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III. METABOLIC BIOCHEMISTRY

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1 III. METABOLIC BIOCHEMISTRY
§3.6 Oxidative Phosphorylation §3.6a Redox Reactions §3.6b Electron Transport §3.6c ATP Synthesis

2 §3.6a Redox Reactions

3 Synopsis 3.6a Oxidation of food to generate energy essentially involves a series of redox reactions—the energy in the food is essentially in the form of electrons! Redox reactions involve the transfer of electrons between compounds—ie they couple reduction and oxidation The free energy of electrons is captured via generation of reduced compounds and released upon their oxidation—release of such free energy is needed to drive many metabolic reactions The tendency of a compound to gain electrons (or become reduced) is rationalized in terms of the so-called reduction potential ()—the higher the , the greater the tendency to be reduced! Free energy and reduction potential are negatively related—the greater the reduction potential difference (+) between two compounds, the more negative the free energy change (-G)

4 Oxidizing and Reducing Agents
Reducing agent (electron donor)—loses electrons (or hydrogen) and becomes oxidized (its oxidation state increases!)—eg Fe2+ loses an electron and becomes oxidized to Fe3+ Oxidizing agent (electron acceptor)—gains electrons (or hydrogen) and becomes reduced (its oxidation state decreases!)—eg Fe3+ gains an electron and becomes reduced to Fe2+

5 Redox Half-Reactions Consider the following redox reaction:
Fe3+ + Cu+ <=> Fe2+ + Cu2+ which occurs during the oxidation of cytochrome c oxidase in the mitochondrion In the context of an electrochemical cell—a device capable of generating electrical energy from chemical reactions—the above redox reaction can be divided into two half-reactions: Cu <=> Cu2+ + e- Fe3+ + e- <=> Fe2+ Simply put, the copper half-cell undergoes oxidation by liberating electrons (electron-rich), while the iron half-cell experiences reduction by consuming electrons (electron-deficient) Such redox pair of half-reactions thus sets up an “electron gradient” across the two half-cells resulting in an electrical potential difference or electromotive force () that drives the flow of electrons from electron-rich half-cell (copper) to electron-deficient half-cell (iron) through an external circuit (eg a conducting wire)—how can we measure ? A salt bridge (eg a soaked filter paper in an electrolyte such as KNO3) is necessary to prevent the build-up of charge difference across the two half-cells by enabling the flow of ions (eg K+ toward the iron half-cell, and NO3- toward the copper half-cell) to maintain electronic neutrality—failure to do so will impede the flow of electrons between the half-cells! KNO3 K+ NO3- Copper half-cell Iron half-cell

6 Reduction Potential Difference ()
Consider the generalized redox reaction: A + B+ <=> A+ + B  = ox - red =>  = B - A where = Indicative of the oxidized state of corresponding species A = Reduction potential of half-reaction of species A (vide infra) B = Reduction potential of half-reaction of species B (vide infra)  = Reduction potential difference between oxidizing (ox) and reducing (red) agents—ie species B and A, respectively - The half-reactions can be expressed as follows (electrons flow from A to B): A <=> A+ + e A B+ + e- <=> B B The reduction potential () is a measure of the tendency of a species to undergo reduction (or to gain electrons)—the higher the value (more positive) of , the greater the reduction tendency Thus, B > A, since species B (acting as an oxidizing agent) displays higher tendency than species A to undergo reduction How can we relate the reduction potential difference () to the standard reduction potential difference () and free energy change (G)?

7 Nernst Equation -  is related to  by the so-called Nernst equation:  =  + [RT/zF]lnQ  [1] where  = Reduction potential difference (V)  = Standard reduction potential difference (V) R = Universal molar gas constant (1.99 cal/mol/K, or 8.32 J/mol/K) T = Absolute temperature (K) z = Number of electrons transferred in the redox reaction F = Faraday constant (96,485 C/mol, or 96,485 J/V/mol) => 1C=1J/V Q = Reaction quotient (similar to equilibrium constant) Q is defined as: Q = [A][B+] / [A+][B] [2]  is related to the free energy change (G) released by the redox reaction as follows: G = -zF [3] Thus, a positive  indicates a favorable redox reaction and vice versa

8 Standard Reduction Potentials ()
Half-reactions with large positive  are strong electron acceptors (oxidizing agents) and energetically favorable Half-reactions with large negative  are strong electron donors (reducing agents) Electrons spontaneously pass from half-reactions with lower  to increasing  Thus, both NADH and FADH2 serve as electron donors to reduce O2 to H2O during oxidative phosphorylation—the free energy released is harnessed for ATP synthesis For example, free energy (G) released by the reduction of O2 by NADH via the following redox reaction is: 0.5O2 + NADH + H+ <=> H2O + NAD+ G = -zF where  = (+0.815V) - (-0.315V) = 1.13V F = 96,485 J/V/mol z = 2 thus G = -(2).(96,485 J/V/mol).(1.13V) => G = -218 kJ/mol

9 Redox Players Major redox players, or cofactors, involved in mediating electron transfer from reduced metabolites to other compounds include: (1) Nicotinamide adenine dinucleotide (NAD+) (2) Flavin adenine dinucleotide (FAD) (3) Flavin mononucleotide (FMN) (4) Coenzyme Q (CoQ) (5) Heme (Haem in Imperial English) (6) Iron-sulfur clusters (Fe-S)

10 Redox Players: NAD+ STRUCTURE REDUCTION Nicotinamide Phospho- anhydride bond Ribose Adenine Only the nicotinamide moiety of NAD+ serves as the site of reversible reduction NAD+ adopts only two oxidation states (oxidized NAD+ and reduced NADH)—implying that it can only accept a pair of electrons as opposed to an unpaired electron phosphorylated in NADP Nicotinamide adenine dinucleotide (NAD+)

11 Redox Players: FAD STRUCTURE REDUCTION Flavin AMP Phosphoanhydride bond Ribose Ribitol is the reduced form of ribose—cf aldose vs alditol (§1.1) Flavin adenine dinucleotide (FAD) Unlike NAD, FAD can adopt three oxidation states (oxidized FAD, radical FADH. and reduced FADH2)—it can accept both paired and unpaired electrons!

12 Flavin mononucleotide (FMN)
Redox Players: FMN STRUCTURE REDUCTION Phosphoester bond Flavin Flavin mononucleotide (FMN) FMN differs from FAD in that it lacks AMP! Like FAD, FMN can also adopt three oxidation states (oxidized FMN, radical FMNH. and reduced FMNH2)—it can accept both paired and unpaired electrons!

13 also known as ubiquinone
Redox Players: CoQ STRUCTURE REDUCTION n where n = 6-10 Coenzyme Q (CoQ) also known as ubiquinone (lipophilic) Like FAD and FMN, CoQ also adopts three oxidation states (oxidized CoQ, radical CoQH. and reduced CoQH2)—it can accept both paired and unpaired electrons!

14 Redox Players: Heme Heme is comprised of an heterocyclic ring called porphyrin with an iron ion at its center—the redox properties of heme are largely owed to the ability of iron to undergo transition between an oxidized (Fe3+) and reduced (Fe2+) state Heme exists in several biologically important forms such as heme a, heme b and heme c Heme occurs as a co-factor in a wide variety of proteins such as Mb and Hb (heme a— see §2.4) and cytochromes a/b/c (hemes a/b/c—see §3.6a) By virtue of the ability of iron to transition between an oxidized (Fe3+) and reduced (Fe2+) state, heme plays a key role in orchestrating a plethora of redox reactions

15 Redox Players: Fe-S Clusters
Iron-sulfur (Fe-S) clusters are comprised of iron ions bridged between sulfide ions and further coordinated by either cysteine or cysteine/histidine sidechain groups within protein chains In iron-sulfur proteins, the two most commonly occurring Fe-S clusters are [2Fe-2S] and [4Fe-4S] In [2Fe-2S] cluster, two iron ions are bridged between two sulfide ions—and each iron ion is further coordinated to either two CYS or CYS/HIS residues to adopt a tetrahedral geometry In [4Fe-4S] cluster, four iron ions are bridged between four sulfide ions carving out a cube-like structure—and each iron ions is further coordinated to a CYS or HIS to adopt a tetrahedral geometry By virtue of the ability of iron to transition between an oxidized (Fe3+) and reduced (Fe2+) state, the Fe-S clusters play a key role in orchestrating the transfer of electrons from reduced to oxidized compounds

16 Exercise 3.6a What are the metabolic roles of the coenzymes NAD+ and FAD? Explain why NADH and FADH2 are a type of energy currency in the cell Explain the terms of the Nernst equation When two half-reactions are combined, how can you predict which compound will be oxidized and which will be reduced? How is  related to G?

17 §3.6b Electron Transport

18 Synopsis 3.6b During processes such as glycolysis and Krebs cycle, oxidation of macronutrients results in the release of electrons that are ultimately captured in the form of reduced NADH/FADH2 In order to recover the free energy of these electrons stored in NADH and FADH2, they are funneled into a series of redox protein complexes collectively referred to as the “electron transport chain (ETC)”—located within the inner mitochondrial membrane (IMM) ETC in turn couples the free energy of incoming electrons with the transfer of protons (H+) across the IMM culminating with the reduction of O2 to H2O via a series of redox reactions—the electrochemical energy stored in the resulting proton gradient across the IMM is then utilized to synthesize ATP directly from ADP and Pi Simply put, the energy released from the oxidation of NADH and FADH2 (ultimately from the nutrients) is coupled to phosphorylation of ADP directly with Pi (HPO42-) to generate ATP in a process referred to as “Oxidative Phosphorylation” Oxidative phosphorylation (OP) is in stark contrast to “substrate-level phosphorylation”—whereby the transfer of a phosphoryl group from a “high-energy” compound (eg phosphoenol pyruvate) to ADP is used to synthesize ATP (see §3.1 and §3.2) Be aware that ETC and OP are often used synonymously, or sometimes considered as two distinct processes—but, in reality, oxidative phosphorylation is ultimately an overall consequence of ETC!

19 Mitochondrion: The Cell’s Power Plant
OMM harbors large non-selective channels such as voltage-dependent anion channels /porins, which enable facilitated diffusion of most metabolites into the intermembrane space! IMM is much more restrictive with respect to the non-selective diffusion of metabolites but harbors metabolite-specific transporters! Both the Krebs cycle and oxidative phosphorylation occur within the mitochondrial matrix! IMM  Inner (Mitochondrial) Membrane OMM  Outer (Mitochondrial) Membrane

20 Oxidative Metabolism NADH and FADH2 produced during glycolysis and Krebs cycle enter the ETC While glycolysis occurs in the cytosol, Krebs cycle and ETC take place within the mitochondrial matrix How does glycolytic NADH enter the mitochondrial matrix so that it can inject its electrons into the ETC?

21 Regeneration of Cytosolic NAD+
IMM Glycerol-3-phosphate Shuttle Glycolytic NADH cannot diffuse through IMM but transfers its electrons to: Mitochondrial FAD, thereby reducing it to FADH2 via the “glycerol-3-phosphate shuttle” Mitochondrial NAD+, thereby reducing it to NADH via the “malate-aspartate shuttle” In each case, glycolytic NADH is reoxidized to cytosolic NAD+ so that it can be reused in glycolysis—while the resulting mitochondrial pools of FADH2 and NADH enter the ETC

22 Coupling of ETC to ATP Synthesis
NADH and FADH2 essentially act like miniature “batteries” in that the flow of electrons from them “powers up” various transmembrane vehicles (eg complexes I, III and IV) that in turn “pump” protons across the IMM against their concentration gradient in a manner akin to active transporters (see §1.5) Krebs cycle V

23 ETC Components: A Closer Look
ETC is primarily comprised of protein complexes I-IV, coenzyme Q (CoQ), and cytochrome c (CytC)—all of which are highly dynamic and move freely within the IMM Krebs cycle 2 NADH NAD+ FADH2 FAD The efflux of protons (H+) is associated with complexes I, III and IV (that essentially act as proton pumps)—the free energy captured in the resulting electrochemical proton gradient is ultimately harnessed to synthesize ATP

24 ETC Components: Standard Reduction Potentials ()
ETC components are arranged such that their  increases progressively from electron injectors NADH and FADH2 to the terminal electron acceptor O2 ETC thus essentially acts like an “electron gradient”—with the electrons having the tendency to flow “downhill” (in the direction of increasing ) in a manner akin to the rolling of a stone down a hill (or the flow of electrons across an electrochemical cell—see §3.1) The free energy resulting from the dissipation of such electron gradient is usurped to pump protons across the IMM—thereby setting up a proton gradient, which is ultimately harnessed to synthesize ATP H2 Direction of Electron Flow

25 ETC Components: Points of ATP Synthesis
 / V Parallel Pathways for injecting electrons into ETC NADH and FADH2 do not directly donate electrons to O2 but rather via a series of membrane-bound protein complexes called I-IV located within the IMM While NADH donates electrons to Complex I, Complex II serves as an electron acceptor for FADH2—both cofactors ultimately donate their electrons to coenzyme Q (CoQ) Electron transport from NADH and FADH2 to O2 via complexes I-IV is an highly exergonic process Free energy (G) of electron transport is coupled to the generation of a proton gradient across the IMM Recall that G is given by (see §1.5): G = -zF Complex II ’ = V G = kJ.mol-1 FADH2 FAD ( V)

26 Complex I (NADH Dehydrogenase): Structure
Peripheral arm  Electron transport Transmembrane arm  Proton pump Mitochondrial Matrix IMM Intermembrane Space

27 Complex I (NADH Dehydrogenase): Function
The peripheral arm of Complex I harbors various cofactors such as flavin mononucleotide (FMN) and nine iron-sulfur clusters—two conforming to [2Fe-2S] geometry (N1a and N1b), while the rest to [4Fe-4S] By virtue of such cofactors, the peripheral arm of Complex I facilitates the transfer of electrons from NADH to coenzyme Q (CoQ)—also known as ubiquinone—in a series of redox steps (from FMN via iron-sulfur clusters N1-N7) culminating with the overall reaction: NADH + CoQ + H+  NAD+ + CoQH2  = V => G = -70 kJ/mol The reaction generates sufficient free energy to pump four protons across the IMM—how?! The transfer of electrons from NADH to CoQ via the peripheral arm of Complex I induces conformational changes within its transmembrane arm Such structural transition is coupled to an alteration of hydrogen bonding network that allows protons to “hop” across the transmembrane arm of Complex I within the IMM from the mitochondrial matrix to the intermembrane space, thereby setting up a proton gradient—that will be ultimately coupled to ATP synthesis (next section) NADH CoQ

28 Complex II (Succinate Dehydrogenase): Structure
Hydrophilic domain (electron transport) FAD(H2) [2Fe-2S] [4Fe-4S] Mitochondrial Matrix [3Fe-4S] CoQ IMM Transmembrane domain (facilitates electron transport by directly binding to CoQ) Heme b Intermembrane Space

29 Complex II (Succinate Dehydrogenase): Function
The hydrophilic domain of Complex II harbors various cofactors such as flavin adenine dinucleotide (FAD) and three iron-sulfur clusters designated [2Fe-2S], [4Fe-4S], and [3Fe-4S] Complex II catalyzes the oxidation of succinate to fumarate (Step 6 of Krebs cycle) by virtue of its succinate dehydrogenase activity coupled with the reduction of its FAD cofactor to FADH2 Next, Complex II facilitates the transfer of electrons from FADH2 to CoQ—in a series of redox steps (from FADH2 via three iron-sulfur clusters) culminating with the overall reaction: FADH2 + CoQ  FAD + CoQH2  = V => G = -16 kJ/mol This reaction does not generate sufficient free energy to pump protons across the IMM via the transmembrane domain of Complex II—this step is however important in that it injects electrons directly into CoQ so that the energy carried by electrons can be utilized by Complexes III and IV in generating the proton gradient Importantly, the transmembrane domain directly binds to CoQ and thus ensures its proximity to the iron-sulfur clusters which pass the electrons downhill to CoQ Transmembrane domain also binds heme b, which is believed to attune electron transit between Complex II and CoQ H2

30 Complex III (Cytochrome c Reductase): Structure
Homodimeric Complex Mitochondrial Matrix IMM CoQH2 Cytochrome bH Cytochrome bL [2Fe-2S] Stigmatellin Transmembrane domain Intermembrane Space Cytochrome c Cytochrome c1

31 Complex III (Cytochrome c Reductase): Function
Q Cycle CytC IMM Complex III harbors four redox centers: cytochrome bH, cytochrome bL, cytochrome c1, and a single [2Fe-2S] iron- sulfur cluster within the so-called Rieske iron-sulfur protein (ISP)—recall that cytochromes harbor heme (Fe3+) cofactor! Owing to such redox centers, Complex III mediates the transfer of electrons from CoQH2 (QH2) to cytochrome c (CytC) via a series of redox steps—collectively referred to as the Q Cycle—culminating with the overall reaction: CoQH2 + 2CytC(Fe3+)  CoQ + 2CytC(Fe2+) + 2H+  = V => G = -37 kJ/mol The Q cycle is critical in that while CoQH2 is a 2e- carrier, CytC (Fe3+) is a 1e- carrier—thus one molecule of CoQH2 reduces TWO molecules of CytC! In order to facilitate the above reaction, CytC transiently binds to Complex III on its intermembrane face lying between IMM and OMM The free energy released by the above reaction is used to pump four protons across the IMM via Complex III—how?! Unlike Complex I, proton pumping by Complex III is primarily mediated by virtue of the ability of CoQ to act as a proton carrier across the IMM

32 Complex IV (Cytochrome c Oxidase): Structure
Cytochrome a Cytochrome a3 Mitochondrial Matrix IMM CytC e- CytC e- Intermembrane Space CuB CuA Homodimeric Complex

33 Complex IV (Cytochrome c Oxidase): Function
Complex IV contains four redox centers: cytochrome a, cytochrome a3, a single Cu+ ion (CuB), and a pair of Cu+ ions (CuA)—recall that cytochromes harbor heme (Fe3+) cofactor! Owing to such redox centers, Complex IV catalyzes the transfer of electrons from cytochrome c (CytC) to the terminal electron acceptor O2 via a series of redox steps—culminating with the overall reaction: 2CytC(Fe2+) + 0.5O2 + 2H+  2CytC(Fe3+) + H2O  = V => G = -112 kJ/mol The free energy released by the above reaction is used to reinforce proton gradient across the IMM in two ways by Complex IV: Two protons are pumped across the IMM, thereby increasing proton concentration in the intermembrane space (2) Depletion of two matrix protons by supplying them for reduction of each half-molecule of O2 Like Complex I, proton pumping by Complex IV is mediated by conformational changes that facilitate protons to “hop” along the transmembrane domain to the intermembrane space Intermembrane Space CytC e- e- Redox Centers e- Matrix

34 Exercise 3.6b Describe the route followed by electrons from NADH/FADH2 to O2 Write the net equation for electron transfer from NADH to O2 For each of the electron-transport complexes, write the overall redox reaction Position the four electron-transport complexes on a graph showing their relative reduction potentials, and indicate the path of electron flow List the types of cofactors in Complexes I, II, III, and IV Describe the different mechanisms for translocating protons during electron transport

35 §3.6c ATP Synthesis SPINDLE

36 Synopsis 3.6c Free energy of electrons in the form of NADH/FADH2 released via ETC is coupled to the generation of an electrochemical proton gradient across the IMM—the electrochemical potential of such a proton gradient is subsequently harnessed to drive ATP synthesis directly from ADP and Pi (strictly, HPO42-) via “oxidative phosphorylation” Coupling the free energy stored in the proton gradient to ATP synthesis is carried out by an enzyme called “ATP synthase”—which can be essentially viewed as Complex V located downstream of Complexes I-IV in the ETC Embedded within the IMM and protruding into the mitochondrial matrix, ATP synthase is comprised of two components: (1) Fo component (IMM-embedded) (2) F1 component (Matrix) Fo (capitalized F subscripted with small letter o not zero!) component includes a c-ring whose rotation is driven by the dissipation of the proton gradient and drives conformational changes in the F1 component—which in turn catalyzes ATP synthesis by the so-called “binding change” mechanism

37 Coupling of ETC to ATP Synthesis
Fo F1 Complex V (ATP Synthase) Coupling the free energy stored in the proton gradient to ATP synthesis is carried out by an enzyme called “ATP synthase”—which can be essentially viewed as Complex V located downstream of Complexes I-IV in the ETC ATP synthase can also act as an ATPase—ie it can catalyze exergonic hydrolysis of ATP to pump protons against their electrochemical gradient! Simply put, oxidative phosphorylation involves coupling the free energy released by the oxidation of nutrients to directly synthesize ATP from ADP and Pi (strictly, HPO42-)

38 ATP Synthase: Mushroom-Like Structure
Matrix Intermembrane space IMM c12 Embedded within the IMM and protruding into the mitochondrial matrix, ATP synthase is comprised of two components: (1) IMM-embedded Fo component (2) Matrix F1 component Fo component (a1b2c12) 1 a subunit 2 b subunits 12 c subunits (but may vary) F1 component (33) 3  subunits (catalytic) 3  subunits (catalytic) 1  subunit 1  subunit 1  subunit The b subunits together with the  subunit form a peripheral stalk that tethers F1 to the a subunit of Fo—a key feature that underscores the rotation of c12 subunits (called c-ring) of Fo relative to F1

39 ATP Synthase: Rotary Engine
ATP synthase can be envisioned as a: Rotor (c12)—the C-ring Spindle ()—spinning inside the …. Stator (ab233) —so what sets the rotor spinning? Protons enter via a hydrophilic channel located between the a-subunit and the c-ring at the intermembrane space: Proton binding to one of the 12 c-subunits causes a conformational change that makes the c-ring rotate counter-clockwise (as viewed from the matrix) by one c-subunit Binding of protons to successive c-subunits makes the c-ring spin smoothly and continuously Such rotary action converts electrochemical energy stored in the proton gradient across IMM—a form of potential energy or the proton motive force (pmf)—into mechanical energy of the spinning rotor Spinning of the rotor is coupled to ATP synthesis occurring at the interface of the 33 catalytic subunits of F1 component (by virtue of yet another conformational change within 33)—the mechanical energy is ultimately converted back to chemical energy! Stator Spindle IMM Matrix c-ring Rotor Intermembrane space Hydrophilic channel

40 ATP Synthase: Basis of Rotation
The conformational change that spins the rotor is driven by a mutual attraction between a cationic arginine on the a-subunit and an anionic aspartate on the c-subunit—essentially a “salt bridge” or an “ion pair” As the proton concentration rises in the intermembrane space (thanks to ETC), such union of opposites is disrupted by the entry of a proton—since it competes with the arginine for binding to the aspartate—via the hydrophilic channel Upon binding to aspartate, the proton induces a structural change within the c-subunit that drives it in a counter-clockwise manner (as viewed from the matrix side) so as to allow the next c-subunit to engage in an arginine-aspartate “bridge” with the stationary a-subunit The above process is repeated to drive the rotary action of the c-ring in a continuous manner until the proton gradient is fully discharged How many protons are required to drive one full turn of the rotor (c-ring)?! Stator Spindle IMM Matrix c-ring Rotor Intermembrane space Hydrophilic channel

41 ATP Synthase: In Action
One full turn of the rotor (c-ring) requires 12 protons—one for each c-subunit! Each full turn produces 3 ATP molecules—one by each of the three  catalytic protomers of 33 subunits of F1 component Simply put, 3/12 (or 0.25) ATP molecules are produced for the discharge of every proton during oxidative phosphorylation How is the spinning of the rotor coupled to ATP synthesis occurring at the interface of the 33 catalytic subunits of F1 component? Enter the “binding change” mechanism

42 ATP Synthase: Binding Change Mechanism
In the so-called “binding change” mechanism, each of the three  catalytic protomers of the 33 subunits of F1 component is envisioned to adopt three distinct conformations designated O, L and T that are in equilibrium exchange with each other: O  catalytically-inactive / low affinity for substrates (ADP and Pi) L  catalytically-inactive / moderate affinity for substrates (ADP and Pi) T  catalytically-active / high affinity for substrates (ADP and Pi) In the absence of the spinning action of the rotor (c12), the  protomer largely equilibrates between the O and L states—it cannot synthesize ATP from ADP and Pi—with the latter being able to accommodate ADP/Pi with moderate affinity Upon the spinning action of the rotor (c12), the free energy released shifts the conformational equilibrium of the  protomers from the L state to the catalytically-active T conformation, enabling it to “stick” together ADP and Pi to generate ATP Upon the synthesis of ATP, the T state undergoes conformational change to O state (with low affinity for substrates), thereby releasing ATP and enabling the  protomer to return to its initial state in order to undergo another catalytic cycle

43 ATP Synthesis Via OP: Oxidation of Carbs
Free energy of electron transport from NADH/FADH2 drives ATP synthesis NADH Oxidation 10 H+/NADH pumped across the IMM 10 H+/NADH * 0.25 ATP/H+ => 2.5 ATP/NADH FADH2 Oxidation 6 H+/FADH2 pumped across the IMM 6 H+/FADH2 * 0.25 ATP/H+ => 1.5 ATP/FADH2 Aerobic Conditions Glycolysis => 7 ATP/glucose Acetyl-CoA Synthesis => 5 ATP/glucose Krebs Cycle => 20 ATP/glucose Total => 32 ATP/glucose Anaerobic Conditions Glycolysis => 2 ATP/glucose Acetyl-CoA Synthesis => 0 ATP/glucose Krebs Cycle => 0 ATP/glucose Total => 2 ATP/glucose

44 ATP Synthesis Via OP: Oxidation of Fats
6 Palmitic Acid (16:0) Palmitoyl-CoA 8 Acetyl-CoA 7 NADH 7 FADH2 8 FADH2 24 NADH 8 GTP 10.5 ATP 17.5 ATP 60 ATP 12 ATP 8 ATP Total Energy = 108 ATP Krebs cycle ETC -Oxidation Palmitic acid is a saturated fatty acid harboring 16 carbon atoms (16:0) It is the most commonly occurring fatty acid in living organisms So how much energy does -oxidation of a single chain of palmitic acid (16 C atoms) generate? Complete degradation of palmitic acid would require 7 rounds of -oxidation producing 7 FADH2, 7 NADH and 8 acetyl-CoA—the final round produces 2 acetyl-CoA! Further oxidation of each acetyl-CoA via the Krebs cycle produces 3 NADH, 1 FADH2 and 1 GTP (enzymatically converted to ATP) per molecule (and there are 8 acetyl-CoA!)—see §3.5 Oxidation of each NADH and FADH2 via the ETC respectively produces 2.5 and 1.5 molecules of ATP—as noted in §3.3 Fat Is hypercaloric!

45 ATP Comparison: Carbs vs Fats
6 Palmitate Glucose Mr = 256 g.mol-1 but: NA = 6x1023 mol-1 thus: m = 256 g.mol-1 / 6x1023 mol-1 m = 43x10-23 g Mr = 180 g.mol-1 but: NA = 6x1023 mol-1 thus: m = 180 g.mol-1 / 6x1023 mol-1 m = 30x10-23 g What is the mass (m) of one molecule of palmitate or glucose?! Complete Oxidation via the Krebs cycle Glucose => ATP => 32 ATP / 30x10-23 g => 1.07x1023 ATP/g Plamitate => 108 ATP => 108 ATP / 43x10-23 g => 2.51x1023 ATP/g Fats generate more than two-fold greater ATP per unit mass compared to carbs—ie they serve as more energy-efficient fuels The energy-efficient nature of fats in particular is not lost on heart (virtually devoid of glycogen reserves)—an organ that primarily uses fats to meet its energy needs (see §3.1)

46 Exercise 3.6c Explain why an intact and impermeable IMM is essential for ATP synthesis Describe the overall structure of the F1 and F0 components of ATP synthase. Which parts move? Which are stationary? Which are mostly stationary but undergo conformational changes? Summarize the steps of the binding change mechanism Describe how protons move from the intermembrane space into the matrix. How is proton translocation linked to ATP synthesis?


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