Cellular Respiration PDF

CELLULAR RESPIRATION Process in which cells consume O2 and produce CO2 Provides more energy (ATP) from glucose than glycolysis Also captures energy stored in lipids and amino acids Evolutionary origin: developed about 2.5 billion years ago Used by animals, plants, and many microorganisms Occurs in t...

CELLULAR RESPIRATION Process in which cells consume O2 and produce CO2 Provides more energy (ATP) from glucose than glycolysis Also captures energy stored in lipids and amino acids Evolutionary origin: developed about 2.5 billion years ago Used by animals, plants, and many microorganisms Occurs in three major stages: - Stage 1: acetyl CoA (activated acetate) production - Stage 2: acetyl CoA oxidation - Stage 3: electron transfer and oxidative phosphorylation CELLULAR RESPIRATION: THREE STAGES Catabolism of proteins, fats, and carbohydrates in the three stages of cellular respiration The breakdown of glucose in the glycolysis pathway leads to the production of 2 molecules of pyruvate, ATP and NADH each. In most eukaryotic organisms, and in many bacteria that live under aerobic conditions, the pyruvate is further oxidized to CO2 and H2O. This aerobic phase is called respiration, more precisely cellular respiration, because in a macroscopic sense, respiration means uptake of O2 and release of CO2 by an organism. Cellular respiration occurs in three major stages: Stage 1: Acetyl-CoA production: oxidation of fatty acids, glucose, and some amino acids yields acetyl-CoA. Generates some ATP, NADH and FADH2 Stage 2: Acetyl-CoA oxidation in the citric acid cycle (4 steps in which electrons are abstracted). Generates more NADH and FADH2 and one GTP Stage 3: Oxidative phosphorylation: electrons carried by NADH and FADH2 are funneled into a chain of mitochondrial (or, in bacteria, plasma membrane-bound) electron carriers - the respiratory chain ultimately reducing O2 to H2O. This electron flow drives the production of ATP. Generates a lot of ATP IN EUKARYOTES, CITRIC ACID CYCLE OCCURS IN MITOCHONDRIA Glycolysis occurs in the cytoplasm Citric acid cycle occurs in the mitochondrial matrix* Oxidative phosphorylation occurs in the mitochondrial inner membrane *Except succinate dehydrogenase step, which is located in the inner membrane (see ETC) STAGE 1 OF CELLULAR RESPIRATION: PRODUCTION OF ACETYL-CoA (ACTIVATED ACETATE) Organic molecules (glucose and other sugars, fatty acids and most amino acids) are oxidized to yield 2-carbon fragments that form the acetyl group in Acetyl Coenzyme-A (Acetyl Co-A): Pyruvate derived from glucose and other sugars (Glycolysis), is oxidized to Acetyl CoA and CO2 by Pyruvate Dehydrogenate (PDH) Complex Fatty acid oxidation leads to acetyl CoA Some amino acid oxidation leads to acetyl CoA STAGE 1 OF CELLULAR RESPIRATION: CONVERSION OF PYRUVATE TO ACETYL-CoA Net Reaction: – Oxidative decarboxylation of pyruvate – First carbons of glucose to be fully oxidized – Formation of NADH (1 NAD+ is reduced to NADH) – ΔG negative: highly spontaneous, because of high energy thioester bond that is formed in intermediate step Catalyzed by the pyruvate dehydrogenase complex (PDH) – Requires 5 coenzymes (4 of which are derived from vitamins) – TPP, lipoyllysine, and FAD are prosthetic groups – NAD+ and CoA-SH are co-substrates The products acetyl-CoA, a high energy thioester, and NADH conserve some of the energy resulting from the decarboxylation of pyruvate. PYRUVATE DEHYDROGENASE COMPLEX (PDH or PDC) PDH complex is a large (up to 10 MDa) multienzyme complex - pyruvate dehydrogenase (E1) - dihydrolipoyl transacetylase (E2) - dihydrolipoyl dehydrogenase (E3) Advantages of multienzyme complexes: ‒ short distance between catalytic sites allows channeling of substrates from one catalytic site to another ‒ channeling minimizes side reactions ‒ regulation of activity of one subunit affects the entire complex Note: Deficiency in PDH (on E1), although rare, causes congenital lactic acidosis (decreased ability to convert pyruvate into acetyl CoA à pyruvate is directed to lactate formation (LDH reaction) à affects the brain (neurodegeneration, or early death in neonatal=onset form STRUCTURE OF THE PDH COMPLEX (FROM CRYO-ELECTRON MICROSCOPY0 The PDH complex consists of 3 distinct enzymes Pyruvate Dehydrogenase - E1 Dihydrolipoyl Transacetylase - E2 Dihydrolipoyl Dehydrogenase - E3 The core (green) consists of 60 molecules of E2, arranged in 20 trimers to form a pentagonal dodecahedron. The lipoyl domain of E2 (blue) reaches outward to touch the active sites of E1 molecules (yellow) arranged on the E2 core. Several E3 subunits (red) are also bound to the core, where the swinging arm on E2 can reach their active sites OVERALL REACTION OF PDH Oxidative decarboxylation of pyruvate to acetyl-CoA by the PDH complex Lipoic acid is attached with an amide link to a side chain of lysine on E2. This provides a long arm that can swing from the active site of E1 to the active sites of E2 and E3 and back. (See also pyruvate decarboxylase reaction) From pyruvate to acetyl CoA Step 1 pyruvate reacts with the bound thiamine pyrophosphate (TPP) of pyruvate dehydrogenase (E1), undergoing decarboxylation to the hydroxyethyl derivative. Step 2, the transfer of two electrons and the acetyl group from TPP to the oxidized form of the lipoyllysyl group of the core enzyme, dihydrolipoyl transacetylase (E2), to form the acetyl thioester of the reduced lipoyl group. Step 3 is a transesterification in which the -SH group of CoA replaces the —SH group of E2 to yield acetylCoA and the fully reduced (dithiol) form of the lipoyl group. Step 4 dihydrolipoyl dehydrogenase (E3) promotes transfer of two hydrogen atoms from the reduced lipoyl groups of E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group of E2. Step 5 the reduced FADH2 of E3 transfers a hydride ion to NAD+, forming NADH. The enzyme complex is now ready for another catalytic cycle. SEQUENCE OF EVENTS IN OXIDATIVE DECARBOXYLATION OF PYRUVATE Enzyme 1: Pyruvate Dehydrogenase Step 1: Decarboxylation of pyruvate to an aldehyde, formation of CO2 (product 1) Step 2: Oxidation of aldehyde to a carboxylic acid ‒ Electrons reduce lipoamide and form a thioester Enzyme 2: Dihydrolipoyl Transacetylase Step 3: Formation of acetyl-CoA (product 2) Enzyme 3: Dihydrolipoyl Dehydrogenase Step 4: Reoxidation of the lipoamide cofactor Step 5: Regeneration of the oxidized FAD cofactor ‒ Forming NADH (product 3) REMINDER: ROLE OF THIAMINE PYROPHOSPHATE (TPP) IN PYRUVATE DECARBOXYLATION (IN STEP 1) Role of TPP in pyruvate decarboxylation.TPP is the coenzyme form of vitamin B1 (thiamine). The reactive carbon atom in the thiazolium ring of TPP is shown in red. In the reaction catalyzed by pyruvate decarboxylase, two of the three carbons of pyruvate are carried transiently on TPP in the form of a hydroxyethyl, or "active acetaldehyde," group, which is subsequently released as acetaldehyde. The thiazolium ring of TPP stabilizes carbanion intermediates by providing an electrophilic (electrondeficient) structure into which the carbanion electrons can be delocalized by resonance. Structures with this property, often called "electron sinks," play a role in many biochemical reactions here, facilitating carboncarbon bond cleavage. ROLE OF LIPOYL-LYSINE IN ACCEPTING THE ACETYL GROUP (IN STEP 2) The lipoyllysyl moiety is the prosthetic group (strongly bound to the protein) of dihydrolipoyl transacetylase (E2 of the PDC complex). The lipoic acid is covalently linked to the enzyme via a lysine residue The lipoyl group occurs in oxidized (disulfide) and reduced (dithiol) forms and acts as a carrier of both hydrogen and an acetyl (or other acyl) group. The oxidized form of the lipoyl residue can accept electrons to form two sulfhydryl groups, one of these can subsequently act as an acyl carrier. The oxidation state of the acetyl group is that of a carboxylic acid. The transfer of the “active aldehyde” group to the reduced form of the lipoyl residue involves the oxidation of the former. Two electrons and the acetyl group are transferred from TPP to the oxidized form of the lipoyllysyl group of the core enzyme, dihydrolipoyl transacetylase (E2), to form the acetyl thioester of the reduced lipoyl group. BIOLOGICAL TETHERS ALLOW FLEXIBILITY (RECALL BIOTIN) The cofactors lipoate, biotin, and the combination of βmercapto-ethylamine and pantothenate form long, flexible arms (blue) on the enzymes to which they are covalently bound, acting as tethers that move intermediates from one active site to the next. The group shaded light red is in each case the point of attachment of the activated intermediate to the tether. ROLE OF LIPOYL-LYSINE IN TRANSFER OF THE ACETYL GROUP TO COENZYME A (IN STEP 3). FORMATION OF ACETYL CoA Lipoyl-lysine After a swing of the long arm of Lipoyl-lysine (not shown), the acetyl group is transferred to the SH group of CoA forming acetylCoA ROLE OF FAD IN THE REOXIDATION OF LIPOAMIDE (IN STEP 4) AND ROLE OF NAD (IN STEP 5) In step 4, after a further swing of the reduced lipoyllysine arm, the two -SH groups come in contact with the oxidized form of another coenzyme, FAD which is reduced to FADH2, while the two –SH groups once again form the disulfide bridge of the initial stage. In Step 5, FADH2 is reoxidized to FAD as the electrons are transferred to NAD+ to form NADH and H+. Central to the mechanism of the pyruvate dehydrogenase reaction are the swinging arms of the lipoyllysine group of E2, which accept the acetyl group and the electrons derived from pyruvate and passes the acetyl group to CoA and the electrons to FAD of E3. All enzymes and coenzymes are clustered, allowing the intermediates to react quickly, never leaving the complex. This mechanism is an example of substrate channeling. SUBSTRATE CHANNELING IN THE THE PDH: INTERMEDIATES NEVER LEAVE THE ENZYME SURFACE Substrate channeling: Movement of the chemical intermediates in a series of enzyme-catalyzed reactions from the active site of one enzyme to that of the next enzyme in the pathway, without leaving the surface of a protein complex that includes all enzymes. Another example: substrate channeling in the Citrate Synthase and Malate DHase metabolon in the CAC Intermediates react quickly because: - they never leave the surface of enzyme complex, - they do not diffuse away from enzyme complex, - their local concentration is high, - the loss of activated groups to other enzymes is prevented. STAGE 2 OF CELLULAR RESPIRATION: ACETYL-CoA OXIDATION (REACTIONS OF THE CITRIC ACID CYCLE) In the second stage of cellular respiration, the acetyl groups of acetyl-CoA are fed into the citric acid cycle, which enzymatically oxidizes them to CO2. The energy released is conserved in the reduced electron carriers NADH and FADH2. Citric acid cycle (CAC) = Tricarboxylic acid cycle = TCA cycle = Krebs cycle Primary role is energy conservation in form of NADH, FADH2, and GTP (ATP) Cycle with no net gain or loss of carbon (2 carbon in Acetyl Co-A enter the cycle, 2 carbon in CO2 molecules exit the cycle) THE CITRIC ACID CYCLE (CAC) The citric acid cycle has 8 steps: The carbon atoms shaded in pink are those derived from the acetate of acetyl-CoA in the first turn of the cycle; these are not the carbons released as CO2 in the first turn. The red arrows show where energy is conserved by electron transfer to FAD or NAD+, forming FADH2 or NADH + H+. Steps 1, 3, and 4 are essentially irreversible in the cell; all other steps are reversible. The product of step 5 may be either ATP or GTP, depending on which succinyl-CoA synthetase isozyme is the catalyst. SEQUENCE OF EVENTS IN THE CITRIC ACID CYCLE Step 1: C-C bond formation to make citrate (irreversible) Step 2: Isomerization via dehydration/rehydration Steps 3 and step 4: Two oxidative decarboxylations to give 2 NADH (irreversible) Step 5: Substrate-level phosphorylation to give GTP Step 6: Dehydrogenation to give reduced FADH2 Step 7: Hydration Step 8: Dehydrogenation to give NADH STEP1: C-C BOND FORMATION BY CONDENSATION OF ACETYL-CoA AND OXALOACETATE TO FORM CITRATE Acetyl-CoA is condensed (Claissen condensation) with oxaloacetate to form citrate. The reaction is highly exergonic because the condensation is coupled to the hydrolysis of the high energy thioester of acetyl-CoA. CITRATE SYNTHASE Condensation of acetyl-CoA and oxaloacetate The only reaction with C-C bond formation Uses Acid/Base Catalysis – Carbonyl of oxaloacetate is a good electrophile – Methyl of acetyl-CoA is not a good nucleophile… – …unless activated by deprotonation Rate-limiting step of CAC Activity largely depends on [oxaloacetate] Highly thermodynamically favorable/irreversible – Regulated by substrate availability and product inhibition STEP 2: ISOMERIZATION BY DEHYDRATION/REHYDRATION. FORMATION OF ISOCITRATE VIA CIS-ACONITATE The formation of isocitrate from citrate is catalyzed by aconitase that can reversibly form either citrate or isocitrate through the intermediate formation of cisaconitate, which normally does not leave the enzyme. The equilibrium mixture contains less than 10% of isocitrate, but in the cells it is rapidly removed lowering the steady state concentration. ACONITASE Elimination of H2O from citrate gives a cis C=C bond (cis-aconitate). Addition of H2O to cis-aconitate gives isocitrate Citrate, a tertiary alcohol, is a poor substrate for oxidation Isocitrate, a secondary alcohol, is a good substrate for oxidation Addition of H2O to cis-aconitate is stereospecific Thermodynamically unfavorable/reversible – Product concentration kept low to pull forward STEP 3: OXIDATION OF ISOCITRATE TO a-KETOGLUTARATE AND CO2 Isocitrate is oxidatively decarboxylated (oxidative decarboxylation # 2 starting from pyruvate, # 1 being PDH reaction) with the capture of the electrons by either NAD or NADP (see next slide). Note: Carbon lost as CO2 DO NOT come from acetyl-CoA. ISOCITRATE DEHYDROGENASE Oxidative decarboxylation (number 1 of CAC) – Lose a carbon as CO2 –Generate NADH Oxidation of the alcohol to a ketone –Transfers a hydride to NAD Isoenzyme use of NAD or NADP+ as a cofactor – Two separate enzymes are involved in the reactions: NAD- dependent enzyme found in the mitochondria (for CAC). NADP-dependent enzyme found in both mitochondria and cytoplasm is likely used for reductive, biosynthetic reactions (second source of NADPH in the cytosol beside PPP). Highly thermodynamically favorable/irreversible –Regulated by product inhibition and ATP STEP 4: OXIDATION OF A-KETOGLUTARATE TO SUCCINYL-CoA AND CO2 α-Ketoglutarate is converted to succinyl-CoA in a reaction that is very similar to the pyruvate dehydrogenase reaction. Energy is conserved in the high energy thioester, succinyl-CoA, and in NADH. α-Ketoglutarate dehydrogenase complex is very similar to PDH complex in structure and function (E1, E2, E3, bound TPP, bound lipoate, CoA, NAD, FAD) a-KETOGLUTARATE DEHYDROGENASE Oxidative decarboxylation (# 2 of the CAC #3 starting from pyruvate) and also the final in the oxidation of pyruvate) – Lose a carbon as CO2 – Generate NADH – Net full oxidation of all carbons of glucose After two turns of the cycle Carbons lost not directly from glucose (because the carbons lost came from oxaloacetate) Succinyl-CoA is another higher-energy thioester bond Highly thermodynamically favorable/irreversible – Regulated by product inhibition a-KETOGLUTARATE DEHYDROGENASE Complex similar to pyruvate dehydrogenase – Same coenzymes, identical mechanisms – Active sites different to accommodate different-sized substrates A conserved mechanism for oxidative decarboxylation. The pathways shown employ the same five cofactors (TPP, coenzyme A, lipoate, FAD, and NAD+), closely similar multienzyme complexes, and the same enzymatic mechanism to carry out oxidative decarboxylations of pyruvate (by the pyruvate dehydrogenase complex), α-ketoglutarate (in the citric acid cycle), and the carbon skeletons of the three branched-chain amino acids, isoleucine, leucine, and valine. STEP 5: CONVERSION OF SUCCINYL-CoA TO SUCCINATE Generation of GTP through thioester: Succinyl-CoA is in turn converted into succinate and the energy of thioester bond is conserved by the synthesis GTP from GDP + Pi. GTP is equivalent to ATP. GTP can donate its phosphate group to ADP to form ATP + GDP (with Nucleotide DiPhosphate Kinase, NDPK). SUCCINYL-CoA SYNTHETASE Substrate level phosphorylation Energy of thioester allows for incorporation of inorganic phosphate Goes through a phospho-enzyme intermediate Produces GTP, which can be converted to ATP Slightly thermodynamically favorable/reversible – Product concentration kept low to pull forward STEP 6: OXIDATION OF SUCCINATE TO FUMARATE Oxidation of an alkane to an alkene Succinate is converted to fumarate by the oxidative removal of two hydrogens, catalyzed by the enzyme succinate dehydrogenase, that convert FAD to FADH2. Succinate dehydrogenase is tightly bound to the mitochondrial membrane in eukaryotes (in cell membrane in bacteria); FAD as well remains tightly bound to the enzyme. SUCCINATE DEHYDROGENASE Bound to mitochondrial inner membrane – Part of Complex II in the ElectronTransport Chain (ETC) Reduction of the alkane to alkene requires FAD (because reduction potential of NAD is too low) à formation of FADH2 FAD is covalently bound to enzyme Near equilibrium/reversible – Product concentration kept low to pull forward STEP 7: HYDRATION OF FUMARATE TO MALATE Hydration across a double bond: Fumarate is then hydrated to malate by fumarase, highly stereospecific for the trans-double bond (see next slide). FUMARASE Stereospecific – Addition of water is always trans and forms L-malate – OH- adds to fumarate… then H+ adds to the carbanion – Cannot distinguish between inner carbons, so either can gain –OH Slightly thermodynamically favorable/reversible – Product concentration kept low to pull reaction forward Not a substrate Not a product STEP 8: OXIDATION OF MALATE TO OXALOACETATE (Mitochondrial) Oxydation of an alcohol to a ketone: Malate is oxidized to oxaloacetate. The equilibrium for the malate dehydrogenase reaction lies far to the left, but in intact cells, oxaloacetate is continually removed by the highly exergonic citrate synthase reaction, so reaction goes to the right. The concentration of oxaloacetate in cells is extremely low (