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MetabolismOutline:• Part I: Fermentations• Part II: Respiration• Part III: Metabolic DiversityLearning objectives are:• Learn about respiratory metabolism,• ATP generation by respiration linked

(oxidative) phosphorylation,• Electron transport

Fermentation vs. Respiration• End products of fermentations are __________

waste products and not fully __________.• Still some useful left in the Still some useful __________ left in the

products

•How can a microorganism get more energy from glucose?

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Use respiration and the tricarboxylic acid cycle

d l b l• TCA, citric acid cycle, Krebs cycle• Aids in oxidizing ____________ to CO2

• Stores H+/electrons in reduced _________:• NADH/H+ and FADH2

• One SLP step produces GTP (gets converted to ATP)

• Major pathway in aerobic respiration

How many ATPs are produced?

1 Glucose

(2 pyruvates)

2 ATP + 2 NADHTCA (2 pyruvates)

2

2

Cash ‘em in

NADH = 8FADH = 2GTP = 2

Glycolysis/TCA38 ATP

per glucose

2

2

2

2

3 ATP : 1 NADH

2 ATP : FADH

1 ATP : GTP

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Fate of reduced coenzymes generated by TCA cycle…

…they are oxidized by enzymes arranged in an electron transport chain.

NADH2 NAD+

2H+

2H+

2H+

2H+

2H+

2H+

1/2 O2

H2O

ADP ATPH+

H+H+H+H+H+H+

Chemiosmotic hypothesis: proton gradient is used to for chemical, osmotic, and mechanical work.

Overview of Respiration

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Electron transport and Oxidative Phosphorylation

cytoplasmic _____________

Required components:

Electron transport & oxidative phosphorylation

__________________proton + charge gradientmembrane-bound ___________

Electron transport phosphorylation occurs during:

redox reaction

_____

Respiration: An aerobic or anaerobic catabolic process. An organic or inorganic electron donor is oxidized using O2 ( or an O2substitute) as the final electron acceptor

Photosynthesis: Capture and use of Light Energy to fix [i.e. Incorporate] carbon into biomass.

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What is the proton motive force (Δp)?

∆p = ∆Ψ - Z∆pH

∆Ψ l t i lH+ H+

H+ develops ∆Ψ = electrical membrane potential, in mV

∆pH = pH gradient

Z = 2.3RT/F = 60 mV

H+A-

H+

H+

H+H+

H+H+

____ develops

____ developsA-

A-

A-

In Out

e-____ develops+

-

1. _________________: protons are pumped from

inside the cell to the outside.

Eg. NADH dehydrogenase

2._____________: protons are transferred to quinones on CM inner leaflet and released on the outer leaflet

Ways to make a proton motive force

3. _______________: protons are consumed

Eg. NADH dehydrogenase and cytochrome c oxidase

leaflet.

1

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pby reduction reactions on the inner leaflet of

the CMEg. O2 reduced to H2O

3

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• Some of the energy liberated during electron transport is used to drive the synthesis of ATP in a process called

id ti h h l ti

Structure and function of ATP synthase (ATPase).

oxidative phosphorylation• Uses the electrochemical proton

gradient (part of the PMF)• Can run in reverse to generate a

proton gradient: 1 mol of F1 >> hydrolyzes 104 ATP to ADP + Pi

• 3-4 protons per ATP synthesized

There is evidence for conformational changes and molecular rotation in the ATP synthase complexduring proton movement across the membrane

Redox reactions are essential to the function of electron transport chains

e-

• In respiration the TEA is usually obtained from the external environment.

• The reduced TEA is usually secreted

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Oxidation-Reduction Reactionsand Electron Carriers

• Oxidation-reduction (redox) reactions involve the transfer of electrons from a donor (reducing agent or reductant) to an acceptor (oxidizing agent or oxidant)

• The equilibrium constant for the reaction is called the standard reduction potential (E0) and is a measure of the tendency of the reducing agent to lose electrons

• Prokaryotes use electron carriers to transfer electrons from a reductant to an acceptor with a more positive (higher) reduction potential, and they thereby allow the release of free energy, which is often used in the formation of ATP.

• Biological cells have a variety of electron carriers, and each is used in particular types of redox reactions; the particular carrier used in any given reaction will depend on the nature and location of the reaction

Quinone/Quinol

• Hydrophobic, non-protein molecules (Fig. 5.18)

• Accepts 2e- and 2H+ but only donates 2e- to next redox partner

•Q-loop or Q-cycle for proton translocation•Many different types, ubiquinone, menaquinone

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The Q-Loop and PMF

Cytochromes In ShewanellaCymA tetraheme

cytochrome

• Contain iron porphyrin ring (heme)• Fe2+ Fe3+ during oxidation.• Electron transfer only (1 e-)• Many different types, numbers of hemes, called cyta, cytb, cytc

CXXCHX = amino acidMotif for c-type cytochromes

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CXXCH in the protein structure

C

Cys

Cys

His

Heme

Iron-Sulfur Cluster

•Electron transfer only•Exist as Fe-S clusters of different types•Ferridoxin is an example of one.

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Flavins

• Found in membrane proteins (integral or peripheral).

• Accepts electrons and protons from NADH

•Flavins only donate electrons

Redox reactions & growth substrates

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Redox reactions and reduction potentials (ERedox reactions and reduction potentials (Eoo’’))

• Eo’ is the tendency of a substrate to donate or accept electrons given.g

• Measured in Volts and determined under standard conditions: pH 7.0, 1 M, 25˚C

• Electrons do not exist in solution so half reactions must be coupled to other ones

The difference in reduction potentials can be compared for various respiratory reactions.

This is useful because we can calculate a ∆G for the reaction.

Electron Tower and EnergyElectron Tower and Energy

H2 + ½ O2 => H2OThe Electron Tower:

Reduction potentialsof half reactions

SeeSection 8.3

H2 2H+ + 2e- (Oxidation)(Reduction) 2H+ + 2e- + ½ O2 H2O

e-(Overall Rnx) H2 + ½ O2 => H2O

∆Eo’ = _____________

• Electrons flow from low (more neg.) to high (more positive) potential e- donors.

• The greater the fall of electrons the more potential energy can be harvested in the balanced reaction.

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Hydrogen Oxidation coupled to Oxygen reduction Example

H2 2H+ + 2e- (Oxidation)(Reduction) 2H+ + 2e- + ½ O2 H2O

∆Eo’ = Eo’(e- acceptor) - Eo’(e- donor)Nernst Equationto calculate ∆Go’

(Overall Rnx) H2 + ½ O2 => H2O

∆Go’ = -nF∆Eo’

Free Energy and Reactions

• Free energy change (ΔG) is the amount of energy in a system that is available to do gy ywork– A _________ ΔG indicates that the reaction is

favorable and will proceed spontaneously (i.e., the reaction is exergonic)

– A _________ ΔG indicates that the reaction is unfavorable and will only proceed if energy is unfavorable and will only proceed if energy is supplied (i.e., the reaction is endergonic)

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Electron transport chains and their relation to E0'.

2H+ H+

O 2 H O

H+

per

cyto

low Eo’ electron flow hi Eo’

Q

• Transfer electrons from an electron donor to an acceptor with a greater, (more positive) reduction potential.

• Electrons from NADH and FADH2 are transported in a series of redox

2NADH 2NAD+2H+ H+

O24H+

2 H2O

H+

are transported in a series of redox reactions to a terminal electron acceptor

• Conserve some of the energy released during electron transfer in PMF

• Use PMF to synthesize ATP

Example: Aerobic electron transport chainExample: Aerobic electron transport chain

NADH + H+ NAD+

2e-, 2H+

1/2O2 H2O

Eo’ = - 0.32 V

Eo’ = + 0.818 V/ O2 2O o 0 8 8

How much energy is released?

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NADH + H+ NAD+

2e-, 2H+

Eo’ = - 0.32 V

1/2O2 H2O Eo’ = + 0.818 V

How much energy does it take to make 1 ATP?

If ∆Go’ = -31 kJ/mol for ATP >>> ADP + Pi, how many ATPs can be made from -220 kJ/mol rxn with NADH oxidation coupled to oxygen reduction?NADH oxidation coupled to oxygen reduction?

Theoretical:

Reality:

Efficiency:

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Anaerobic∆Eo’

Electron Tower

0.45 Vn= 2e-

0.84 V

Aerobic

calculate the ∆Eo’ and ∆Go’

n= 2e-

1.24 Vn= 2e-

Electron transport and ATP synthesisElectron transport and ATP synthesis

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Inhibitors of Respiration

• __________: block the flow of ________ through the system, which blocks formation of ____________. – Carbon monoxide and cyanide bind to cytochromes– DCCP (dicyclohexylcarbodiimide) binds to ATP synthase

• __________: Prevent ___________ without ff ti affecting _______________.– Dinitrophenol (DNP), lipid soluble make membrane leaky;

destroys the PMF; shuts down ATP production by oxidative phosphorylation.

PMF can be used for lots of processes

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Three Important Processes to Remember in Respiration

Three Important Processes to Remember in Respiration

1. Carbon Flow As an organic compound is oxidized to CO2, reducing power (NADH, NAD(P)H, FADH)

and carbon intermediates are generated. These intermediates will be used in biosynthesis and/or secreted.These intermediates will be used in biosynthesis and/or secreted.

2. Electron FlowElectrons in a chemical energy source are

transferred by the membrane-bound intermediate electron carriers of an ETSto a final electron acceptor (e.g. O2, NO3

-, SO42-,CO2,…);

The electron flow generates PMF.The reduced products are secreted

3. Oxidative PhosphorylationEnergy generated by electron flow is captured as PMF and, then, used to synthesize

ATP.

Carbon and Electron Flow explain how Glycolysis and the TCA Cycle are linked to Oxidative Phosphorylation in Respiration.

Summary• Catabolism-the breakdown of larger, more complex molecules into smaller, simpler ones, during

which energy is released, trapped, and made available for work

• Catabolism is a Multi-stage process– Stage 1-breakdown of large molecules (polysaccharides, lipids, proteins) into their component

constituents with the release of little (if any) energy– Stage 2-degradation of the products of stage 1 aerobically or anaerobically to even simpler

molecules with the production of some ATP, NADH, and/or FADH2St 3 l t bi id ti f t 2 d t ith th d ti f ATP NADH – Stage 3-complete aerobic oxidation of stage 2 products with the production of ATP, NADH, and FADH2; the latter two molecules are processed by electron transport to yield much of the ATP produced

• Substrate level Phosphorylation – transfer of Pi from a high energy phosphorylated intermediate to ADP by a kinase enzyme– fermentations are important pathways for SLP reactions.

• Respiration, Electron Transport, and Oxidative Phosphorylation– Electrons from NADH and FADH2 are transported in a series of redox reactions to a terminal

electron acceptor– Electron carriers are located within the plasma membrane in prokaryotesp p y– Some of the energy liberated during electron transport is used to drive the synthesis of ATP

in a process called oxidative phosphorylation

• Redox reactions– Oxidation-reduction (redox) reactions involve the transfer of electrons from a donor

(reducing agent or reductant) to an acceptor (oxidizing agent or oxidant)