Outline:
Metabolism
• Part I: Fermentations
• Part II: Respiration
• Part III: Metabolic Diversity
Learning 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
products
•How can a microorganism get more energy from glucose?
1
Use respiration and the
tricarboxylic acid cycle
• 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?
TCA
NADH = 8
FADH = 2
2 ATP + 2 NADH
1 Glucose
(2 pyruvates)
2
GTP = 2
2
Glycolysis/TCA
38 ATP
per glucose
Cash ‘em in
3 ATP : 1 NADH
2
2 ATP : FADH
2
1 ATP : GTP
2
2
2
Fate of reduced coenzymes
generated by TCA cycle…
…they are oxidized by enzymes arranged in an electron
transport chain.
2H+
2H+
NADH2 NAD+
2H+
ADP
2H+
2H+
H+
2H+ H2 O
1/2 O2
ATP
H+ H+H+H+ H+H+
Chemiosmotic hypothesis: proton gradient is used to for
chemical, osmotic, and mechanical work.
Overview of Respiration
3
Electron transport and Oxidative
Phosphorylation
Electron transport & oxidative
phosphorylation
Required components:
cytoplasmic _____________
__________________
redox reaction
proton + charge gradient
membrane-bound ___________
_____
Electron transport phosphorylation occurs during:
Respiration: An aerobic or anaerobic catabolic process. An
organic or inorganic electron donor is oxidized using O2 ( or an O2
substitute) as the final electron acceptor
Photosynthesis: Capture and use of Light Energy to
fix [i.e. Incorporate] carbon into biomass.
4
What is the proton motive force (∆p)?
H+
∆p = ∆Ψ - Z∆pH
H+
H+
H+
H+
____ develops
AH+ +
H
H+ A+
H
A-
H+ A -
e-
____ develops
∆Ψ = electrical
membrane potential, in
mV
∆pH = pH gradient
Z = 2.3RT/F = 60 mV
+ ____ develops
-
Out
In
Ways to make a proton motive force
1. _________________:
protons are pumped from
inside the cell to the
outside.
Eg. NADH dehydrogenase
and cytochrome c oxidase
2
1
2._________: protons
are transferred to
quinones on CM inner
leaflet and released on
the outer leaflet.
1
3
3. _______________:
protons are consumed
by reduction reactions
on the inner leaflet of
the CM
Eg. O2 reduced to H2O
5
Structure and function of ATP
synthase (ATPase).
•
•
•
•
Some of the energy liberated during
electron transport is used to drive the
synthesis of ATP in a process called
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 complex
during 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
6
Oxidation-Reduction Reactions
and 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
7
The Q-Loop and PMF
Cytochromes
In Shewanella
CymA
tetraheme
cytochrome
CXXCH
•
•
•
•
Contain iron porphyrin ring (heme)
Fe2+
Fe3+ during oxidation.
Electron transfer only (1 e-)
Many different types, numbers of hemes, called cyta , cytb, cytc
X = amino acid
Motif for c-type
cytochromes
8
CXXCH in the protein structure
Cys
His
Cys
Heme
Iron-Sulfur Cluster
•Electron transfer only
•Exist as Fe-S clusters of different types
•Ferridoxin is an example of one.
9
Flavins
• Found in membrane proteins
(integral or peripheral).
• Accepts electrons and protons
from NADH
• Flavins only donate
electrons
Redox reactions & growth
substrates
10
Redox reactions and reduction potentials (E
(Eo’)
• Eo’ is the tendency of a substrate to donate
or accept electrons given.
• 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.
See
Section 8.3
Electron Tower and Energy
The Electron Tower:
Reduction potentials
of half reactions
H2 + ½ O2 => H2O
H2  2H+ + 2e- (Oxidation)
(Reduction) 2H+ + 2e- + ½ O2  H2O
(Overall Rnx) H2 + ½ O2 => H2O
e-
∆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.
11
Hydrogen Oxidation coupled to
Oxygen reduction Example
H2  2H+ + 2e- (Oxidation)
(Reduction) 2H+ + 2e- + ½ O2  H2O
(Overall Rnx) H2 + ½ O2 => H2O
∆Eo ’ = Eo ’(e- acceptor) - Eo’(e- donor)
Nernst Equation
to calculate ∆Go’
∆Go’ = -nF∆Eo’
Free Energy and Reactions
• Free energy change (∆G) is the amount of
energy in a system that is available to do
work
– A _________ ∆G indicates that the reaction is
favorable and will proceed spontaneously (i.e.,
the reaction is exergonic)
– A _________ ∆G indicates that the reaction is
unfavorable and will only proceed if energy is
supplied (i.e., the reaction is endergonic)
12
low Eo ’
Electron transport
chains and their
relation to E0'.
2H+
electron flow
hi E o ’
H+
H+
per
Q
cyto
2NADH
2H+
2NAD+
H+
O2
4H+
2 H2 O
H+
• 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
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 chain
NADH + H+
NAD+
Eo’ = - 0.32 V
2e-, 2H+
1/2O2
H2O
Eo’ = + 0.818 V
How much energy is released?
13
NADH + H+
NAD+
Eo’ = - 0.32 V
2e-, 2H+
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?
Theoretical:
Reality:
Efficiency:
14
Anaerobic
Electron Tower
∆Eo’
0.45 V
n= 2e-
0.84 V
n= 2e-
Aerobic
1.24 V
n= 2e-
calculate the ∆Eo’ and ∆Go’
Electron transport and ATP synthesis
15
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
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
16
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.
2. Electron Flow
Electrons in a chemical energy source are
transferred by the membrane-bound intermediate electron carriers of an ETS
to a final electron acceptor (e.g. O2, NO3-, SO42-,CO2 ,…);
The electron flow generates PMF.
The reduced products are secreted
3. Oxidative Phosphorylation
Energy 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 FADH2
– 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 prokaryotes
– 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)
17