CELLULAR RESPIRATION

aerobic respiration

• the process that extracts energy from food in the presence of oxygen

• energy is used to synthesize ATP from ADP and P_i

• ATP is then used to supply energy directly to cells

• C₆H₁₂O₆ + 6O₂ —> 6CO₂ + 6H₂O

• oxygen is the final electron acceptor

obligate aerobes

• most eukaryotes are obligate aerobes

• they cannot live without oxygen, and use aerobic cellular respiration almost exclusively

ways ATP is produced in aerobic respiration (2)

• substrate-level phosphorylation

• oxidative phosphorylation

substrate-level phosphorylation

the formation of ATP by the direct transfer of a phosphate group from a substrate to ADP

oxidative phosphorylation

the formation of ATP using energy transferred indirectly from a series of redox reactions, involving a final electron acceptor

stages of aerobic cellular respiration (4)

1. glycolysis

2. pyruvate oxidation

3. citric acid cycle (Krebs cycle)

4. oxidative phosphorylation

mitochondrion structure

• two membranes: outer and inner membrane

• outer membrane is smooth, but the inner membrane has foldings of cristae

  • cristae give the inner membrane a larger surface area, thus enhancing productivity of cellular respiration

• intermembrane space is between the membranes

• mitochondrial matrix (includes enzymes, mitochondrial DNA, ribosomes) is the interior aqueous environment

• # of mitochondria in a cell correlates with the cell’s level of metabolic activity

most common anaerobic pathways (2)

• anaerobic respiration: process that uses a final inorganic oxidizing agent other than oxygen to produce energy

• fermentation: process that uses an organic compound as the final oxidizing agent to produce energy

  • yields significantly less free energy compared to aerobic respiration

• both are catabolic processes

obligate & facultative anaerobes

• obligate anaerobe: an organism that cannot survive in the presence of oxygen

• facultative anaerobe: an organism that can live with or without oxygen (e.g. yeast and E. coli bacteria that live in our gut)

glycolysis

• a catabolic pathway (releases energy by breaking down molecules)

• oxidizes glucose to produce two molecules of pyruvate

• potential energy and electrons released leads to synthesis of ATP and NADH

• most fundamental and probably most ancient of metabolic pathways

• two phases: initial energy investment phase and energy payoff phase (5 steps each)

facts that support glycolysis being the most fundamental metabolic pathway (3)

1. nearly universal (found in almost all organisms)

2. does not require O₂: oxygen became abundant in the atmosphere ~2.5 billion years ago, about 1.5 billion years after life began

3. glycolysis occurs in the cytosol and involves soluble enzymes: does not require more sophisticated cellular enzymes to operate

glycolysis energy investment steps

1. phosphorylation

2. isomerization

3. phosphorylation

4. lysis

5. isomerization

glycolysis energy payoff steps

6. redox reaction

7. substrate-level phosphorylation reaction

8. mutase reaction

9. dehydration reaction

10. substrate-level phosphorylation reaction

mutase reaction

the shifting of a chemical group to another within the same molecule

glycolysis energy investment: (1) phosphorylation reaction

glucose receives a phosphate group from ATP to produce glucose-6-phosphate (G6P), making it more chemically active

glycolysis energy investment: (2) isomerization reaction

G6P is rearranged into its isomer, fructose-6-phosphate (F6P)

glycolysis energy investment: (3) phosphorylation reaction

F6P receives a phosphate group from ATP (to ADP + P_i) to produce fructose 1,6-bisphosphate (F16P)

glycolysis energy investment: (4) lysis reaction

F16P is split into two different 3-carbon sugars: glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP)

glycolysis energy investment: (5) isomerization reaction

DHAP is rearranged into its isomer G3P, giving a total of two G3P molecules per molecule of glucose

glycolysis energy payoff quantities

as two molecules of G3P are produced in reaction 5, all the reactions from 6 to 10 are doubled

glycolysis energy payoff: (6) redox reaction

• G3P is oxidized; two electrons and two H⁺ are released

  • both electrons and one H⁺ are transferred to NAD⁺ to form NADH

  • one proton is released to the cytosol

• using energy from the redox reaction, a phosphate (not from ATP) attaches to the oxidized substrate to form two molecules 1,3-bisphosphoglycerate (1,3-BPG) and two molecules of NADH

glycolysis energy payoff: (7) substrate-level phosphorylation reaction

• one phosphate group of each 1,3-BPG is transferred to ADP to produce ATP

• results in two molecules of ATP and two molecules of 3-phosphoglycerate (3-PG)

glycolysis energy payoff: (8) mutase reaction

3-PG is rearranged, shifting the phosphate group from the 3-carbon to the 2-carbon to produce 2-phosphoglycerate (2-PG)

glycolysis energy payoff: (9) dehydration reaction

• a double bond is formed in the 2-PG by extracting an H₂O molecule

• forms two molecules of phosphoenolpyruvate (PEP) and two molecules of water

glycolysis energy payoff: (10) substrate-level phosphorylation reaction

• the remaining phosphate group is transferred from PEP to ADP

• forms two molecules of pyruvate and two molecules of ATP

energy investment and payoff in glycolysis

• initially, two ATP are consumed as glucose and F6P become phosphorylated

• in the energy investment phase, the two ATP increase the free energy of the chemicals in the glycolytic pathway

• however, even more free energy is released in the payoff phase, as four ATP and two NADH molecules are synthesized

net equation for glycolysis

glucose + 2 ADP + 2 P_i + 2 NAD⁺ —> 2 pyruvate + 2 ATP + 2 NADH + 2 H⁺ + 2 H₂O

extraction of free energy in pyruvate

• glycolysis releases less than a quarter of the chemical energy in glucose that can be harvested by cells

• most of the energy remains in the two molecules of pyruvate

• the extraction of the remaining free energy in pyruvate continues via pyruvate oxidation and the citric acid cycle

pyruvate oxidation: pyruvate movement

• citric acid cycle occurs in the mitochondrial matrix, so pyruvates produced in glycolysis must pass through mitochondrial membranes

• pyruvate diffuses through the large pores of the outer membrane

• as pyruvate is a charged molecule, a pyruvate-specific membrane carrier (mitochondrial pyruvate carrier) is required for it to enter the mitochondrion

pyruvate oxidation steps (3)

• decarboxylation reaction

• dehydrogenation reaction

• formation of acetyl CoA

pyruvate oxidation: decarboxylation reaction

• pyruvate is decarboxylated by the pyruvate dehydrogenase complex

• the carboxyl group of pyruvate is removed and forms one molecule of CO₂ as waste

• this reaction produces one-third of the CO₂ we exhale

pyruvate oxidation: dehydrogenation reaction

• the remaining two-carbon molecule is oxidized to produce an acetyl group

• two electrons and one H⁺ are transferred to NAD⁺, forming NADH, and an H⁺ ion is released into solution

pyruvate oxidation: formation of acetyl CoA

• coenzyme A (CoA) is a sulfur-containing compound derived from a B vitamin

• CoA is attached via its sulfur atom to the two-carbon intermediate, forming acetyl coenzyme A

net reaction for pyruvate oxidation

2 pyruvate + 2 NAD⁺ + 2 CoA —> 2 acetyl-CoA + 2 NADH + 2 H⁺ + 2 CO₂

citric acid cycle

• consists of eight enzyme-catalyzed reactions

• seven reactions take place in the mitochondrial matrix, and one binds to the matrix side of the inner mitochondrial membrane

citric acid cycle: (1) citrate production

acetyl CoA adds its two-carbon acetyl group to the four-carbon molecule oxaloacetate to form the six-carbon molecule citrate

acetyl-CoA

• contains a high-energy, unstable thioester bond between acetyl group and CoA which stores a lot of potential energy

• an energy releasing reaction drives the cycle forward

• commits the acetyl group from acetyl-CoA to the cycle for oxidation and energy production

citric acid cycle: (2) isomerization

• citrate is rearranged to its isomer isocitrate through the removal of one H₂O molecule and addition of H₂O

• the hydroxyl group from the third carbon is rearranged to the fourth carbon

• makes the molecule a better substrate for oxidative decarboxylation in the next step

citric acid cycle: (3) oxidative decarboxylation

• isocitrate is oxidized; two electrons and one H⁺ are transferred to NAD⁺ to produce NADH

• oxidized substrate is decarboxylated; an -OH group is converted to a carbonyl group, and H⁺ is released into solution

• forms 5-carbon ɑ-ketoglutarate

citric acid cycle: (4) oxidative decarboxylation

• ɑ-ketoglutarate is oxidized

  • NAD⁺ is reduced to NADH and H⁺

• oxidized substrate is decarboxylated; CO₂ is released

• four-carbon is attached to coenzyme A, forming succinyl-CoA, a high energy intermediate that will drive GTP/ATP synthesis in next step

citric acid cycle: (5) GTP/ATP synthesis

• high-energy, unstable thioester bond in succinyl-CoA is broken by a phosphate group which displaces it

• forms four-carbon succinate

• energy released is used to add a phosphate group to GDP/ADP, forming GTP/ATP

• most GTP is used to make ATP; some reactions use GTP directly for energy

citric acid cycle: (6) succinate oxidation

• succinate is oxidized

  • two electrons and two H⁺ are transferred to FAD (an energy carrier), thereby reducing it to FAD₂

• forms the four-carbon fumarate

citric acid cycle: (7) production of malate

fumarate is converted into malate by the addition of a water molecule (hydroxyl group on second carbon)

citric acid cycle: (8) production of oxaloacetate

• malate is oxidized; two electrons and one proton transferred to NAD⁺, reducing it to NADH

• hydroxyl group is converted into a carbonyl group and H⁺ is released into solution

• forms oxaloacetate, regenerating the molecule that started the cycle

citric cycle allows the following (5)

• complete oxidation of acetyl CoA (releases CO₂)

• generation of high energy electron carriers

• substrate-level phosphorylation

• regeneration of oxaloacetate

• intermediates for biosynthesis

citric acid net equation

2 acetyl CoA + 6 NAD⁺ + 2 FAD + 2 GDP/ADP + 2 P_i + 4 H₂O —> 2 CoA + 6 NADH + 2 FADH₂ + 6 H⁺ + 4 CO₂ + 2 GTP/ATP

“electron escorts” produced by glycolysis and Calvin cycle

• 4 molecules of ATP (though substrate-level phosphorylation)

• 10 molecules of NADH

• 2 molecules of FADH₂

• energy is used by oxidative phosphorylation to power ATP synthesis

electron transport chain (ETC)

• a collection of molecules embedded in the inner membrane of the mitochondrion

• consists of four protein complexes:

  • complex I (NADH dehydrogenase)

  • complex II (succinate dehydrogenase)

  • complex III (cytochrome complex)

  • complex IV (cytochrome oxidase)

• complex II is a single peripheral membrane protein, the rest are composed of multiple proteins

cristae for ETC

• cristae increases surface area to provide space for thousands of copies of each component of ETC

• infolded membrane with its concentration of electron carrier molecules is well-suited for the series of sequential redox reactions that take place along the ETC

electronegativity in ETC complexes

• complexes I, III, and IV are protein complexes with increasing electronegativity along the chain

• complexes have cofactors that alternate between reduced and oxidized states as they pull electrons from upstream molecules and subsequently donate electrons to more electronegative downstream molecules

• not the protein themselves that transfer electrons but rather non-protein groups bound to the proteins of each complex

ETC: (1) complex I reduction

• electrons from glycolysis and citric acid cycle are transferred from NADH to the first molecule of the ETC in complex I; Complex I is reduced

• NADH is oxidized to NAD⁺; hydride ion splits into two electrons (passed to complex I) and 1 H⁺ released into mitochondrial matrix

ETC: (2) complex I oxidation

• Complex I passes electrons to ubiquinone (UQ), a hydrophobic molecule in the membrane core

• Complex I is oxidized and UQ is reduced

ETC: (3) complex II reduction

• FADH₂ passes electrons to complex II

• FADH₂ is oxidized to FAD, and Complex II is reduced

• (separate from complex I processes)

ETC: (4) complex III reduction

• UQ (having received electrons from Complex I and II) passes electrons to Complex III

• UQ is oxidized and Complex III is reduced

ETC: (5) complex III oxidation

• complex III passes electrons to cytochrome c and is oxidized

• cytochrome c is reduced

ETC: (6) complex IV reduction

• cytochrome c passes electrons to complex IV

• cytochrome c is oxidized, complex IV is reduced

ETC: (7) complex IV oxidation

• complex IV passes electrons to the final electron acceptor, O₂ (highly electronegative)

• complex IV is oxidized and O₂ is reduced

• O₂ requires four electrons to be fully reduced

mobile electron shuttles in ETC (2)

• ubiquinone (UQ): shuttles electrons from complexes I and II to complex III

• cytochome c: transfers electrons from complex III to complex IV

O₂ reduction in ETC

• final product is water

• O₂ + 4 e^- + 4 H⁺ —> 2 H₂O

• four electrons come from cytochrome c, four protons come from mitochondrial matrix

• formation of H₂O is necessary as O₂^- is reactive and toxic to cells

free energy in ETC

• electron carriers alternate between reduced and oxidized states

• each component of the chain becomes reduced when it accepts electrons from “uphill”, less electronegative neighbour

• returns to oxidized form as it passes electrons to “downhill”, more electronegative neighbours

• ETC makes no ATP directly; instead passes electrons in a series of small steps that release energy in manageable amounts

• mitochondrion couples electron transport an energy release to ATP synthesis through chemiosmosis

proton-motive force

• as electrons are passed through complexes, energy is released from NADH or FADH₂

• energy is used to active transport H⁺ against its concentration gradient (from matrix to intermembrane space)

• creates a proton gradient called proton-motive force

chemiosmosis

• an energy-coupling mechanism that uses potential energy stored in the form of an H⁺ gradient across a membrane to drive cellular work

• H⁺ move back across the membrane, diffusing down its gradient

• passage of H⁺ through ATP synthase drives phosphorylation of ADP + P_i —> ATP

energy flow in cell respiration

glucose —> NADH —> ETC —> proton-motive force —> ATP

ATP synthesis through NADH and FADH₂

each NADH that transfers a pair of electrons to ETC contributes enough to the proton-motive force to generate about 3 molecules of ATP

• 1 NADH results in 10 H⁺ being transported into intermembrane space

• 4 H⁺ re enter the matrix via ATP synthase

• therefore, 1 NADH generates enough proton motive force for the synthesis of 2.5 ATP

• 1 FADH₂ generates enough proton-motive force for the synthesis of 1.5 ATP

what ATP synthesis numbers take into account (3)

• the slight energetic cost of moving the ATP from mitochondrion to cytosol (where it will be used)

• ATP yield varies slightly depending on the type of shuttle used to transport electrons from cytosol to mitochondrion

• (also reduces ATP yield) use of proton-motive force generated by the redox reactions of respiration to drive other kinds of work (e.g. bringing pyruvate into mitochondrion)

energy and protons released in ETC is determined by

• determined by structure and function of each protein complex, and amount of energy released

• the greater the difference in redox potential between electron donors and acceptors, the more energy is released

why ATP yield varies depending on electron shuttle

• two electrons of NADH captured in glycolysis must be conveyed into mitochondrion by one of several electron shuttle systems; depending on the type, the electrons are passed to either NAD⁺ or FAD in matrix

• because FADH₂ skips complex I, it misses the opportunity to contribute to the four protons being pumped

• thus, fewer protons (H⁺) are pumped into intermembrane space, leading to less ATP production

why oxygen is needed in oxidative phosphorylation

• most of the ATP generated by cellular respiration is due to the work of oxidative phosphorylation

• aerobic respiration depends on an adequate supply of O₂ into the cell

• without the electronegative oxygen to pull electrons down the ETC, oxidative phosphorylation eventually ceases

anaerobic respiration: sulfur

• anaerobic respiration requires electronegative final electron acceptor

• some “sulfate-reducing” marine bacteria use the sulfate ion (SO₄^2-) at the end of their respiratory chain

• the operation of this chain builds up a proton-motive force to produce ATP, where H₂S is made as a byproduct rather than H₂O

fermentation

• a way of harvesting chemical energy without using either O₂ or any ETC (without cellular respiration)

• an extension of glycolysis that allows the continuous generation of ATP by the substrate-level phosphorylation

• for this to occur, there must be a sufficient supply of NAD⁺ to accept electrons during oxidation step of glycolysis

• fermentation consists of glycolysis plus reactions that regenerate NAD⁺ by transferring electrons from NADH to pyruvate or derivatives of pyruvate

why fermentation needs to replenish NAD⁺

without some mechanism to recycle NAD⁺ from NADH, glycolysis would soon deplete cell’s pool of NAD⁺ and would shut itself down for lack of an oxidizing agent

how fermentation replenishes NAD⁺

• under aerobic conditions, NAD⁺ is recycled from NADH by the transfer of electrons to the ETC

• an anaerobic alternative is to transfer electrons from NADH to pyruvate, the end product of glycolysis

alcohol fermentation

pyruvate is converted to ethanol in two steps:

1. pyruvate produced by glycolysis is decarboxylated to form the two-carbon compound acetaldehyde; this releases CO₂

2. acetaldehyde is reduced by NADH to ethanol; this generates the supply of NAD⁺ needed for continuation of glycolysis

CO₂ bubbles generated by baker’s yeast during alcohol fermentation allows bread to rise

alcohol fermentation equations

• pyruvate + NADH + H⁺ —> NAD⁺ + CO₂ + ethanol

• glucose + 2 ADP + 2 P_i —> 2 ATP + 2 CO₂ + 2 ethanol

lactate fermentation

pyruvate is reduced directly by NADH to form lactate as an end product, regenerating NAD⁺ with no release of CO₂

lactic acid fermentation examples (2)

• fermentation by certain fungi and bacteria used in the dairy industry to make cheese and yogurt

• human muscle cells make ATP by lactic acid fermentation when oxygen is scarce

  • occurs during strenuous exercise, when sugar catabolism for ATP production outpaces the muscle’s supply of oxygen from the blood

lactic acid fermentation equations

• pyruvate + NADH + H⁺ —> NAD⁺ + lactate

• glucose + 2 ADP + 2 P_i —> 2 ATP + 2 lactate