylation in glycolysis + ATP formed by oxidative phosphorylation or ETC.	6 ATP + 28 ATP		34 ATP

 

 

22 ATP produced by oxidation of NADH₂ and FADH₂ in Kreb’s cycle and 6 ATP comes from oxidative decarboxylation of pyruvic acid.

 

(b) Formation and use of water

 

Formation of water molecules
Formation of water molecules in glycolysis	2 phosphoglyceric acid (2 mols) \[\xrightarrow{-{{H}_{2}}O}\] 2 phosphoenol pyruvic acid (2 mols) 1, 3-diphosphoglyceraldehyde \[\xrightarrow{-{{H}_{2}}O}\] 1, 3 diphosphoglyceric acid	2H2O   2H2O
	Total water molecules formed in glycolysis	4H2O
Formation of water molecules in kreb?s cycle	One molecule of water in each of the five oxidation reactions (these reactions occur twice as there are two molecules of pyruvic acid). Other than oxidation reaction Citric acid (2 mols) ® Cis-aconitic acid (2 mols)	10 H2O   2H2O
	Total water molecules formed in Kreb?s cycle	12 H2O
	Total water molecules formed in aerobic respiration (Glycolysis + Kreb?s cycle)	16 H2O
Use of water molecules
Use of water in Glycolysis	3-phosphoglyceraldehyde  (2 mols) \[\xrightarrow{+{{H}_{2}}O}\] 1, 3 diphosphoglyceric acid (2 mols)	2H2O
	Total water molecule used in glycolysis	2H2O
Use of water in Kreb?s cycle	Oxaloacetic acid (2 mols) \[\xrightarrow{+{{H}_{2}}O}\] Citric acid (2 mols) Cis aconitic acid (2 mols) \[\xrightarrow{+{{H}_{2}}O}\] Isocitric acid (2 mols) Succinyl CoA (2 mols) \[\xrightarrow{+{{H}_{2}}O}\] Succinic acid (2 mols) Fumaric acid (2 mols) \[\xrightarrow{+{{H}_{2}}O}\] Malic acid (2 mols)	2H2O 2H2O 2H2O 2H2O
	Total water molecules used is Kreb?s cycle	8H2O
	Total water molecules used in aerobic respiration (Glycolysis + Kreb?s cycle)	10H2O
Net gain of water molecules in aerobic respiration	Number of water molecules formed - Number of water molecules used = ( 16 H2O - 10H2O)	6H2O

 

          

 

(c) Evolution of carbon dioxide

Pyruvic acid (2 mols) \[\xrightarrow{-C{{O}_{2}}}\] Acetyl CoA (2 mols) Oxalosuccinic acid  \[\xrightarrow{-C{{O}_{2}}}\] a ketoglutaric acid (2 mols) a Ketoglutaric acid (2 mols) \[\xrightarrow{-C{{O}_{2}}}\] Succinyl CoA (2 mols)	2CO2 2CO2 2CO2
Total CO2 molecules released in aerobic respiration	6CO2

 

 

 

 

 

(d) Use of O₂ (Oxygen)

Use of oxygen in Glycolysis	1, 3-diphosphoglyceraldehyde (2mols) \[\xrightarrow{+\frac{1}{2}{{O}_{2}}}\] 1, 3-diphosphoglyceric acid (2 mols)	1O2
Use of oxygen in Kreb?s cycle	Five oxidation reactions of Kreb?s cycle (2 times)	5O2
	Total O2 molecules required for aerobic respiration	6O2

 

(ix) Energy storage and energy transfer : In respiration energy released takes in the form of chemical energy, stored in a form called ATP . Energy transfer of biological oxidation hinges on the formation of labile high energy phosphate bonds of ATP. Nicotinamide adenine dinucleotide phosphate (NAD), Flavin adenine dinucleotide  (FAD), Guanosine triphosphate are also the product of respiration and converted to ATP by electron transport system.

 

(a) Adenosine triphosphate :  An energy intermediate :

There are several compounds like NAD, FAD, GTP and ATP are known as energy yielding compounds. The best known, and probably the most important of these are adenosine triphosphate (ATP).  It serves as the  energy currency of the cells.

Structure of ATP :  Adenosine triphosphate is a nucleotide consisting of three main constituents;

 

(a) A nitrogen contain purine base

(b) A five carbon sugar ribose

(c) Three inorganic phosphate groups

The bonds attaching the last two phosphate to the rest of the molecule are high energy bonds (~)  contain more than twice the energy of an average chemical bond.

 

• ATP hydrolysis : The energy is usually released from ATP by hydrolysing the terminal phosphate groups. Each molecule on hydrolysis yields ADP, one inorganic phosphate group (Pi) and about 7.28 Kcal energy.

ADP further hydrolysed to AMP and inorganic phosphate, releasing 7.3 Kcal energy per molecule (of ATP). Above process represented by following reactions.

          

\[\text{Adenosine}\,\text{triphosphate}\xrightarrow{\text{hydrolysis}}\text{Adenosine}\,\text{diphosphate}\,(ADP)\underset{\text{hydrolysis}}{\mathop{+Pi+}}\,7.3Kcal.....\]

 \[\text{Adenosine}\,\text{diphosphate}\to \text{Adenosine}\,\text{monophosphate(AMP)}+Pi+7.3Kcal.\]

Phosphorylation :  The ATP hydrolysis reactions are reversible because ATP are synthesized from ADP, Pi and energy (take up for the bond formation). The addition of phosphate group to ADP and AMP called phosphorylation. Energy required for the bond formation is equal to the energy released in hydrolysis.

The significant role of ATP as an intermediate energy transfer compound

 

• Major functions of ATP :  ATP molecules receive the energy, which released in exergonic reactions and make this energy available for various endergonic reactions. Some of the important process in which ATP is utilized are as follows:

(a) Synthesis of carbohydrates, proteins, fats, etc.

(b) Translocation of organic food.

(c) Absorption of organic and inorganic food.

(d) Protoplasmic streaming.

(e) Growth.

(b) Nicotinamide dinucleotide phosphate/ Nicotinamide dinucleotide (NADP/NAD) :  It is called universal hydrogen acceptor, produced during aerobic respiration (glycolysis+ Kreb's cycle) and also in anaerobic respiration, work as coenzyme in ATP generation Via electron transport system. NADP have one additional phosphate.

Structure of NAD = Nicotinamide-adenine-dinucleotide (formerly called coenzyme I or CO-I Diphosphopyridine dinucleotide = DPN) is shown below:

 

          

 

It plays a crucial role in dehydrogenation processes. Some dehydrogenases do not work with NAD, but react with NADP (Nicotinamide adenine dinucleotide phosphate). Formerly called Coenzyme II or Triphosphopyridine nucleotide = TPN Nicotinamide is a vitamin of B group.

 

First NAD and NADP both functions as hydrogen acceptors. Later H ions and electrons (e^_) from these are transported through a chain of carriers and after being released at the end of a chain react with O₂ and from H₂O (see Electron Transport chain). During the release of 2 electron from 2H⁺ atoms from NAD. 2H and their reaction with O₂ to form water, 3 ATP molecules are synthesized.

 

 

Important Tips

 

 

• Krebs cycle is the central pathway of the cell respiration where the catabolic pathways converge upon it an anabolic pathways diverge from it, so called amphibolic pathway.
• Acetyl Co-A, also called active acetate.
• Number of oxidation steps(dehydrogenation) for pyruvic acid is 5.
• In Kreb’s cycle, acetyl CoA undergoes two decarboxylation and four dehydrogenation. Krebs cycle catabolises about 80-90% of glucose.
• ATP discovered by Lohmann (1929), term was coined by Lohmann (1931) while ATP cycle was discovered by Lipmann (1941).
• Allosteric inhibition or negative feedback by accumulation of NADH
• Production of 36 ATP molecules from the oxidation of glucose is only an estimate as :

(i) NADH₂  may be used in some other metabolic pathway.

(ii) NADH₂ do not always produces 3 ATP molecules. More active muscle cells produces more while less active fat cell produce less ATP molecules.

(iii) Not all the proteins are routed through F₀ -F₁ channel.

(iv) So realistic aerobic respiratory efficiency ranges between 22% to 38%, as realistic power limit is  21 ATP molecule.

 

 

(3) Electron transport system :  The electron transmitter system is also called electron transport chain (ETC), or cytochrome system (CS), as  four out of these seven carriers are cytochrome. It is the major source of cells energy, in the respiratory breakdown of simple carbohydrates intermediates like phosphoglyceraldehyde, pyruvic acid, isocitric acid, \[\alpha -\]ketoglutaric acid, succinic acid and malic acid are oxidised. The oxidation in all these brought about by the removal of a pair of hydrogen atoms (2H) from each of them. This final stage of respiration is carried out in  ETS, located in the inner membrane of mitochondria (in prokaryotes the ETS is located in mesosomes of plasma membrane). The system consists of series of precisely arranged seven electron carriers (coenzyme) in the inner membrane of the mitochondrion, including the folds or cristae of this membrane. These seven electron-carriers function in a specific sequence and are:

 

Nicotinamide adenine dinucleotide (NAD), Flavin mononucleotide (FMN), Flavin adenine dinucleotide (FAD), Co-enzyme-Q or ubiquinone,  Cytochrome-b, Cytochrome-c, Cytochrome-a and Cytochrome-a₃,

 

 

 

 

The first carrier in the chain is a flavoprotein which is reduced by NADH₂. Coenzyme passes these electron to the cytochromes arranged in the sequence of b-c-a-a₃, finally pass the electron to molecular oxygen. In this transport, the electrons tend to flow from electro-negative to electro-positive system, so there is a decrease in free energy and some energy is released so amount of energy with the electrons goes on decreasing. During electron-transfer, the electron-donor gets oxidised, while electron-acceptor gets reduced so these transfers involve redox-reaction and are catalysed by enzymes, called  reductases.  Oxidation and reduction are complimentary. This oxidation-reductiion reaction over the ETC is called  biological oxidation.

\[\text{Electron}-\text{donor}\to {{\text{e}}^{\text{-}}}+\text{electron}-\text{acceptor}\]

here, electron-donor and electron -acceptor form redox pair.

 

During the electron transfers, the energy released at some steps is so high that ATP is formed by the phosphorylation of ADP in the presence of enzyme ATP synthetase present in the head of F₁-particles present on the mitochondrial crista. This process of ATP synthesis during oxidation of coenzyme is called oxidative phosphorylation, so ETS is also called  oxidative phosphorylation pathways.

\[\text{ADP}+\text{Pi}\xrightarrow{\text{ATP Synthetase}}\text{ATP}\]

 

From the cytochrome a₃, two electrons are received by oxygen atom which also receives two proton (H⁺) from the mitochondrial matrix to form water molecule. So the final acceptor electrons is oxygen.  So the reaction

\[{{H}_{2}}+\frac{1}{2}{{O}_{2}}\to {{H}_{2}}O\](called metabolic water) is made to occur in many steps through ETC, so the most of the energy can be derived into a storage and usable form.

 

(i)  Two route systems of ETC:  The pairs of hydrogen atoms from respiratory intermediates are received either by NAD⁺ or FAD coenzymes which becomes reduced to NADH₂ and FADH₂. These reduced coenzyme pass the electrons on to ETC. Thus, regeneration of NAD⁺ or FAD takes place in ETC. There are two routes ETC :

(a) Route 1 : NADH₂  passes their electrons to Co-Q through FAD . In route 1 FAD is the first electron carrier. 3 ATP molecules are produced during the transfer of electron on following steps :

NAD to FAD

Cyt b to Cyt c  and

Cyt a to Cyt a₃

(b)  Route 2 :  FADH₂ passes their electron directly to FAD. 2 ATP  molecules are produced during the transfer of electron on following steps.

Cyt b to Cyt c and

Cyt a to Cyt a₃

(ii) Structure of mitochondria in relation to oxidative function :  On inner side of mitochondria elementary particles or F₀-F₁ complex of ATPase complex or elementary particle (oxysomes) are found. Previously it was considered that elementary particles contain all the enzyme of oxidative phosphorylation and electron transport chain.

Component of electron transport chain are located in the inner membrane in the form of respiratory chain complexes. For complexes following theories are given :

(a) Four complex theory :  According to Devid green electron transport chain contains 4 complexes-

Complex I :  Comprises NADH  dehydrogenase and its 6 Iron Sulphur centers (Fe-S).

Complex II : Consists of Succinate dehydrogenase and its 3 Iron Sulphur centers.

Complex III : Consists of cytochrome b and c, and a specific Iron-Sulphur centers.

Complex IV :  Comprises cytochromes a and a₃.

 

 

(b) Five complex theory : According to Hatefi, (1976), Complex I to Complex IV are related to the electron transport.

• Complex V related to mainly with ATP synthesis, so it is called ATPase /ATP syntheses complex.
• The head piece (F₁) of the oxysome consists of 5 hydrophobic subunits (\[\alpha ,\beta ,\gamma ,\delta ,\varepsilon \]), which are responsible for ATPase functioning.
• The stalk (F₀) contain F₅ (oligomycin sensitivity conferring protein) e. CSCP and F₆. F₀ are related to the proton channel and embeded fully in thickness of inner mitochondrial membrane.
• Five complex i.e. I, II, III, IV, V, have been isolated from mitochondrial membrane by chemical treatment.
• Complex I : NADH/NADPH : CoQ reductase

                Complex II : Succinate : CoQ reductase

                Complex III : Reduced CoQ (CoQH₂) : cytochrome C reductase

                Complex IV : Cytochrome C oxidase

                Complex V : ATPase

Cytochrome C  and Q are mobile components of the respiratory chain.

 

(iii)  Oxidative phosphorylation : The process of ATP synthesis during oxidation of reduced coenzymes in ETC is called  oxidative phosphorylation. Peter Mitchell (1961) proposed the chemiosmotic mechanism  of ATP synthesis (Noble prize in 1978) which states that ATP synthesis occurs due to H⁺- flow through a membrane. It involves two steps :

(a) Development of proton gradient. At each step of ETC, the electron- acceptor has a higher electron -affinity than the electron-donor. The energy from electron-transport is used to move the proton (H⁺) from the mitochondrial matrix to inter-membranous or outer chamber. Three pairs of protons are pushed to outer chamber during the movement of electrons along route I while two pairs of protons are moved to outer chamber during the movement of electrons along route-II. This generates a pH-gradient across the inner mitochondrial membrane with protons  (H⁺) concentration higher in the outer chamber than in the mitochondrial matrix. This difference in H⁺ concentration across the inner mitochondrial membrane is called proton-gradient(\[\Delta \]pH). Due to proton gradient, an electrical potential (Dy) is developed across the inner mitochondrial membrane as the matrix is now electronegative with respect to the intermembranous (outer) chamber.  The proton gradient and membrane electric potential collectively called proton motive force.

(b) Proton flow : Due to proton-gradient, the protons returns to the matrix while passing through proton channel of F₀-F₁ ATPase. This proton gradient activates the enzyme ATP synthetase or F₀ - F₁ ATPase

 

ATP synthetase controls the formation of ATP from ADP and inorganic phosphate in the presence of energy.

 

 

Important Tips

• Cytochromes were discovered by MacCunn and term cytochrome given by P.Kailin.
• Iron-Sulphur is the component of ETC (complex I, II, III) and helps in transfer of electrons from FMNH₂ to coenzyme Q.  Thus, deficiency of iron direct affect ETC or oxidative phosphorylation.
• Cytochromes are Iron-containing (Iron porphyrin protein) electron transferring (electrons picked up and release by Fe) except cytochrome a₃. Cytochrome a₃ contains both Iron and Copper, in this Fe picks the electrons and through  Cu it hands over electron to oxygen, so cytochrome a₃  called terminal electron donar.
• Cytochrome P-450 : It occurs in E R and takes part in hydroxylation reaction.
• ETC inhibitors

         (i) Dinitrophenol (2,4-DNP) : It prevents synthesis of ATP from ADP because it directs electrons from CoQ to Q_2.

         (ii) Cyanide : It prevents flow of electrons from Cyt a₃ to oxygen.

         (iii) Carbon monoxide : It functions like cyanide.

         (iv) Antimycin A: Transfer of electron from Cyt b to Cyt c₁ is prevented.

         (v) Rotenone :  It checks flow of electrons from NADH /FADH₂ to CoQ.

• Action of ATPase needed Na⁺ and K⁺.
• Amount of energy released in ETC :

         (i) 12.2 Kcal during transfer of electrons from NAD to FMN.

         (ii) 15.2 Kcal during transfer of electrons from Cyt b to Cyt c.

         (iii) 24.5 Kcal during transfer of electrons from  Cyt a to Cyt a_3.

 

(iv) Role of shuttle system in energy production : Glycolysis occurs in the cytoplasm outside the mitochondrion in which 2\[NAD{{H}_{2}}\] molecules are produced but ETC is located along inner mitochondrial membrane, so \[NAD{{H}_{2}}\]of glycolysis must enter inside the mitochondrion to release energy. But the inner mitochondrial membrane is impermeable to NADH₂. In mitochondrial membrane, there are 2 shuttle-system, each formed of carrier-molecule.

These shuttle systems are :

(a) Malate-Aspartate shuttle : It is more efficient and results in the transfer of electron from NAD. 2H in cytosol to NAD inside the mitochondrion,  via NAD. 2H dehydrogenase as follows :

 

Electrons are transferred from NAD. 2H in cytosol to malate which traverses the inner mitochondrial membrane and reoxidised to form NAD. 2H thus resulting in the formation of oxaloacetate . Oxaloacetate does not readily cross the inner mitochondrial membrane and so a transamination reaction is needed to form aspartate which does traverse this barrier. As a result 3 ATP molecules are generated  for each pair of electrons. Thus if this shuttle is predominant there is a gain of 38 ATP molecules by complete oxidation of one molecule of glucose.

 

 

(b) Glycerol-Phosphate shuttle : It is less efficient and results in the reduction of FAD inside the mitochondrion.

 

If this shuttle predominates the electrons from NAD. 2H are transferred to FAD inside the mitochondrion as follows. NAD. 2H reacts with dihydroxyacetone phosphate (DHAP) in cytosol to form glycerol phosphate which diffuses through outer mitochondrial membrane to the outer surface of inner membrane. There glycerol phosphate reacts with membrane dehydrogenase to form dihydroxyacetone phosphate (DHAP) which returns to cytosol.  In this process FAD is reduced to FADH₂. Electrons from FADH₂ directly pass to Q and other components of ETC and results in the synthesis of 2 ATP for each molecule of FADH₂. In this case complete oxidation of glucose will result in a gain of 36 ATP molecule.

 

Which shuttle predominates depends on the particular species and tissues envolved, for example: 38 ATP are formed in kidney, heart and liver cell while 36 ATP molecules are formed in muscle cells and nerve cells. In these cells glycerol-phosphate shuttle is predominant and 2 ATP formed from NADH_2.

 

Other pathways of glucose oxidation

 

(1) Entner-Doudoroff pathway

(i) Discovery: Entner-Doudoroff path discovered by Entner & Doudoroff. This pathway is also called glycolysis of bacteria.

Certain bacteria such as Pseudomonas sacchorophila, P. fluorescens, P. lindeneri and P. averoginosa lack phosphofructokinase enzyme. They cannot degrade glucose by glycolytic process.

 

(ii) Description:  In this pathway the glucose molecule first phosphorylated to Glucose-6-phosphate by ATP. Then it oxidised to 6-phosphogluconic acid by NADP which itself reduced to NADPH_2­ by the electrons released. The NADPH₂ is channeled through ETS system to produce 3-molecules of ATP per NADPH₂ molecule through ETS system to produce 3 molecules of ATP per NADH₂ molecule and 1, 6-phosphogluconic acid is channeled to pyruvic acid. The main reaction are:

(a) \[\text{Glucose-6-phosphate}+\text{NADP }\to \text{6-Phosphogluconolactone }+\text{NADP}{{\text{H}}_{\text{2}}}\]

(b) 6-phosphoglyconolactone ® 2-Keto-3-deoxyphospho-6-gluconic acid 

(c) 2-Keto-3-deoxyphosphogluconic acid ® Pyruvic acid+Glyceraldehyde-3-phosphate

(d) Glyceraldehyde-3-phosphate ® Pyruvic acid

The glyceraldehyde 3-phosphate by EMP pathway gets converted into pyruvic acid which can be further used up in the process.

 

(2) Pentose phosphate pathway

(i) Discovery : It is also called as Hexose monophosphate (HMP) shunt or Warburg Dickens pathway or direct oxidation pathway. It provides as  alternative pathway for breakdown of glucose which is independent of EMP pathway (glycolysis) and Krebs cycle. Its existence was suggested for the first time by Warburg et al. (1935) and Dickens (1938). Most of the reaction of this cycle were described by  Horecker et al.(1951) and  Racker (1954).

(ii)  Occurrence :  Pentose phosphate  pathway that exists in many organisms.  This pathway takes place in the cytoplasm  and requires oxygen for its entire operation.

 

(iii) Description :  There are two types of evidences is support of the existence of such an alternative pathway-works on the inhibiting action of malonic acid on the Krebs cycle and studies with the radioactive (C¹⁴).

Twelve molecules of NADH₂ formed in the reaction can be oxidised back to 12 NADP with the help of the cytochrome system and oxygen of the air.

           \[12\text{ NADP}{{\text{H}}_{\text{2}}}+6{{O}_{2}}\underset{\text{System}}{\mathop{\xrightarrow{\text{Cytochrome}}}}\,\text{12}{{\text{H}}_{\text{2}}}\text{O}+\text{12NADP}\]

In this electron transfer process, 36 molecules of ATP are synthesized.

The reaction can be summarised as follows :

 

• \[\text{6 Glucose }+\text{ 6 ATP}\xrightarrow{\text{hexokinase}}6\,\text{Glucose-6-phosphate}\]
• \[\text{6 Glucose-6-phosphate }+\text{6 NADP}+\text{6}{{\text{H}}_{\text{2}}}\text{O}\xrightarrow{\text{dehydrogenase}}6\text{ phosphogluconic acid }+\text{6NADP}{{\text{H}}_{\text{2}}}\]

Image pending

 

• \[\text{2 Ribulose-5-phosphate}\xrightarrow{\text{isomerase}}2\text{ ribose-5-phosphate}\]
• \[\text{5 Ribulose-5-phosphate}\xrightarrow{\text{isomerase}}4\text{ xylulose-5-phosphate}\]
• \[2\text{ Xylulose-5-phosphate}+\text{2 ribose-5-phosphate}\]

\[\xrightarrow{\text{transketolase}}2\,\text{sedoheptulose}-\text{7phosphate}+\text{2 glyceraldehyde-3-phosphate }\]

• \[2\text{ Sedoheptulose-7-phosphate }+\text{2 glyceraldehyde 3-phosphate}\]

\[\xrightarrow{\text{transketolase}}\text{2 fructose-6-phosphate}+\text{2 erythrose-4-phosphate}\]

 

• \[\text{2 Erythrose-4-phosphate }+\text{2 xylulose-5-phosphate}\]

\[\xrightarrow{\text{transketolase}}2\text{ fructose-6-phosphate}+\text{2 glyceraldehyde}\,\,\text{3-phosphate}\]

• \[\text{2 Glyceraldehyde-3-phosphate}+{{\text{H}}_{\text{2}}}\text{O }\xrightarrow{\text{aldolase}}\text{fructose-6-phosphate}+{{\text{H}}_{\text{3}}}\text{P}{{\text{O}}_{\text{4}}}\]

      Sum total of the reaction :

      \[6\text{ }Glucose-phosphate\text{ }+\text{ }12NADP+7{{H}_{2}}O5\text{ }Fructose-6-phosphate+6C{{O}_{2}}+\text{ }12NADP{{H}_{2}}+{{H}_{2}}P{{O}_{4}}\]

      (iv) Significance of PPP

• It is the only pathway of carbohydrate oxidation that gives NADPH₂, Which is needed for synthetic action like synthesis of fatty acid (in adipose tissues) and amino acids (in liver).
• It synthesizes 3C-glyceraldehyde-3-P, 3C-dihydroxy acetone phosphate, 4C-erythrose-4-P, 5C-ribulose phosphate, 5C-xylulose phosphate, 5C-ribose phosphate, 6 C-Fructose 6-phosphate, 7C-sedoheptulose-7-phosphate.
• It is the major pathway by which necessary ribose and deoxyribose are supplied in the biosynthesis of nucleotides and nucleic acid.
• Erythrose 4 phosphate for the synthesis of lignin, oxine, anthocyanine and aromatic amino acid (phenylalanine, tyrosine, and tryptophan).
• Young growing tissues appears to use to the Krebs cycle as the predominant pathway for glucose oxidation, while aerial parts of the plants and other tissues seem to utilise the PPP as well as the Krebs cycle.
• It gives 6 CO₂, required for photosynthesis.
• Ribulose five phosphate is used in photosynthesis to produce RuBP which act as primary CO₂ acceptor in C₃

(3) Comparison of the different pathway of glucose metabolism

 

(4) Cyanide resistant pathway : Cyanide-resistant respiration seems to be widespread in higher plant tissues. Cyanide prevents flow of electron from Cyt a₃ to oxygen, so called ETC inhibitor. In these plant tissues resistance is due to, a branch point in the ETS preceeding the highly cyanide-sensitive cytochromes. The tissues lacking this branch point, or alternate pathway and blockage of cytochromes by cyanide, inhibits the electron flow.

 

Significance

(a) The role of alternative pathway is that it may provide a means for the continued oxidation of NADH and operation of the tricarboxylic acid cycle, even through ATP may not be sufficiently drained off.

 

 

 

 

 

(b) It is significant in respiratory climateric of ripening fruits and leads to the production of hydrogen peroxide and super oxide, which in turn enhances the oxidation and breakdown of membranes.

(c) Necessary activities in the ripening process because peroxides are necessary for ethylene biosynthesis.