Metabolism and Fermentation: Notes PDF

Rajarshi Shahu Mahavidyalaya Latur, (Autonomous) Course Title : Metabolism Course Teacher: Dr. Manisha Patil  Metabolism  All cells function as biochemical factories.  Within a living cell, biomolecules are constantly being synthesized and trans...

Rajarshi Shahu Mahavidyalaya Latur, (Autonomous) Course Title : Metabolism Course Teacher: Dr. Manisha Patil  Metabolism  All cells function as biochemical factories.  Within a living cell, biomolecules are constantly being synthesized and transformed into some other biomolecules through enzyme catalyzed reactions.  All the interconnected chemical reactions occurring within a cell are called Metabolism.  Metabolism is derived from Greek Word for Change.  Each Chemical compound involved in the Process is known as Metabolites.  Majority of Metabolic Reactions do not occur in isolation.  They are always linked to some other reactions.  A series of linked chemical reactions called as Metabolic Pathway  A Starting molecule Reaction 1 Enzyme 1 B Reaction2 Enzyme 2 C Reaction 3 Enzyme 3 D Product Metabolic pathways can be 1. Linear (Glycolysis) 2. Cyclic ( Citric Acid Cycle) 3. Spiral ( Biosynthesis of Fatty Acid)  Flow of Metabolites through Metabolic Pathway has a definite rate and direction  Because the biomolecules within the cell are in continual state of degradation and resynthesis.  This is called as dynamic state of body constituents  Purpose of metabolism 1. Generation of energy to drive vital functions 2. Synthesis of biological molecules  Metabolic pathway fall into two categories 1.Anabolic PW 2.Catabolic PW Anabolic Pathways  They are involved in synthesis of Compounds  They consume energy (Endergonic in nature)  Example :Synthesis of amino acid Catabolic Pathways  They are involved in oxidative breakdown of larger complex molecules  They release energy (Exergonic in nature)  Example: When Glucose is breakdown to Lactic acid in skeletal muscle  Some Pathways can be either catabolic or anabolic, They are referred as amphibolic pathways.  Example: Citric acid cycle.  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya Latur, (Autonomous) Course Title : Metabolism Course Teacher: Dr. Manisha Patil  Fermentation  Pyruvate, the end product of glycolysis has two fates 1. In the presence of oxygen it undergoes aerobic respiration 2. In the absence of oxygen it undergoes anaerobic respiration and Fermentation. Anaerobic Respiration 1. It is different from fermentation 2. The final electron acceptor are inorganic molecules (Nitrate, Carbonate, Sulfate etc) Fermentation 1. It is anaerobic energy yielding process. 2. The final electron acceptor are organic molecules. 3. It is a self contained process and no outside electron acceptor is involved. 4. It does not involve an electron transport system. 5. In absence of oxygen during fermentation NAD+ is regenerated from NADH by the transfer of electron to organic molecule.(ethyl alcohol, lactic acid)  Examples of Fermentation 1.Lactic Acid Fermentation Glucose 2 NAD+ Glycolysis 2 NADH 2 Pyruvate 2 NADH Lactate dehydrogenase 2 NAD+ 2 Lactic Acid  Muscle cells and and certain bacterial species (eg. Lactobacillus) oxidize NADH by transforming Pyruvate to lactate.  This process is known as Lactic Acid Fermentation.  This reaction is catalyzed by LDH.  LDH exists in multiple forms in animal tissue.  Cori Cycle Glycolysis Gluconeogenesis Glucose Glucose 2 Pyruvate 2 Pyruvate 2 Lactate 2 Lactate Skeletal muscle Blood Liver  In animals lactic acid formed in the muscle is recycled to glucose in the liver.  Lactic acid produced in muscle is transported from muscle to liver, where it is reoxidized by Liver LDH to pyruvate.  By the process of gluconeogenesis, pyruvate is finally converted to glucose in the liver  Liver again exports/transports glucose to muscle for glycolysis.  This cycle is known as Cori cycle  It is also known as lactic acid cycle  The cori cycle is named for Carl and Gerty Cori.  They received the noble prize in physiology or medicine in 1947 for their studies on glycogen metabolism and blood glucose regulation.  Examples of Fermentation 2.Alcoholic Fermentation Glucose 2 NAD+ Glycolysis 2 NADH 2 Pyruvate Pyruvate 2CO2 Decarboxylase 2 Acetaldehyde 2 NADH Alcohol 2 NAD+ Dehydrogenase 2 Ethanol  In Alcoholic fermentation, pyruvate is converted to ethanol.  It occurs in yeast and some bacterial species  In yeast, pyruvate is decarboxylated to form acetaldehyde is catalyzed by pyruvate decarboxylase.  Acetaldehyde is then reduced to form ethanol by alcohol dehydrogenase.  The first step is non-oxidative decarboxylation and second step is NADH dependent reduction.  Pyruvate decarboxylase require TPP as a cofactor.  Comparison Between Parameters Aerobic Anaerobic Fermentation Respiration Respiration Condition Oxygen Oxygen Oxygen dependent independent independent Final electron Molecular Inorganic Organic Molecule acceptor oxygen Molecule Type of 1.Substrate level 1.Substrate level Substrate level Phosphorylation Phosphorylation Phosphorylation Phosphorylation used to generate 2.Oxidative 2.Oxidative ATP Phosphorylation Phosphorylation  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya, Latur (Autonomous) Course Title : Metabolism Course Teacher: Dr. Manisha Patil  Pyruvate Oxidation  In the presence of oxygen, further oxidation of Pyruvate occurs in mitochondrial matrix (in Eukaryotes)  In cytosol in case of prokaryotes.  In mitochondrial matrix Pyruvate first oxidizes into acetyl-CoA Life Sciences : fundamental and practice, sixth edition, pathfinder publication Conversion of Pyruvate to acetyl –CoA. It is the junction between glycolysis and citric acid cycle. Pyruvate is a charged molecule, so it enters in mitochondria with the help of transport protein.  The Pyruvate Dehydrogenase Complex catalyzes the process of oxidative decarboxylation. In the overall reaction  In the reaction , the carbonyl group of pyruvate is lost as CO2.  While remaining two carbons forms acetyl –CoA.  The reaction is highly exergonic.  It is irreversible in vivo.  Pyruvate dehydrogenase complex is assemly of three enzymes.  Pyruvate Dehydrogenase Complex (E.coli) Enzyme Function No. of Cofactors Polypeptide Pyruvate Decarboxylation 24 TPP dehydrogenase and oxidation of (E1) pyruvate Dihydrolipoyl Catalyzes transfer 24 Lipoic acid, transacetylase of acetyl group to CoA (E2) CoA Dihydrolipoyl Reoxidizes 12 NAD+, FAD dehydrogenase dihydrolipoamide (E3) In Eukaryotes, this enzyme contains small amount of two regulatory enzymes 1.Kinase:phosphorylates serine 2.Phosphatase: removes phosphate Inhibitors of enzyme :Arsinite and Mercuric ions Mechanism of action of Pyruvate dehydrogenase complex Step 1:Decarboxylation of pyruvate occurs with formation of hydroxy ethyl-TPP. Step 2: Transfer of two carbon unit to lipoic acid. Step 3: formation of acetyl-CoA. Step 4:Lipoic acid is re-oxidized. Life Sciences : fundamental and practice, sixth edition, pathfinder publication  Regulation of Pyruvate Dehydrogenase Complex Enzyme Activator Inhibitor E2 CoA Acetyl CoA (Allosteric inhibition) E3 NAD+ NADH (Allosteric inhibition) Covalent Modification occurs only in Eukaryotes (Phosphorylation/Dephosphorylation) :E1  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya, Latur (Autonomous) Course Title : Metabolism Course Teacher: Dr.Manisha Patil  Oxidative Phosphorylation  Most of the free energy released during oxidation of glucose to CO2 is retained in the reduced coenzymes NADH and FADH2 generated during glycolysis and TCA cycle.  Electrons are released from NADH and FADH2 and eventually transferred to O2 Life Sciences : fundamental and practice, sixth edition, pathfinder publication  The Standard Free energy change for these exergonic reactions are -52.6 kcal/mol (NADH) and -43.14 kcal/mol (FADH2 ).  The large amount of energy released during oxidation of NADH and FADH2 is used in the formation of ATP.  This energy conversion process is termed as Oxidative Phosphorylation. Electron Transport Chain  Electrons are transferred from NADH/FADH2 to O2 through a series of electron carriers  Electron carriers are present in inner mitochondrial membrane.  In prokaryotes they are present in plasma membrane.  In 1948 Eugene Kennedy and Albert Lehninger discovered mitochondria are site of oxidative phosphorylation in eukaryotes.  The process of electron transport begins when hydride ion is removed from NADH and converted to proton and two electrons.  Electron carriers involved are grouped into four large respiratory enzyme complexes each containing transmembrane proteins that hold the complex.  The Four Major Enzyme complex in ETC are  NADH- CoQ reductase or NADH dehydrogenase (Complex I)  Succinate-CoQ reductase (Complex II)  CoQ- Cytochrome C reductase or Cyt.bc1 complex (Complex III)  Cyt C Oxidase (Complex IV)  Complex I, II and III are associated in a supramolecular complex termed respirasome. Enzyme Complex Prosthetic group Complex I (46 subunits) FMN, Fe-S Complex II (4 subunits) FAD, Fe-S Complex III (11 subunits) Heme, Fe-S Complex IV (13 subunits) Heme, Cu+ Overview of electron flow through respiratory chain Life Sciences : fundamental and practice, sixth edition, pathfinder publication Complex I  It is a large , multisubunit complex  it causes oxidation of NADH and passes electron from NADH to Coenzyme Q.  It contain 1 molecule of FMN (flavin mononucleotide) and 6 to 7 Fe-S centers.  During transport of each pair of electron from NADH to CoQ , Complex 1 pumps four protons across inner mitochondrial membrane Life Sciences : fundamental and practice, sixth edition, pathfinder publication   Fe-S Prosthetic group consist of non-heme iron complexed with sulphur.  There are two common types of Fe-S centers 1. [2Fe-2S] 2. [4Fe-4S]  These Fe-S centers consist of equal numbers of iron and sulphide ions  They are coordinated to four Cys sulfhydryl group of proteins Life Sciences : fundamental and practice, sixth edition, pathfinder publication Coenzyme Q (CoQ)  It is also known as ubiquinone  It is a benzoquinone linked to a number of isoprene units.  The name ubiquinone is for ubiquitous nature of quinone.  Q refers to quinone Chemical group.  There are redox state of COQ  Fully oxidized (ubiquinone, Q)  Partially reduced semiquinone(ubisemiquinone)  Fully Reduced (Ubiquinol, QH2) Life Sciences : fundamental and practice, sixth edition, pathfinder publication  CoQ is the only electron carrier in ETC that is not a protein bound prosthetic group.  It is a carrier of Hydrogen atoms ( Proton + electrons) Complex II  Succinate dehydrogenase converts succinate to fumarate during Krebs cycle.  it is a inner mitochondrial bound Enzyme.  It is a integral component of Complex II.  The 2 electrons released during the conversion are transferred to FAD , then Fe-S center and finally to CoQ.  Thus CoQ draws electrons from both NADH and FADH2 in ETC. Life Sciences : fundamental and practice, sixth edition, pathfinder publication Complex III Life Sciences : fundamental and practice, sixth edition, pathfinder publication  Within Complex III , the electrons are transferred from CoQ to Fe-S center [2Fe-2S] to Cyt c1 to Cyt C  Subsequently electrons are transferred to b type cytochromes.  b type cytochromes 1. bL or b566- Low affinity (L) 2.bH or b562- High affinity (H)  Complex III pumps four protons across inner mitochondrial membrane  The Mechanism involved in Proton pumping is called proton motive Q cycle. Life Sciences : fundamental and practice, sixth edition, pathfinder publication  It was proposed by Peter Mitchell.  Ubiquinone are hydrophobic and uncharged  Diffusion of one ubiquinol (QH2) takes place at Qp site,  In the first half cycle, 2e- are transferred from QH2.  one electron is transferred to Fe-S then to Cyt C1 and other to bL then to bH and finally to Q to form semiquinone  In the second half cycle, second QH2 gives 2e-  one electron is transferred to Fe-S then to Cyt C1 and other to bL then to bH and finally to Semiquinone to form ubiquinol Note: Cytochromes 1. They are heme proteins 2. They are classified as b, c, a depending on wavelength  Two b type cytochrome:b566 (bL) and b562 (bH)  Two c type cytochrome : c and c1  Two a type cytochrome :a and a3  Complex IV Life Sciences : fundamental and practice, sixth edition, pathfinder publication  Complex IV catalyzes transfer of electrons from Cyt c reductase to molecular oxygen.  It consist of 13 subunits, 2 Heme groups and 3 copper ions arranged as two copper centers  The two heme groups are called heme a and heme a3  They are located in different environments within complex IV, therefore have different properties.  Two copper centers are a and b (Cua and Cub)  Cua : two copper ions linked by two Cys residues  Cub: one copper ions linked by three Histidine residues The electron transfer pathway Cyt c Cua Cyt a Cub Cyt a3 O2  The final electron acceptor is O2, yielding H2O.  Complex IV pumps two protons (2H+)  Inhibitors of Electron Transport Chain Types of Interference Compound Target/Mode of action Inhibitors of electron Rotenone (Complex I) transfer Prevents electon transfer from Fe-S center to Amobarbital (Amytal) ubiquinone Piericidin A Myxothiazol Antimycin A (Complex III) Blocks Electron transfer from Cytochrome b to Cytochrome C1 Cyanide (Complex IV) Inhibit Cytochrome oxidase Carbon monoxide Inhibits terminal transfer of Azide electrons to oxygen Rotenone  It is a plant product  It is used as fish poison and as an insecticide. Piericidin A  It is an antibiotic  It blocks the transfer of of electrons at complex I by competing with Q. Antimycin A  It is also antibiotic Cyanide, Azide and CO  Cyanide and azide binds to Fe3+ form of Cyt a3 and CO binds with the Fe2+ form of cyt a3  Rotenone Antimycin A CN- or CO Cyt NADH CoQ Cyt b Cyt c1 Cyt c O2 (a.a3) Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya, Latur (Autonomous) Course Title : Metabolism Course Teacher: Dr Manisha Patil  Krebs Cycle  It is also Known as Citric acid cycle or Tricarboxylic acid cycle.  It was discovered by H.A.Kreb a German British Biochemist.  He received Noble Prize in 1953.  The cycle occurs in matrix of mitochondria.( In Eukaryotes)  The cycle occurs in Cytosol. ( In Prokaryotes) Step 1: Formation of citrate  Oxaloacetate reacts with acetyl CoA and water to yields Citrate and CoA.  The reaction is catalyzed by Citrate Synthase Step 2a and 2b: Formation of Isocitrate via cis-Aconitate  The enzyme aconitase catalyzes the reversible transformation of citrate to isocitrate.  An isomerization reaction in which water is first removed and added Back.  Fluoroacetate is an toxic compound (pesticide)  It Blocks Citric acid Cycle by its metabolic conversion of fluorocitrate (inhibitor of aconitase) Step 3: Oxidation of Isocitrate to α-Ketoglutarate and CO2  Isocitrate is oxidized and decarboxylated to α-ketoglutarate.  The carbon carrying the hydroxyl group is converted to carbonyl group.  The product is unstable losing CO2.  The oxidative decarboxylation is catalyze by isocitrate dehydrogenase. Step 4: Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2  A second oxidative decarboxyIation, in which α-ketoglutarate is converted to succinyl-CoA and CO2 by the action of the α- ketoglutarate dehydrogenase complex.  This step produce NADH, CO2 and High Energy thioester bond to CoA. Step 5: Conversion of Succinyl-CoA to Succinate  Conversion of succinyl-CoA to succinate.  This reaction is catalysed by the enzyme succinyl-CoA synthetase or succinic thiokinase.  The reaction releases sufficient energy to form ATP (in plants) or GTP (in animals).  GTP can form ATP through a coupled reaction.  It is Substrate level Phosphorylation Step 6: Oxidation of Succinate to Fumarate  The succinate is oxidized to fumarate by the flavoprotein succinate dehydrogenase.  In eukaryotes succinate dehydrogenase is tightly bound to the mitochondrial inner membrane, in bacteria, to the plasma membrane.  FAD removes two hydrogen atoms from.  The enzyme is strongly inhibited by Malonate.  Classical example of competitive inhibitor. Step 7: Hydration of Fumarate to Malate  The reversible hydration of fumarate to malate is catalyzed by fumarase. Step 8: Oxidation of Malate to Oxaloacetate  In the last reaction of the citric acid cycle, NAD-linked malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate.  The carbon carrying hydroxyl group is converted to carbonyl group.(regeneration of oxaloaceatate) Overall reaction Acetyl-CoA + 3NAD+ + FAD + GDP+3H2O 2CO2 + 3NADH + FADH2 + GTP + H2O  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya , Latur (Autonomous) Course Title : Metabolism Course Teacher: Dr Manisha Patil  Shuttle Systems  The glycolytic pathway is the primary source of NADH formation.  NADH transfers electron to Electron transport chain but NADH cannot cross inner mitochondrial membrane.  So there are two shuttle systems which help to transfer electron from NADH to ETC. 1.Malate –Aspartate Shuttle system 2. Glycerol 3-Phosphate Shuttle system  1.Malate –Aspartate Shuttle system NADH oxaloacetate NAD+ Malate Matrix Aspartate Malate Aspartate Cytosol NAD+ NADH oxaloacetate  Mechanism: Movement of NADH from Cytoplasm to Mitochondrial Matrix.  The electron (e- ) are carried into mitochondrial matrix in the form of malate.  In the cytoplasm oxaloacetate is converted to malate by malate dehydrogenase.  Malate is first transferred into cytoplasm to mitochondrial matrix where the reverse reaction takes place by mitochondrial Malate Dehydrogenase and NADH is regenerated.  Glycerol 3-Phosphate Shuttle system Mitochondrial glycerol Glycerol 3-phosphate dehydrogenase 3-Phosphate NADH Cytosolic FAD glycerol 3-phosphate dehydrogenase NAD+ FADH2 Dihydroxyacetone phosphate Cytosol Matrix  Electrons from NADH enter into ETC by being used to reduced DHAP to Glycerol 3 –Phosphate.  Glycerol 3 –Phosphate is again reqxidized Mitochondrial glycerol 3-phosphate dehydrogenase.  Electrons are transferred to FAD  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya Latur, (Autonomous) Course Title : Metabolism Course Teacher: Dr Manisha Patil  Anaplerotic Reaction  Anaplerotic Reactions replenish Citric Acid Cycle intermediates as they serve as biosynthetic precursors.  Kornberg proposed the term anaplerotic or filling up reactions. First Anaplerotic reaction In animals,(mammalian liver and kidney) the most important anaplerotic reaction is catalyzed by pyruvate carboxylase, a mitochondrial enzyme Second Anaplerotic reaction In plants and bacteria ,an alternative route leads directly from Third Anaplerotic reaction In heart and muscles, PEP PEP Carboxykinase Oxaloacetate Fourth Anaplerotic reaction In Prokaryotes and Eukaryotes  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya, Latur (Autonomous) Course Title : Metabolism Course Teacher: Dr. Manisha Patil  Electrochemical Proton Gradient  Transfer of electrons through electron transport chain is accompanied by pumping of protons across inner mitochondrial membrane. From the mitochondrial matrix to intermembrane space  Total 10 H+ ions are translocated from the matrix per electron pair from NADH to O2.  This movement of H+ generates  pH gradient  Voltage gradient pH gradient across the inner mitochondrial membrane (with the pH higher in the matrix than in the intermembrane space) Life Sciences : fundamental and practice, sixth edition, pathfinder publication Voltage Gradient (membrane potential) across inner mitochondrial membrane(with inside negative and outside positive) Life Sciences : fundamental and practice, sixth edition, pathfinder publication  The pH gradient (∆pH) and voltage gradient together constitute electrochemical proton gradient.  It exerts a proton motive force (pmf)  A mitochondrion actively involved in aerobic respiration have membrane potential of about 160mV (Negative inside the matrix)  pH gradient = 1 pH unit (Higher on matrix side)  Membrane Potential can be determined by adding radioactive K+  Ions and trace amount of valinomycin Note: Extra points  Valinomycin is a ionophore  Inner membrane is impermeable to K+ ions but valinomycin binds K+ ion and carries across membrane Rajarshi Shahu Mahavidyalaya, Latur (Autonomous) Course Title : Metabolism Course Teacher: Dr. Manisha Patil  Pasteur Effect Louis Pasteur observed that during alcohol fermentation , A. Yeast when grown under aerobic condition:  Glucose consumption drops  Ethanol production drops B. Yeast when grown under anaerobic condition:  Glucose consumption increases several fold  Ethanol production increases Reason for decrease in consumption of glucose is that  fermentation results in production of 2 ATPs per Glucose  Where as aerobic respiration yields 32 ATPs per glucose.  hence for generation of same amount of ATP to perform essential metabolic activities more consumption of glucose is needed in anaerobic condition  Pasteur’s observation : yeast consumes more glucose when growing anaerobically than aerobically is called pasteur effect  Warburg Effect  Most cancer cells exhibit increased glycolysis followed by Lactic Acid Fermentation even in presence of Oxygen and used this metabolic pathway for generation of ATP as a main source of energy supply.  This phenomenon is known as Warburg effect (Aerobic Glycolysis)  Respiratory quotient  Respiration involves oxidation of respiratory substrates such as glucose and fats.  The oxidation involves release of CO2 along with release of energy.  The organic substances which are catabolised in the living cells to release energy are called respiratory substrates.  Respiratory quotient / Respiratory coefficient is defined as ratio of moles of CO2 produced to the moles of oxygen consumed during complete oxidation of metabolic fuel to CO2 and H2O.  mol of CO2 produced RQ = mol of O2 consumed For example 1. For carbohydrates (Glucose) C6H12O6 + 6O2 6CO2 +6H2O RQ = 6 mol of CO2 produced 6 mol of O2 consumed Therefore RQ for carbohydrates is 1 2. For Fats (Palmitate) C16H32O2 + 23O2 16CO2 +1 6H2O RQ = 16 mol of CO2 produced 23 mol of O2 consumed Therefore RQ for Palmitate is 0.7 2. For organic acids (Malic acid) C4H6O5 + 3O2 4CO2 + 3H2O RQ = 4 mol of CO2 produced 3 mol of O2 consumed Therefore RQ for Malic acid is 1.3  Name of the substance and their RQ Carbohydrates 1 Proteins 0.8-0.9 Oleic acid (Lipid) 0.71 Malic acid 1.33 Oxalic acid 4.0  P/O ratio  Estimation of number of ATP molecules formed during aerobic state can be calculated from P/O ratio.  The ratio of ATP synthesized to oxygen reduces to water molecules.  During transport of two electrons from NADH to oxygen about 10 protons are transported into intermembrane space.  During transport of two electrons from FADH2 to oxygen about 6 protons are transported into intermembrane space.  ATP synthase requires 4 protons (H+) for 1 ATP (3 protons for ATP synthesis and 1 proton is used to transport ATP,ADP and Pi across mitochondrial membrane.  The P/O ratio for the oxidation of NADH is 2.5 and for FADH2 is 1.5  NADH --- 10H+ 10/4 =2.5  FADH2 --- 6H+ 6/4 =1.5  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya, Latur (Autonomous) Course Title : Metabolism Course Teacher: Dr. Manisha Patil  ATP Synthase Life Sciences : fundamental and practice, sixth edition, pathfinder publication  The use of proton motive force for ATP synthesis is catalyzed by ATP Synthase.  It is also Known as FoF1 complex/Complex V.  It consist of two components 1.F0 component 2.F1 ATPase Fo component  It is embedded in inner mitochondrial membrane  It contain one a subunit, Two b subunit, 9-12 c subunit  C subunit consist of two α helices that span the membrane  Aspartic acid residues lies in the center of second helix  It regulates proton channel (H+)  Oligomycin (antibiotic) blocks ATP synthesis by blocking flow of protons through Fo ( Fo – “ O” subscript denotes inhibition by oligomycin) 2.F1 ATPase  It is made up of 3α ,3β,γ,δ,ε  It is tightly bound to Fo and protrudes in matrix  3β subunits are site of ATP synthesis  At the center of F1 ATPase is γ subunit which interacts with Fo  γε and C9-12 ring complex is rotor (moving unit)  a,2b,3α,3β,δ complex is stator (stationary unit)  Mechanism of ATP synthesis  Binding change mechanism model is widely accepted for ATP synthesis.  Paul Boyer developed this model.  It is also known as Flip Flop mechanism.  Due to proton translocation through Fo γ subunit of F1 ATPase rotates at 120°, which leads to confirmation change of nucleotide binding site (F1 ATPase – β subunits).  The three F1 β subunits alternate between three conformational state that differ in the binding affinity for ATP, ADP and Pi. They are  An “O” state (open state) that binds ATP, ADP & Pi very weakly.  An “L” state (Loose state) that binds ADP & Pi loosely.  An “T” state (Tight state) that binds ADP & Pi very tightly.  The Phosphoanhydride bond of ATP a synthesized in T state & ATP is released in “O” state.  A 120° rotation of γ subunit in counter clockwise direction changes one confirmation state to another. O→L → T → O Life Sciences : fundamental and practice, sixth edition, pathfinder publication  In stage 1, the open state “ O” is empty, The L sate contains ADP + Pi and T state contains ATP.  In logical intermediate stage : due to rotation of γ , L sate is converted to T, the T state is converted O and O state converted to L, the L state can now accept new substrate.  In Stage II ATP is fallen of O State, new ADP and Pi bound to L State and ATP is synthesized at T state Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Uncoupling agents and Ionophores Uncoupling agents  They uncouple oxidation from phosphorylation.  They allow oxidation of NADH and FADH2 and reduction of oxygen.  But block ATP synthesis Uncouplers Mode of action DNP (2-4-Dinitrophenol) Inhibits ATP Dicoumarol synthesis FCCP (carbonyl cyanide –p- (trifluromethoxy) phenylhydrazone Thermogenin Ionophores  They are lipophilic molecules that binds specific cations and facilitate their transport through the membrane  Example Valinomycin is an antibiotic an example of Ionophore  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya, Latur (Autonomous) Course Title : Metabolism Course Teacher: Dr. Manisha Patil  Chemiosmotic Theory  In 1961 Peter Mitchell Proposed Chemiosmotic theory of oxidative phosphorylation.  This model propose that energy from electron transport drives an active transport system, which pumps protons from matrix to inter membrane space which generates proton gradient.  When protons flow back to matrix, energy is dissipated and some of it is used for synthesis of ATP. Life Sciences : fundamental and practice, sixth edition, pathfinder publication  General Mechanism for Oxidative Phosphorylation  As a high energy electron is passed along the ETC  Some of the energy released is used to drive three respiratory enzyme complex that pumps H+ out of the matrix space  The resulting proton gradient across inner membrane drives H+ back through ATP synthase.  Experimental proof of chemiosmotic hypothesis  It was provided by Andre Jagendorf and Ernest uribe (1966)  Isolated chloroplast thylakoid vesicle containing F0F1 particles were equilibrated in the dark with a buffered solution at pH 4.0.  When the pH of thylakoid membrane became 4.0, the vesicles were mixed with a solution at pH 8.0 containing ADP and Pi  Conclusion :Synthesis of ATP by F0F1 depend on a pH Gradient Life Sciences : fundamental and practice, sixth edition, pathfinder publication  Rajarshi Shahu Mahavidyalaya (Autonomous), Latur Course Title : Metabolism Course Teacher: Dr Manisha Patil  UNIT II Fate of Light absorbed by photosynthetic pigments  Fate of Light absorbed by photosynthetic pigments  Each photon represents a quantum of light energy  A molecule of chlorophyll on the absorption of light becomes excited to higher energy state.  An excited chlorophyll molecule is not stable.  It returns rapidly to the ground state (unexcited state) in three possible ways Life Sciences : fundamental and practice, sixth edition, pathfinder publication  By converting extra energy into heat or some combination of heat and light of longer wavelength ( fluorescence)  By transferring the energy directly to neighboring chlorophyll molecule  By transferring the high energy electron to another nearby molecule (electron acceptor) and returning to its original state by taking low energy electron from other molecule (electron donor)  By converting extra energy into heat or some combination of heat and light of higher wavelength (fluorescence)  An excited molecule in a singlet state has maximum lifetime of ≈ 10-8 sec.  An excited molecule can undergo various modes of non radioactive as well as radioactive decay.  Radioactive decay is called luminescence  If the luminescence arises because of transition of electrons from a singlet excited state to ground state, it is also called fluorescence.  If the luminescence arises because of transition of electrons from a triplet excited state to ground state, it is also called phosphorescence. S0 represents ground state S1 and S2 lowest vibrational energy level of first and second excited singlet state T1 lowest vibrational energy level of triplet state. Horizontal lines represents vibration energy level associated with electronic States  By transferring the energy but not the electrons directly to neighboring chlorophyll molecule this process is called resonance energy transfer.  It is also known as Forster transfer  The excited energy is trapped by the reaction center because the excited state of chlorophyll has lower energy than that of antenna molecule  By transferring high energy electron to another nearby molecule ( electron acceptor ) and then returning to its original state by taking low-energy electron from some other molecules (electron donor ) this is known as photochemical reaction.  photochemical reaction is one of the fastest Known chemical reaction  Absorption and action spectra  Photosynthetic pigments absorbs electromagnetic radiation in visible and infrared region.  The graph showing degree of absorption of light by a pigment as a function of wavelength is referred as absorption spectrum for that pigment.  Chlorophyll absorbs strongly in the blue violet and red region of spectrum while carotenoids absorbs in the blue and green region.  A graph showing the degree to which different wavelengths affects photochemical reaction is called action spectrum for photosynthesis Pigment Absorption maxima (nm) Chlorophyll a 430, 660 Chlorophyll b 463, 643 Bacteriochlorophyll a 364, 770 Bacteriochlorophyll b 373, 795 Bacteriochlorophyll c 434, 666 In 1883 Theodor W. Engelmann determine the first action spectra by measuring oxygen evolution at different wavelengths of light  He projected a spectrum of light on filamentous chloroplast of green alga spirogyra and observe that oxygen seeking bacteria introduced into the system concentrated in the region of spectrum where chlorophyll pigments absorbs.  This action spectrum give the first indication of effectiveness of light absorbed by accessory pigments in driving photosynthesis  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya (Autonomous), Latur Course Title : Metabolism Course Teacher: Manisha Patil  UNIT II Photosynthesis Cyclic Electron Flow  In cyclic electron flow only PSI is involved.  The photoexcited electrons from P700 of PSI moves through Cytb6f complex and back to P700.  It is coupled to proton pumping in the thylakoid lumen and proton flows down their electrochemical gradient through ATP synthase complex.  ATP synthesis occurs.  Formation of ATP due to light induced cyclic electron flow is called cyclic photophosphorylation.  There is no production of NADPH.  No release of oxygen  In Plants cyclic electron flow is utilized only when concentration of NADPH is sufficient but still require ATP to power the activities of chloroplast.  it is more productive as compared to non cyclic photophosphorylation with regard to ATP synthesis.  Absorption of photons by PSI results in release of 8 protons in the lumen of cytb6f.  This protons flow through ATP synthase to yield three molecules of ATP Difference between Cyclic and Non Cyclic Electron Flow Non Cyclic Electron Flow Cyclic Electron Flow Both PSI and PSII Only PSI Photolysis of water No Photolysis of water Formation of Oxygen No Oxygen Formation ATP synthesis occurs ATP synthesis occurs NADPH synthesis occurs No NADPH synthesis Difference between Oxidative Phosphorylation and Photophosphorylation Oxidative Phosphorylation Photophosphorylation ATP synthesis driven by oxidation ATP synthesis driven by Light of NADH and FADH2 Light-independent Process Light-dependent process Occurs during aerobic respiration Occurs during Photosynthesis In Eukaryotes, Occurs in In Eukaryotes, Occurs in Mitochondria Photosynthesis Involves the reduction of O2 to H2O Involves the oxidation of H2O to O2 with electrons donated by NADH with NADP+ as electron acceptor and FADH2  Prokaryotic Photosynthesis  There are three groups of photosynthetic bacteria 1. Purple bacteria 2. Green bacteria 3. Cyanobacteria  Cyanobacteria : 1. They carry oxygenic photosynthesis 2. They have PSI and PSII similar to plants 3. They also use water as electron donor and generate oxygen during photosynthesis  Purple and green bacteria: 1. They have only one type of reaction centre they perform anoxygenic photosynthesis 2. It is also called bacterial photosynthesis  In purple bacteria the reaction center is similar to photosystem II and in green bacteria photosystem I  Comparison Property Oxygenic Photosynthesis Anoxygenic Photosynthesis Photosynthetic Chl a BChl Pigment Photosynthetic H2 O H2,H2S,S, Organic Electron Donor matter O2 Production Present Absent Primary Product of ATP + NADPH ATP energy Conversion Carbon Source CO2 Organic Compound and/or CO2  Purple Photosynthetic Bacteria There are two divisions of photosynthetic purple bacteria 1. non-sulfur purple bacteria ex. Rhodobacter sphaeroides and Rhodopseudomonas viridis 2. sulfur purple bacteria ex. Chromatium vinosum  Purple Sulfur Purple Non Green Sulfur Green Non Sulfur Sulfur Photosynthetic BChl a and b BChl a and b BChl a plus c,d BChl a and c Pigments or e Location Plasma Plasma Plasma Plasma membrane and membrane and membrane and membrane and chromatophore chromatophore chlorosomes chlorosomes Electron H2,H2S,S Organic H2,H2S,S H2,H2S, Donor molecules, Variety of sometimes sugars, amino reduced sulfur acids and compounds or organic acids H2 Metabolic Photolitho Photoorgano Photolitho Photo Type autotrophs heterotrophs autotrophs heterotrophs Non chlorophyll based photosynthesis  oxygenic and anoxygenic photosynthesis are chlorophyll based photosynthesis.  Pigment chlorophyll or bacteriochlorophyll are the major pigment used to absorb light and initiate conversion of light energy to chemical energy.  However some archaea instead of chlorophyll use membrane-bound purple pigment called bacteriorhodopsin.  Ex. Halophile Halobacterium salinarum.  bacteriorhodopsin is similar to pigment rhodopsin found in the mammalian eye.  its function is to light driven proton pump.  it has two distinct components chromophore that is caretenoids derivative, retinal and multipass (seven pass) transmembrane protein.  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication  Rajarshi Shahu Mahavidyalaya (Autonomous), Latur Course Title : Metabolism Course Teacher: Dr Manisha Patil  UNIT II Photosynthesis  Stages of Photosynthesis It is a two stage process  one stage is dependent on light (Light Reactions)  and other independent of light (Dark Reactions)  Light Reactions occur in the Grana of chloroplast and require the direct energy of light to make NADPH and ATP that are used in the dark reaction.  Light reactions are also known as thylakoid reactions because almost all reactions take place in thylakoid( Oxidation of Water, Reduction of NADP+ and ATP formation).  A process of formation of ATP from ADP and inorganic phosphate by utilizing light energy is called for photophosphorylation  Dark reactions occurs in the stroma of the chloroplast where the products of the light reaction ATP and NADPH are used to make Glyceraldehyde-3-phosphate (a triose phosphate) from reduction of carbon dioxide.  Difference Between Light and Dark Reactions Light Reactions Dark Reactions Light Dependent Reaction Light-Independent Reaction Occurs in the Grana of Occurs in the Stroma of Chloroplast Chloroplast Photochemical Reaction Chemical Reaction Formation of ATP and NADPH Utilization of ATP and NADPH Oxidation of H2O Reduction of CO2  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication  Rajarshi Shahu Mahavidyalaya (Autonomous), Latur Course Title : Metabolism Course Teacher: Dr Manisha Patil  UNIT II Photosynthesis  Light Reactions (Photochemical Reaction)  In Oxygenic Photosynthetic Organisms flow of electron is of two types 1.Non-Cyclic 2.Cyclic  Noncyclic electron flow It is the light induced electron transport from water to NADP along with evolution of oxygen. It involves two photosystems PSII and PSI. Electron moves from water through PSII to PS1 and then to NADP+. Electron transport leads to generation of proton motive force and synthesis of ATP Formation of ATP due to light induced non cyclic electron flow is called non cyclic photophosphorylation. It is also called z scheme because of its outline. Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Non cyclic electron flow begins with absorption of photon by PSII (P680) I. It gets excited (P680 to P680*) and rapidly transfers the electron to nearby pheophytin and changes to P680+  Pheophytin is a chlorophyll in which Central magnesium atom is replaced by two hydrogen atoms.  The positively charged P680+ ( P680* to P680+ ) is a strong oxidizing agent.  It attracts electron from electron donor to regenerate original P680. II. Reduced Pheophytin transfers electron to plastoquinone.  Plastoquinone is tightly bound and loosely bound electron carrier  QA (primary quinone, bound to D2)  QB (secondary quinone, bound to D1)  QA is photoreduced only to plastosemiquinone but QB can accept two electrons and two protons forming fully reduced plastoquinol (Plastohydroquinone) III. Plastoquinol diffuses in the membrane and binds with Cyt b6f complex.  Cytb6f function to oxidize plastoquinol and pump proton from the stroma in the thylakoid lumen.  Cytochromes are electron transfer protein containing heme, as prosthetic group.  They are divided into three groups cytochrome a b and c.  In photosynthetic organisms mainly b and c type cytochromes are found.  Cytb6f complex possesses one Rieske Fe-S protein , 2 Cyt b and Cyt c. Cyt C (historically called Cyt f ) IV. Cytochrome f then transfers electron to blue colored protein plastocyanin  plastocyanin is a small water soluble and copper containing protein it is a monomer and located on the luminal side of thylakoid membrane. V Plastocyanin transfers electron to PSI  PS1 contains special pair of chlorophyll known as P700.  It absorbs light (photon) and get excited.(P700 to P700*) VI The excited P700* transfers electron to A0  A0 (modified chlorophyll) VII A0 transfers electron to A1  A1 (phylloquinone , it is also known as vitamin k1). VIII A1 then transfers electron to Fe-S center.  Fe-S centers Fx, FA and FB  They are 4Fe-4S type IX The electrons are then transfer to ferredoxin.  it is a small water soluble Fe-S protein [2Fe-2S]  The soluble flavoproteins ferrodoxin- NADP+ reductase (FNR) catalyzed transfer of electron from reduced ferrodoxin to NADP+.  Two electrons are required for reduction of NADPH.  For each electron transferred from water to NADP two photons are absorbed one by each photosystem.  To form one molecule of oxygen, it requires transfer of four electrons from two water molecules to two NADP+  total 8 photons are absorbed ,4 by each photosystem  In oxygenic photosynthetic organisms water act as electron donor.  Splitting of two water molecules yields 4 electrons, 4 protons and molecular oxygen. 4 hv 2H2O 4H+ + 4e- + O2  Splitting of water is catalyzed by protein complex (oxygen evolving complex).  it is located in luminal surface of thylakoid membrane  it contains several proteins (such as manganese stabilizing protein PsbO, PsbP and PsbQ) as well as 4 manganese together with Calcium ion, Chloride ion and bicarbonate ion.  The four electrons do not pass directly to P680+ ,One electron at a time is accepted.  The oxygen evolving complex oxidized water and passes four electrons one at a time from water molecules and finally release oxygen. The sequential absorption of four photons, each causing the loss of one electron from the Mn center , produces an oxidizing agent that can take four electrons from 2H2O producing O2. The electron lost from the Mn center pass one at atime to Tyr residue (Yz) in the D1 Protein  There is some sort of gear wheel that collects four electron from two water molecule and transfer them one at a time to the reaction centre. Manganese ion cycles through five different state S0 to S4. Each transition is a photon driven redox reaction. This model of photooxidation of water is called a S- State mechanism. Inhibitors 1. DCMU (3-(3,4 dichlorophenyl)-1, 1 dimethyl urea) or Diuron  it is herbicide.  It abolishes oxygen production because it competes with plastoquinone for Qb binding site of PSII and blocks the electron flow from PSII to cytb6f. 1. Paraquat  it acts as Herbicide  It blocks photosynthetic electron flow by intercepting electrons between ferredoxin acceptors and NADP+ and then reduces oxygen to superoxide. ATP synthesis (Photophosphorylation)  In oxygenic photosynthetic organism water act as electron donor.  Photooxidation of 2 water molecule gives 4 electrons, 4 protons and oxygen electrons are transported down the electron transport chain.  Some of the energy released is used to pump protons across the thylakoid membrane from the stroma of the chloroplast to the thylakoid lumen producing electrochemical proton gradient.  electrochemical gradient generate proton motive force.  Absorption of eight Photon by PSII and PS1( 4 photons each)  photooxidation of two water molecules yields 4 protons and one molecule of oxygen in the thylakoid lumen. Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  it was also estimated that transport of four electrons to cytochrome b6f result in formation of 8 protons from the stroma of thylakoid lumen hence total 12 protons are released in the lumen.  The accumulating protons in the thylakoid lumen pass back across thylakoid membrane to the stroma through ATP synthase.  It catalyse ATP synthesis.  ATP synthase produce 1 ATP for every three protons.  Therefore results in the production of four molecules of ATP per molecule of oxygen evolved  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication Rajarshi Shahu Mahavidyalaya (Autonomous), Latur Course Title : Metabolism Course Teacher: Manisha Patil  UNIT II Photosynthesis  Concept of pigment system  In 1943 Robert Emerson and Charlton Lewis examined action spectra in the visible region for oxygen evolution in the green alga Chlorella pyrinoidosa.  Quantum yield remained constant upto 680nm and further declines (decrease) sharply.  This drop in Quantum yield in the Far-Red region of spectrum was called Red Drop Phenomenon. Quantum yield is number of oxygen molecules produce per Photon absorbed Quantum Requirement : Reciprocal of quantum yield is Quantum Requirement. ( i.e number of photons needed for each oxygen molecule produced)  Emerson enhancement effect  Emerson and his colleagues set up two beams of light 1.one beam in Red region (wavelength less than 680nm) 2.other in in far-Red region (wavelength greater than 680nm)  They found at when the two beams were applied simultaneously the rate of photosynthesis was two to three times greater than sum of the rate obtained with each beam separately,  This phenomenon is known as Emerson enhancement effect  This enhancement effect suggests that photosynthesis involves two photosystems 1 one driven by light of longer wavelength (greater than 680nm) 2. other driven by light of shorter wavelength (less than or equal to 680nm  Pigment Systems  In all natural photosynthetic system pigment molecules are bound to proteins forming pigment protein complex called pigment system (photo system) The pigment system has two components 1.Photochemical Reaction Centre 2. Antenna complex  Photochemical reaction centre carries photochemical reaction They are two types of photochemical reaction centre 1.Fe-S type reaction centre (type I) (PSI) 2. Pheophytin – quinone type reaction centre (type II) (PSII) Antenna complex  It is also known as light harvesting complex  It has two components 1. core or inner antenna 2. peripheral or outer antenna Role of antenna complex  It captures light energy and transfer to reaction centre by a process called resonance energy transfer  The size of the antenna complex varies.  Generally 200 to 300 chlorophylls per reaction centre in higher plants.  All photosynthetic organisms possess antenna complex with the exception of Heliobacteria  In oxygenic photosynthetic organism such as plants and cyanobacteria both types of photosystems are present. 1.Photosystem with Fe-S type reaction center. it is called photosystem I (PSI) 2.Photosystem with pheophytin -quinone type reaction center. It is also called photosystem II (PSII) Photosystem I (PSI)  it is a protein pigment complex that uses light energy to transfer electron from plastocyanin (PC) to ferredoxin  it has Fe-S type reaction centre.  it contains several proteins, Chla , carotenoids, Fe-S center, phylloquinones and other cofactors  In cyanobacteria : it consists of 11 proteins PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaI, PsaJ, PsaK, PsaL, and PSaM  PSI is also called P700 (p stands for pigment)  The photochemical reaction centre of PS1 is called P700 because it is most effectively absorbs light of wavelength 700nm. Photosystem II (PSII)  It is a pigment protein complex Known as PSII.  It has Pheophytin-Quinone type reaction center.  The photochemical reaction centre Chl a of PSII is also known as P680 because it absorbs light of wavelength 680nm.  In higher plants the reaction centre contains membrane proteins D1 and D2 (about 39 kDa each), cytochrome b559 and other proteins along with Chl a, pheophytin, plastoquinone and other cofactors.  The reaction centre is associated with light harvesting antenna complexes 1. core antenna 2. peripheral antenna includes chla/b binding proteins called LHCII Life Sciences : fundamental and practice, sixth edition, pathfinder publication Distribution of PSI and PSII  They both are distributed differently in thylakoid membrane within chloroplast.  PSI is found in non-appressed membranes of grana thylakoid and stroma thylakoid.  PSII is found in appressed membrane of grana thylakoid. Life Sciences : fundamental and practice, sixth edition, pathfinder publication  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication  Rajarshi Shahu Mahavidyalaya (Autonomous), Latur Course Title : Metabolism Course Teacher: Dr Manisha Patil  UNIT II Concept of photosynthetic unit  Concept of photosynthetic unit  In 1932 Robert Emerson and William Arnold provided first evidence for the corporation of many chlorophyll molecules in energy conversion during photosynthesis.  They suggested that all chlorophyll molecules in a chloroplast are not directly involved in photochemical reaction.  Experiment : suspension of algae chlorella and light of saturated intensity for very short duration (≈10 μsec)  Observation : They determined minimum amount of light needed to produce maximum oxygen production during photosynthesis.  Based on the number of chlorophyll molecules they calculated that one molecule of oxygen is released after absorption of 8 photons of light by near about 2400 chlorophyll molecule  They explained that 2400 chlorophyll molecule act as a unit. It is called photosynthetic unit. In this unit most of the chlorophyll molecule (light harvesting antenna complex) absorbs light and transfer the excitation energy to reaction centre, where special pair of Chla performs photochemical reaction. The size of photosynthetic unit differ from species to species between 300 to 5000 chlorophyll molecules Life Sciences : fundamental and practice, sixth edition, pathfinder publication  Oxygenic and Anoxygenic Photosynthesis 1. In anoxygenic photosynthesis  Light energy is captured and converted into ATP without production of oxygen.  therefore water is not used as electron donor 2. In oxygenic photosynthesis  Light energy is captured and converted into ATP with production of oxygen.  Synthesis of oxygen occurs due to photooxidation or photolysis of water  Before 1930 researches considered CO2 as a source of oxygen in oxygenic photosynthesis organism.  This idea was challenged in 1930 by C.B. van Niel of Stanford University.  He proved that oxygen produced by plants is derived from water and not from CO2.  He found that photosynthetic bacteria Chromatium vinosum assimilate CO2 in light without evolving oxygen.  Such bacteria use H2S as an electron donor instead of water and form sulphur instead of oxygen. Light CO2 + 2H2S (CH2O) + H2O + 2S Bchl  The chemical similarity between H2S and H2O led van Neil to propose the general Light CO2 + 2H2A (CH2O) + 2A + H2O Bchl  Where H2A is water in green plants and cyanobacteria and H2S in in photosynthetic sulphur bacteria.  He hypothesized that water act as a source of oxygen.  The validity for this hypothesis was published in the year 1941 by Ruben and Kamen.  They demonstrated by isotopic study using 18O labelled water and CO2 on green alga chlorella that the source of oxygen formed in photosynthesis is water Experiment 1 CO2 + 2H2O (CH2O) + H2O + O2 Experiment 2 CO2 + 2H2O (CH2O) + H2O + O2  References  Bruce Alberts et al., 2012, Molecular Biology of the cell, 5th edition, Garland Science, N.Y.  Harvey Lodish et al., 2013, Molecular Cell Biology, 7th edition,W.H. Freeman and Co.,N.Y.  Lehninger, Nelson and Cox, 2008, Principles of Biochemistry, 4th edition, W.H. Freeman and Co.,N.Y.  Pranav Kumar and Usha Mina ,2017 ,Life Sciences : fundamental and practice, sixth edition, pathfinder publication