II. Cellular Respiration A. 3 stages of cellular respiration 1. glycolysis 2. citric acid cycle (Krebs Cycle) 3.

electron transport and phosphorylation 4. for each stage students should know: a. where the process occurs b. what happens in basic chemical terms c. why the cell does the process B. Glycolysis 1. glyco = sugar 2. lysis = splitting 3. occurs in cytoplasm of BOTH prokaryotes and eukaryotes 4. summary of glycolysis a. glucose is split (1) one 6 carbon molecule --> two 3 carbon molecules (2) see the overall reaction b. 2 three carbon pyruvate molecules are produced c. during glycolysis, some energy is conserved as ATP & NADH d. see Fig 7.12 in Brooker et al. e. note: (1) no O2 involved in glycolysis -reactions can occur in absence O2 -reactions can also occur in presence O2 -glycolysis is neither aerobic nor anaerobic (2) NAD+ serves as oxidizing agent -NAD+ is the cofactor (coenzyme) -cofactors are one type of ligand that can bind to a protein -enzyme binds NAD+ -see human GAPDH, gif -GAPDH is enzyme of glycolytic pathway that uses NAD+ to oxidize GAP -GAP is one of the molecules produced as glucose is broken down -enzyme binds substrate (GAP) -Where do you think the GAP binding site is located? -enzyme brings NAD+ and the substrate together such that the NAD+ can oxidize the the substrate - NAD+ --> NADH - see the whole reaction (3) energy released during the glycolysis reactions is conserved as: (a.) NADH (equivalent to NADPH) (b.) ATP (4) Ten Steps of Glycolysis

(a) see Fig. 7.12 in Brooker et al. (b) students should be able to: i) identify where energy put into the glycolytic pathway -look for steps where ATP --> ADP ii) identify where where energy is taken out of the glycolytic pathway -look for steps where ADP --> ATP -look for steps where NAD+ --> NADH iii) summarize the overall energy balance, explaining the input of 2 ATP and the net gain 2 ATP and explaining the 2 NADH/glucose iv) list the starting and ending molecules of the glycolytic pathway C. Citric Acid Cycle = Krebs Cycle = Tricarboxylic Acid Cycle 1. occurs in mitochondria of eukaryotes (Where do these reactions occur in prokaryotes?) a. structure of mitochondrion (EM) b. cartoon of a mitochondrion -possible exam question (1) outer mitochondrion membrane (omm) (a) controls access to inner mito membrane (b) contains many transport proteins i) pyruvate transporter -lets pyruvate into mitochondrion ii) ADP/ATP antiporter -ADP comes in as ATP goes out -why is this critically important? iii) metabolite transporters a) let several K-cycle intermediates out b) K-cycle intermediates serve as precursors in synthesis of amino acids and nucleic acids in the cytoplasm (c) omm does not contain electron transport chain (ETC) (2) inner mitochondrial membrane (imm) (a) has the ETC components (b) has the synthetic ATPase = coupling factor = F1 = CF = "knobs" i) mitochondrial ATPase enzyme (F1) is similar in structure to CF in thylakoids and bacteria ii) F1 (mito) genetically related to CF in thylakoids and bacteria (c) many membrane transporters like the OMM -molecules must cross OMM AND IMM to get to matrix (3) encloses the matrix

(a) "soup" that contains enzymes of Krebs Cycle = Citric Acid Cycle = CAC i) named for Hans Krebs a) illucidated pathway in the 1930's (b) contains many small molecules i) CAC intermediates ii) ADP, Pi, ATP, NAD+, NADH etc 2. CAC = citric acid cycle = Krebs Cycle = TCA a. -see a Krebs Cycle Summary b. pyruvate produced in cytoplasm by glycolysis c. pyruvate transported into matrix (1) must cross omm & imm to get to matrix (2) pyruvate transport proteins found in both membranes d. in matrix, pyruvate oxidized by the enzyme pyruvate dehydrogenase (1) pyruvate dehydrogenase uses two coenzymes (a) NAD+ i) made from the vitamin niacin ii) WHAT HAPPENS IF YOU ARE DEFICIENT IN NIACIN? (b) coenzyme A i) made from another B vitamin (pantothenic acid) ii) WHAT HAPPENS IF YOU ARE DEFICIENT IN PANTOTHENIC ACID? (2) reaction oxidizes pyruvate (a) CO2 produced i) this is part of the C leaving the citric acid cycle (leaves as CO2) i) this is part of the CO2 that will be exhaled into atmosphere (b) acetyl-CoA produced i) 2 carbons of the acetyl group will enter CAC ii) 2 carbons of the acetyl group will also eventually leave CAC as CO2 that will be exhaled into atmosphere -Possible Exam Questions: -In what form does carbon enter the Krebs Cycle? -In what form does carbon leave the Tricarboxylic Acid Cycle? (c) NADH produced as pyruvate is oxidized i) NADH is a high energy molecule ii) one NADH has enough energy to make 3 ATP iii) this is another example of how cell extracts energy from glucose e. energy conservation during complete oxidation of pyruvate to CO2 and H2O in the matrix (includes the CAC)

some NADH produced some GTP produced some FADH2 produced 1,2,3 above means that part of energy in pyruvate molecule goes into NADH, GTP, FADH molecules (5) LATER, ATP is produced from the NADH, GTP, FADH2 f. pyruvate oxidation to acetyl-CoA is not considered part of the CAC g. carbon atoms enter the CAC as acetyl-CoA h. see Fig 7.15 in Brooker et al. -see a Krebs Cycle Summary h. reactions of the CAC i. what to know about the CAC (1) know how C enters CAC (2) know how C leaves CAC (3) be able to follow C skeletons through the CAC (4) be able to follow energy thru CAC -be able to identify the steps where energy is being extracted from the carbon skeletons (5) understand what is happening! j. do not memorize the names or the structures D. Electron Transport (ET) 1. CAC uses up lots of NAD+ and FAD -BUT, NAD+ and FAD present at very low concentrations -How can this be???? 2. CAC occurs ONLY under aerobic conditions a. O2 absolutely required b. BUT, CAC doesn't use O2 directly c. How explain that O2 is absolutely required, but not directly used???? 3. answers to 1 & 2: a. O2 needed to regenerate NAD+ and FAD - NAD+/NADH & FAD/FADH2 only present in catalytic amounts - as NADH and FADH2 are produced, they are reoxidized back to NAD+ and FAD using O2 4. it is the mitochondrial electron transport chain that accomplishes the O2 mediated oxidation of NADH and FADH2 5. Energetics of CAC a. NAD+ & FAD used to extract energy from CAC intermediates -see step 3, step 4, step 6, step 8 b. NAD+ oxidizes the CAC intermediates c. much NADH & FADH produced in oxidation reactions

(1) (2) (3) (4)

(1) NADH & FADH contain the energy that was once in the CAC intermediates d. Questions: (1) How does the cell extract energy from NADH? FADH2? (2) How does the cell regenerate NAD+? FAD? (3) How does the cell get energy in the form of ATP? e. Answers: (1) Electron transport and the associated proton pumping as NADH (or FADH2) is oxidized with O2 via the electron transport chain in the inner mitochondrial membrane (2) Electron transport and the associated proton pumping as NADH (or FADH2) is oxidized with O2 via the electron transport chain in the inner mitochondrial membrane (3) The proton pumping in 1 & 2 above is associated with proton driven oxidative phosphorylation (ATP synthesis) by coupling factor = CF = synthetic ATPase 5. focus on NADH----net reaction (NADH-->O2): a. net reaction is very exergonic b. exergonic reaction drives ATP synthesis (1) recall: ADP + Pi --> ATP is an endergonic reaction

6. components of ETC a. see Figs 7.16 and 7.18 in Brooker et al. b. see an animation of mitochondrial ETC b. NADH dehydrogenase (DH) complex (1) several proteins in a complex (2) catalyzes NADH-->NAD+ (a) note that NAD+ is regenerated (b) e- taken from NADH (c) e- given to ubiquinone (d) H+'s are pumped from matrix to intermembrane space between omm and imm i) energy for H+ pumping comes from highly exergonic reaction (oxidation of reduced NADH to NAD+ via electron transport chain to O2 to make H2O c. the lipid ubiquinone (UQ) familiar form (1) dissolved in the hydrophobic interior of the IMM (2) carries 2e- and 2H+'s from NADH DH complex to cytochrome b-c1 complex -picks up 2e- & 2H+ on matrix side of membrane -carries 2e- & 2H+ across membrane

-gives the 2e- to cytochrome b-c1 complex -release 2H+ into intermembrane space (3) by carrying the 2e- and 2H+ across membrane it pumps 2 H+'s from matrix side to intermembrane space (4) is important part of H+ pumping mechanism (see animation) i) energy for H+ pumping comes from highly exergonic reaction (oxidation of reduced NADH to NAD+ via electron transport chain to O2 to make H2O d. cytochrome (cyt) b-c1 complex (1) several proteins in complex (2) carries 2e- from UQ to cyt c (3) pumps 2 more H+'s from matrix side to intermembrane space (a) mechanism of H+ pumping here too complicated to deal with in this class i) energy for H+ pumping comes from highly exergonic reaction (oxidation of reduced NADH to NAD+ via electron transport chain to O2 to make H2O

e. cytochrome c (1) transfers e- from cytochrome b-c1 complex to cytochrome oxidase complex (2) one of the most thoroughly studied proteins in the world (3) small (10,000 daltons) peripheral protein (4) first protein x-ray crystal structure f. cytochrome oxidase complex (1) several proteins in the complex -Note: crystal shows a dimer -Note: crystal has a bound CO molecule -access cytochrome oxidase with Jmol (2) receives e- from cyt c (3) passes e- to O2 to make H2O -this is the O2 that you need to breathe -exam questions: -Name the enzyme that uses most of the O2 that you are required to breathe. -What subcellular organelle is responsible for using most of the O2 that you breathe? -The O2 that you breathe is needed to ________? (4) pumps 2 H+'s from matrix side to intermembrane space i) energy for H+ pumping comes from highly exergonic reaction (oxidation of reduced

NADH to NAD+ via electron transport chain to O2 to make H2O g. succinate dehydrogenase (SDH) complex (1) several proteins in the complex (2) has a binding site for succinate (3) catalyzes the succinate --> fumerate -NOTE: SDH is a CAC enzyme -is a transmembrane integral protein, gif (4) uses FAD/FADH2 as a cofactor (5) carries e- from succinate to ubiquinone pool (6) the ETC of mitochondria is BRANCHED (7) see more detailed cartoon of mitochondrial ETC -this info will be helpful in the MitochondriaLab -memorize this cartoon for the exam 7. summary of ETC reaction's a. see Figs 7.16 and 7.18 in Brooker et al. -Possible Exam Question: Which electron transport protein complex is missing from this cartoon? Answer b. NADH oxidation by ETC c. e- passed from NADH to O2 d. NADH to O2 is a highly exergonic RXN e. some of energy in NADH conserved by pumping H+'s f. energy in NADH transfered to H+ gradient across imm g. SO: energy once in glucose is now in the transmembrane H+ gradient across the inner mito membrane E. Oxidative Phosphorylation 1. as NADH and succinate are oxidized by O2, energy is liberated 2. this liberated energy is used to pump H+s from the matrix side of imm to the intermembrane space 3. thus, a H+ gradient develops with a high conc. of H+s in the intermembrane space and low conc. of H+s in the matrix 4. some of the energy once in glucose and then in the Krebs Cycle intermediates is now in the the H+ gradient 5. How does the cell use the energy in H+ gradient? 6. cell uses coupling facter (CF) to convert the energy in H+ gradient into ATP -cartoon from Science Magazine -see an early X-ray structure 7. concentration of H+ high in intermembrane space 8. concentration of H+ lower in matrix

9. strong gradient of H+s across membrane 10. H+s MOVE THROUGH CF to return to matrix 11. CF uses energy in H+ gradient to drive synthesis ATP (ATP synthesis = "phosphorylation") a. just like CF of thylakoids in chloroplasts b. just like CF of bacterial plasma membrane 12. see details of mechanism of ATP synthesis 13. see Movie of ATP synthesis

F. Chemiosmotic Energy Coupling 1. electron transport from NADH to O2 is highly exergonic - much energy available to do work 2. electron transport energy used to pump protons 3. proton gradient develops across the membrane (proton concentration much higher on the outside of the inner mitochondrial membrane 4. energy once in NADH transfered to proton gradient 5. coupling factor is a special protein complex that can use the energy in a proton gradient 6. coupling factor allows protons to return to matrix (exergonically move down their concentration gradient) IF the endergonic rxn ADP + Pi --> ATP occurs -coupling factor "couples" exergonic proton return to the matrix to endergonic ATP synthesis -uses the energy in the proton gradient to drive the synthesis of the endergonic reaction ADP + Pi --> ATP 7. see Fig 7.16 and 7.18 in Brooker et al. 8. the whole process (steps 1-6) is an example of chemiosmotic energy coupling 9. the whole process (steps 1-6) is also an example of oxidative phosphorylation -what is being oxidized? -what is being phosphorylated? -what is the immediate energy source for ATP synthesis 10. contrast oxidative phosphorylation with substrate level phosphorylation 11. another example of substrate level phosphorylation

G. ATP yield from complete oxidation of glucose = How many ATPs does a cell get from one glucose molecule under aerobic conditions?

1. see Fig 7.10 in Brooker et al.

H. How efficient is the cell at recovering energy from glucose? -recall the energetics of glucose -recall the number of ATP produced per glucose molecule -recall that it takes about 7 kcal of energy to link Pi with ADP to make ATP -SO: 38 ATP/glucose x 7 kcal/ATP = 266 kcal/glucose is conserved -efficiency = energy conserved/total energy = 266/686 = 0.39 -cells are about 40% efficient at recovery energy from glucose I. Catabolism of other molecules 1. cells get energy from molecules other than glucose 2. all 20 aa can be broken down to: a. pyruvate, or b. acetyl CoA, or c. CAC intermediates d. various intermediates feed into CAC 3. any sugar can be converted to glucose a. then feeds into glycolysis 4. glycerol (from fats) converted to glyceraldehyde-P a. then feeds into glycolotic pathway 5. fatty acids breakdown to acetyl-CoA a. acetyl-CoA feeds into CAC in matrix of mitochondrion 6. see Fig 7.21 in Brooker et al. J. Biosynthesis 1. intermediates of glycolysis and CAC are used as carbon skeletons 2. carbon skeletons used to synthesize other molecules needed by cell a. amino acids b. nucleic acids c. fats d. lipids e. phospholipids 3. eg: alpha-ketoglutarate (a CAC intermediate) is used to make the amino acid glutamatic acid -see the reaction -practice quiz 4. eg: acetyl-CoA (from pyruvate DH) used to make fatty acids -two carbon acetyl groups linked together to make fatty acids -all biological fatty acids have an even number of C atoms -Possible Exam Question: Why do all biological fatty acids have an even number of C atoms?

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NADH is being oxidized back to NAD+ The reaction is: NADH + O2 -------------------> NAD+ + H2O ETC Back to the outline ADP is phosphorylated to ATP The reaction is: ADP + Pi ----------------> ATP CF Back to the outline The electron transport reactions are found in the plasma membrane of aerobic bacteria. The Krebs Cycle enzymes are found in the cytoplasm of the aerobic bacteria. The F1 ATPase is found on the plasma membrane. . The ATP synthesis associated with oxidative phosphorylation is driven by a proton (H+) gradient across the inner mitochondrial membrane.

The missing protein complex is Succinate Dehydrogenase