Quadrant - I
Glycolysis, TCA cycle and Electron transport chain Objectives
Understand the various forms of potential energy utilized in the photosynthetic and respiration processes;
Define the similarities and differences between the electron transport chains utilized in photosynthesis and aerobic respiration and how the oxidation and reduction of molecules and the flow of electrons through these electron transport chains is use to generate stable forms of energy in plants and animals
Elucidate the general flow of electrons from glucose to NAD + and FAD+ through a series of chemical intermediates;
Describe how ATP is produced via substrate-level phosphorylation and by the ATP synthase
Trace the flow of an electron from a water molecule to NADPH to glucose to NADH back to a water molecule during photosynthesis and respiration
3.1 GLYCOLYSIS
The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADH
It is also named as the Embden-Meyerhof Pathway
Glycolysis is a common pathway; present in all organisms: from yeast to mammals.
In eukaryotes, glycolysis takes place in the cytosol.
Glucose enters most cells by a specific carrier that transports it from the exterior of cell into the cytosol. - The enzymes of glycolysis are located in the cytosol.
Glycolysis converts glucose to two C3 units (pyruvate), and released free energy is used to synthesize ATP from ADP and Pi.
Post glycolysis pathways
- There are two major pathways of post glucose metabolism:
- Aerobic oxidation --- 2Pyruvate → 6CO2 + 6H2O (and 30 ATP)
- Anaerobic fermentation - Homolactic in muscle --- 2Pyruvate → 2Lactate (no ATP) - Alcoholic in yeast --- 2Pyruvate → 2CO2 + 2EtOH
Net Reaction:
Glucose + 2NAD+ + 2 Pi + 2 ADP = 2 pyruvate + 2 ATP + 2 NADH + 2 H2O
Glycolysis occurs in three stages:
Priming stage- 1-3 consisting of a phosphorylation (kinase), an isomerization, and a second phosphorylation (kinase). The strategy here is to trap the glucose inside the cell and to form a compound that can be readily cleaved into symmetrical, phosphorylated three-carbon units.
Splitting stage- 4-5 here the fructose is cleaved into two three-carbon units that are readily interchangeable.
Oxidoreduction-phosphorylate stage-6-10 consisting of a redox reaction (dehydrogenase), two dephosphorylation reactions, an isomerase (mutase), and a lyase (enolase). In this stage, ATP is harvested when the three-carbon fragments are oxidized to pyruvate.
Thus, net profit is 2ATPs and 2NADH per glucose. Overall reaction is:
Glucose + 2ADP + 2NAD+ + 2Pi → 2pyruvate + 2ATP + 2NADH + 2H2O + 4H+
The Oxidizing power of NAD+ must be recycled.The number of NAD+ molecules in a cell is limited. Thus in order to continue glycolysis, NADH must be oxidized to NAD+.
1. Under anaerobic condition in muscle:
Pyruvate → lactate and NADH → NAD+ 2. Under anaerobic condition in yeast:
Pyruvate → ethanol + CO2, and NADH → NAD+ + ΔH 3. Under aerobic condition:
Pyruvate → CO2 + H2O, and NADH → NAD+ + 3ATP
- In anaerobic glycolysis, the free energy of oxidation (NADH → NAD+) is wasted as heat (ΔH).
- In aerobic glycolysis, NADH is a “high-energy” compound, and produces 3ATP per NADH.
3.1.1 THE REACTIONS OF GLYCOLYSIS
A. Glucose →Glucose-6-phosphate (G6P), First ATP Utilization Enzyme: Hexokinase (HK)
Reaction: Transfer phosphoryl group
B. Glucose 6-phosphate (G6P) to Fructose 6-phosphate (F6P)
Enzyme: Phosphoglucoseisomerase (PGI) Reaction: Isomerization of an aldose to ketose
C. Fructose 6-phosphate (F6G) to Fructose-1,6-bisphosphate (FBP) , Second ATP Utilization
Enzyme: Phosphofructokinase (PFK) Reaction: Transfer phosphoryl group
D. Fructose-1,6-bisphosphate to two trioses, Glyceraldehyde-3-phosphate (GAP) and Dihydroxyacetone phosphate (DHAP)
Enzyme: Aldolase
Enzyme: Aldolase Reaction: Aldol cleavage
E. Dihydroxyacetone phosphate (DHAP) to Glyceraldehyde-3-phosphate (GAP)
F. Glyceraldehyde 3-phosphate (GAP) to 1,3-Bisphophoglycerate (1,3-BPG) First “High-Energy” Intermediate Formation
Enzyme:
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Reaction: Oxidation and phosphorylation
G. 1,3-Bisphosphoglycerate (1,3-BPG) to 3-Phosphoglycerate (3PG): First ATP Generation
Enzyme: Phosphoglycerate kinase (PGK).
Reaction: Phosphorylation
H. 3-Phosphoglycerate (3PG) to 2-Phosphoglycerate (2PG)
Enzyme:
Phosphoglyceratemutase (PGM)
Reaction: Intramolecularphosphoryl group transfer
BPG binds into the central cavity of the Hb molecule and stabilizes the T-form (deoxy-Hb).
Thus, high [BPG] in blood reduces the oxygen affinity of Hb molecules.
- Hexokinase deficiency results in low [3PG]low [G6P]→→→ low [3PG]→
low [2,3-BPG], thus increase Hb O2 affinity.
- Pyruvate kinase deficiency results in accumulation of [2PG] high [PEP]
→high [2PG] → high [2,3-BPG], thus decrease Hb O2 affinity.
I. 2-Phosphoglycerate (2PG) to Phosphoenolpyruvate (PEP)
Enzyme: Enolase Reaction: Dehydration J. Phosphoenolpyruvate (PEP) to Pyruvate
Enzyme: Pyruvate kinase
Reaction: Hydrolysis to ATP synthesis 3.1.2 Fate of Pyruvate
• NADH is formed from NAD+ during glycolysis.
• The redox balance of the cell has to be maintained for further cycles of glycolysis to continue.
• NAD+ can be regenerated by one of the following reactions /pathways:
• Pyruvate is converted to lactate
• Pyruvate is converted to ethanol
• In the presence of O2, NAD+ is regenerated by ETC.
Pyruvate is converted to acetyl CoA that enters TCA cycle and gets completely oxidized to CO2.
FERMENTATION: ANAEROBIC FATE OF PYRUVATE
- Amount of NAD+ in a cell is limited. Hence, NADH produced by GAPDH must be recycled in order to continue glycolysis.
- Under aerobic condition, NADH is re-oxidized by sending electrons into the mitochondria.
- Under anaerobic condition, the NAD+ is replenished by the reduction of pyruvate by two processes:
- Homolactic fermentation (in muscle) - Alcoholic fermentation (in yeast) A. Homolactic fermentation
The overall process of anaerobic glycolysis in muscle is:
Glucose + 2ADP + 2Pi → 2 lactate + 2ATP + 2H2O +H+
- Much of the lactates in the muscle cells are carried by blood to liver, and they are reconverted to glucose.
- Muscle fatigue and soreness are caused by the accumulation of glycolytically generated acid (H+), but not lactate.
B. Alcoholic Fermentation
- is a two step reaction.
1. Decarboxylation of pyruvate to form acetaldehyde.
2. Reduction to ethanol by NADH.
pyruvate -decarboxylase Acetaldehyde alcoholdehydrogenase Ethanol - Pyruvate decarboxylase needs a cofactor thiamine pyrophosphate (TPP).
Both homolactic and alcoholic fermentation have the same function. That is “anaerobic regeneration of NAD+”, so as to continue glycolysis quickly to produce ATP molecules by glycolysis.
3.1.3.Glycolysis is used for rapid ATP production.
- ATP production of anaerobic glycolysis is ~100 times faster than that of oxidative phosphorylation (aerobic pathway).
- Thus, tissues such as muscle consuming ATP rapidly regenerate it almost entirely by anaerobic glycolysis.
- Since the end product, lactate, is aerobically regenerated to glucose in liver, glucoseis not really wasted in the homolactic fermentation.
Red muscle fiber contains a large amount of mitochondria which produce ATP by oxidative phosphorylation (aerobic pathway).
- White muscle fiber contains less amount of mitochondria, indicating that ATP is generated by anaerobic fermentation (anaerobic pathway).
All the enzymes involved in glycolysis are activated
Three reactions with large -ΔG in muscle are catalyzed by:
Hexokinase (HK); Phosphofructokinase (PFK); Pyruvate kinase (PK)
These three reactions are non-equilibrium. Other reactions are near equilibrium at physiological condition
3.1.4.Regulation of Glycolysis
Glycolysis is a strongly regulated pathway. Only the irreversible enzymes (hexokinase,phosphofructokinase, pyruvate kinase) can be regulated. Phosphofructokinase is by far the most regulatedenzyme. Regulation can occur in several ways:
1. allosteric modulators
2. hormone control (through phosphorylation/dephosphorylation) 3. de novo synthesis
Inhibitor and Toxins
1. Arsenate – affects GAP dehydrogenase. Arsenate looks like Pi and is added to GAP by GAP dehydrogenase instead of Pi. Phosphoglycerate kinase removes the arsenate group in step 7, but ATP is not generated in this step. Thus, get 0 net ATP from glycolysis. Glycolysis is not stopped, but ATP is not produced. This is detrimental to RBC.
2. Iodoacetate (iodoacetamide) – inhibits GAP dehydrogenase :Inhibits GAP dehydrogenase by irreversibly binding to the Cys residue in the active site rendering the enzyme inactive.
3. Fluoride ion – inhibits enolase Fluoride ion complexes with the Mg+2ion at the active site of the enzyme rendering the enzyme inactive.
3.2 Citric acid cycle INTRODUCTION
The citric acid cycle is a central metabolic pathway that completes the oxidative degradation of amino acids, monosaccharides, and fatty acids. In aerobic catabolism, these biomolecules are broken down to smaller molecules that ultimately contribute to a cell’s energetic or molecular needs.
Early metabolic steps, including glycolysis and the activity of the pyruvate dehydrogenase complex, produce a two-carbon fragment named an acetyl group that is linked to a large cofactor known as coenzyme A (or CoA). It is during the citric acid cycle that acetyl-CoA is
oxidized to carbon dioxide which is a waste product, accompanied by the reduction of the cofactors NAD+ and ubiquinone.
The citric acid cycle aids two main purposes:
1. To increase the cell’s ATP-producing potential by generating a reduced electron carriers such as NADH and reduced ubiquinone; and
2. To provide the cell with a variety of metabolic precursors.
The cycle begins with the formation of six-carbon citric acid by the reaction between acetyl- CoA and the four-carbon oxaloacetate. Through the following steps of the cycle, two of the six carbons of the citric acid leave as carbon dioxide to ultimately yield the four carbon product, oxaloacetate that is used again in the first step of the subsequent cycle. During the eight reactions that take place, for every molecule of acetyl-CoA the cycle produces three NADH and one flavin adenine dinucleotide (FAD/FADH2), along with one molecule of ATP.
THE OVERALL REACTIONS OF TCA CYCLE
3.2.1.Reaction 1
The first reaction of the citric acid cycle is catalyzed by the enzyme citrate synthase. Here in this step, oxaloacetate is combined with acetyl-CoA to form citric acid.
Reaction 2: Acontinase
The enzyme acontinase catalyses the next reaction of the citric acid cycle. The molecule isocitrate is yielded in this transformation.
Reaction 3: Isocitrate Dehydrogenase
Two events occur in reaction 3 of the citric acid cycle. The first reaction generates NADH from NAD. the reaction is catalysed by the enzyme isocitrate dehydrogenase to yield an
intermediate which then has a carbon dioxide molecule removed from it to yield alpha- ketoglutarate.
Reaction 4: Alpha-ketoglutaratedeydrogenase
Alpha-ketoglutarate loses a carbon dioxide molecule and coenzyme A is added in its place in reaction 4 of the citric acid cycle. The decarboxylation takes place with the help of NAD that is converted to NADH. Alpha-ketoglutarate dehydrogenase enzyme catalyzes this reaction to form succinyl-CoA.
Reaction 5: Succinyl-CoA Synthetase
The enzyme succinyl-CoA synthetase catalyzes the fifth reaction of the citric acid cycle.
Here, a molecule of guanosine triphosphate (GTP) is synthesized. As a result, succinate is formed.
Reaction 6: Succinate Dehydrogenase
The enzyme succinate dehydrogenase catalyzes the removal of two hydrogens from succinate in the sixth reaction of the citric acid cycle to form fumarate
Reaction 7: Fumarase
In this reaction, the enzyme fumarase catalyzes the addition of a water molecule to the fumarate in the form of an –OH group to yield the molecule L- malate.
Reaction 8: Malate Dehydrogenase
In the last reaction of the citric acid cycle, we regenerate oxaloacetate by oxidizing L–malate with a molecule of NAD to produce NADH.
3.2.2.Three Enzymes of the Citric Acid Cycle Are Regulated
Three factors govern the rate of flux through the cycle: substrate availability, inhibition by accumulating products, and allosteric feedback inhibition of early enzymes by later intermediates in the cycle.
There are three strongly exergonic steps in the cycle, those catalyzed by citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase. Each can become the rate
limiting step under some circumstances.
Conclusion
what the citric acid cycle has generated from one acetyl-CoA molecule.
The acetyl-CoA, has been oxidized to two molecules of carbon dioxide.
Three molecules of NAD were reduced to NADH.
One molecule of FAD was reduced to FADH2.
One molecule of GTP (the equivalent of ATP) was produced.
Reduction is really a gain of electrons. In other words, NADH and FADH2 molecules act as electron carriers and are used to generate ATP in the next stage of glucose metabolism, oxidative phosphorylation. In the next part, we will learn what processes take place to ultimately derive the majority of the ATP we need to fuel our daily activity.
3.3.The Electron Transport Chain
The electron transport chain is the final and most important step of cellular respiration.
Glycolysis and the Citric Acid Cycle make the necessary precursors and the electron transport chain creates majority of the ATP. The Electron Transport System, also called the Electron Transport Chain, converts redox energy available from oxidation of NADH and FADH2, into proton-motive force that is used to synthesize ATP through conformational changes in the ATP synthase complex through a process called oxidative phosphorylation.
The citric acid cycle oxidizes acetate into two molecules of CO2 while capturing the electrons in the form of one molecule of FADH2 and 3 NADH molecules. These reduced molecules contain a pair of electrons with a high transfer potential. These electrons are eventually going to be transferred by a system of electron carriers to O2 to form H2O. The process takes place in the mitochondria and is the major energy source used to produce ATP by oxidative phosphorylation.
ATP synthesis is not an energetically favourable reaction: energy is needed in order for it to happen. This energy is derived from the oxidation of NADH and FADH2 by the four protein complexes of the electron transport chain (ETC). The ten NADH that enter the electron
transport originate from each of the earlier processes of respiration: two from glycolysis, two from the transformation of pyruvate into acetyl-CoA, and six from the citric acid cycle. The two FADH2 is created in the citric acid cycle.
In complex I, electrons are passed from NADH to the electron transport chain, where they flow through the remaining complexes. NADH is oxidized to NAD in this process. FADH isoxidized inComplex II, garnering still more electrons for the chain. No additional electrons enter the chain at complex III, but electrons from complexes I and II flow through it. After the arrival of electrons at complex IV, they are transferred to a molecule of oxygen. Since the oxygen gains electrons, it is reduced to water.
While this oxidation and reduction reactions take place, additional, related event occurs in the electron transport chain. The drive of electrons through complexes I-IV causes protons (hydrogen atoms) to be pumped out of the intermembrane space into the cell cytosol. Thus, a net negative charge (from the electrons) builds up in the matrix space while a net positive charge (from the proton pumping) builds up in the intermembrane space. This differential electrical charge establishes an electrochemical gradient.
3.3.2. The Components of the Electron Transport Chain
The electron transport chain of the mitochondria is the means by which electrons are removed from the reduced carrier NADH and transferred to oxygen to yield H2O.
1) NADH
NADH is generated in the matrix by the reactions of pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehyrogenase.
NAD+ + 2e- + H+ NADH Eo’ = −0.315 V
2.) Flavoproteins: Flavoproteins have either a FAD (flavin adenosine dinucleotide) or a FMN (flavin mononucleotide) prosthetic group. Flavoproteins can accept or donate electrons one at time or two at a time. Therefore they are often intermediaries between two electron acceptors/donors and one electron acceptors/donors.
FAD + 2e- + 2H+ FADH2; FMN + 2e- + 2H+ FMNH2 Eo’ ≈ 0 V 3.) Coenzyme Q (CoQ)
Coenzyme Q is a versatile cofactor because it is a soluble electron carrier in the hydrophobic bilipid layer of the inner mitochondrial membrane. Alike flavoproteins, CoQ can accept/donate electrons one at a time or two at a time.
3.) Cytochromes: Cytochromes are proteins that contain heme prosthetic groups which functions as one electron carriers. The heme iron is involved in one electron transfers involving the Fe2+ and Fe3+ oxidation states. Cytochrome c,c1, a and a3 are the different cytochromes seen.
4.) Iron-Sulfur Proteins
These are non-heme iron-sulfur proteins. The different iron-sulfur proteins are FeS,Fe2S2
,Fe3S4
5.) Copper Proteins: Copper bound proteins participate in one electron transfers involving the Cu+ and Cu2+ oxidation states.
3.3.3. Overview of the Electron Transport Chain.
Electrons move along the electron transport chain going from donor to acceptor until they reach oxygen the ultimate electron acceptor. The typical reduction potentials of the electron carriers are between the NADH/NAD+ couple (-0.315 V) and the oxygen/H2O couple (0.816
V) .The components of the electron transport chain are organized into 4 complexes. Each complex contains several different electron carriers.
1. Complex I also known as the NADH-coenzyme Q reductase or NADH dehydrogenase. 2.
Complex II also known as succinate-coenzyme Q reductase or succinate dehydrogenase. 3.
Complex III also known as coenzyme Q reductase. 4. Complex IV also known as cytochrome c reductase.
Complex I accepts electrons from NADH and serves as the link between glycolysis, the citric acid cycle, fatty acid oxidation and the electron transport chain.Complex I is also called NADH-Coenzyme Q reductase because this large protein complex transfers 2 electrons from NADH to coenzyme Q. Complex I was previously known as NADH dehydrogenase. This complex binds NADH, transfers two electrons in the form of a hydride to FMN to produce NAD+ and FMNH2. The subsequent steps involve the transfer of electrons one at a time to a series of iron-sulfur complexes that includes both 2Fe-2S and 4Fe-4S clusters.
The final step of this complex is the transfer of 2 electrons one at a time to coenzyme Q.
CoQ like FMN and FAD can function as a 2 electron donor/acceptor and as a 1 electron donor/acceptor. The isoprenoid tail of CoQ makes it highly hydrophobic and lipophilic and therefore it is a mobile electron carrier. It can diffuse freely in the bilipid layer of the inner mitochondrial membrane
Complex II includes succinate dehydrogenase and serves as a direct link between the citric acid cycle and the electron transport chain. It is the only enzyme of the citric acid cycle that is an integral membrane protein. The complex is composed of four subunits. Complex II comprises 3 Fe-S centres, 1 4Fe-4S cluster, 1 3Fe-4S cluster and 1 2Fe- 2S cluster.
Succinate is bound and a hydride is transferred to FAD to generate FADH2 and fumarate in the first step of this complex.The electrons from FADH2are transferred one at a time to the Fe-S centers.The last step of this complex is the transfer of 2 electrons one at a time to coenzyme Q to produce CoQH2.
Complexes I and II both produce reduced coenzyme Q. This CoQH2 is the substrate for Complex III.
Complex III transfers the electrons from CoQH2 to reduce cytochrome c which is the substrate for Complex IV. This complex is also known as coenzyme Q-cytochrome c reductase because it passes the electrons from CoQH2 to cyt c through a very unique electron transport pathway called the Q-cycle.
Cytochrome b has the same iron protoporphyrin as hemoglobin and myoglobin. The c cytochromes have heme c through covalent attachment by cysteine residues. Cytochrome a is exist in two forms in complex IV.
Q-Cycle
The Q-cycle is initiated when CoQH2 diffuses through the bilipid layer to the CoQH2 binding site which is near the intermembrane face. The CoQH2 binding site is known as QP site. The electron transfer happens in two steps. In the first step, one electron from CoQH2 is transferred to the Rieske protein (a Fe-S protein) which transfers the electron to cytochrome c1. In this process 2 protons are released to the intermembrane space.
The two electron carrier CoQH2 gives up its electrons one at a time to the Rieske protein and the bL heme both of which are 1 electron carriers.
The electrons that end up on cytochrome c1 are transferred to cytochrome c. Cytochrome c is the only water soluble cytochrome. Cytochrome c is coordinated to ligands that protect the iron contained in the heme from oxygen and other oxidizing agents. Cytochrome c is a mobile electron carrier which diffuses through the intermembrane space shuttling electrons from the c1 heme of complex III to CuA site of complex IV
Complex IV transfers the electrons from cytochrome c to reduce molecular oxygen into water. Complex IV is also known as cytochrome c oxidase because it accepts the electrons from cytochrome c and directs them towards the four electron reduction of O2 to form 2 molecules of H2O. 4 cyt c (Fe2+) + 4 H+ + O2 4cyt c (Fe3+) + 2H2O
Each of these complexes are large multi subunit complexes embedded in the inner mitochondrial membrane. The reduction of oxygen by complex IV involves the transfer of
four electrons. Four protons are abstracted from the matrix and two protons are released into the inter membrane space.
Summary
The electron transport chain is the stepwise process of cellular respiration that is responsible for producing:
Water (with the help of oxygen we breathe)
up to 34 ATP (thanks to the proton gradient)
NAD and FAD (which are recycled to be used again in the Citric acid cycle and glycolysis)
This process happens in the mitochondria of Eukaryotes and cell membrane of Prokaryotes .
The last key point to remember is this only happens in aerobic conditions( oxygen present).
If there is a shortage of oxygen cellular respiration will take an alternative pathway at the end of glycolysis resulting in the production of lactic acid and ATP.
