BI/CH 422/622

OUTLINE:
Introduction and review
Transport
Glycogenolysis
Glycolysis
Other sugars
Pasteur: Anaerobic vs Aerobic Exam-1 material
Fermentations
Exam-2 material Chemiosmotic theory: Phosphorylation
Pyruvate
pyruvate dehydrogenase (ox-decarbox; S-ester) ATPase
Krebs’ Cycle Mitchell Hypothesis
How did he figure it out?
Overview Binding-Change Model
8 Steps Connection to the proton-
Citrate Synthase (C–C)
Aconitase (=, -OH) motive force
Isocitrate dehydrogenase (ox-decarbox; =O)
Ketoglutarate dehydrogenase (ox-decarbox; S-ester)
Succinyl-CoA synthetase (sub-level phos)
Succinate dehydrogenase (=)
Fumarase ( -OH)
Malate dehydrogenase (=O)
Energetics
Regulation
Summary
Oxidative Phosphorylation
Energetics (–0.16 V needed for making ATP)
Mitochondria
Transport (2.4 kcal/mol needed to transport H+ out)
Electron transport: Oxidative
See Achieve:
Discovery
Four Complexes
Ch19: Case Study: The Narrow Window
Complex I: NADH à CoQH2
Complex II: Succinate à CoQH2
Complex III: CoQH2 à Cytochrome C (Fe2+)
Complex IV: Cytochrome C (Fe2+) à H2O

Electron Transport
Summary of the Electron Flow in the Respiratory Chain

DG’ of H+ transport = 2.4 kcal/mol Difference in number of

As a consequence, it will take
~3 protons per ATP. protons transported reflects
differences in ATP
This scheme is for 2e– coming down from 1 NADH or 1 FADH2 synthesized=P/O ratios:
Complex I à Complex IV Substrate P/O ratio
1NADH + 11H+(N) + ½O2 à NAD+ + 10H+(P) + H2O Pyruvate 3
Acetyl CoA 3
Complex II à Complex IV
NADH 3
FADH2 + 6H+(N) + ½O2 à FAD + 6H+(P) + H2O
Succinate 2

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Phosphorylation
(The Chemiosmotic Theory)

Oxidative Phosphorylation

Glycogenolysis

Glycolysis
Pyruvate Oxidation

Krebs' Cycle

Phosphorylation These are

Oxidative tightly
COUPLED

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 The Chemiosmotic Theory

Dr. Kornberg: Lecture 02.17.17 (45:00-48:25)-

mitochondria coupling
(3.5 min)

Oxidative Phosphorylation
Relationship of ETC and ATP Synthesis
The Chance/Williams experiment showed that
fuel oxidation is COUPLED to ATP synthesis

Here, same experiment done

the other way:

• As described, ATP synthesis requires electron transport.

• But electron transport does not require ATP synthesis.

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 Oxidative Phosphorylation
Relationship of ETC and ATP Synthesis
The Chance/Williams experiment showed that
fuel oxidation is COUPLED to ATP synthesis

Here, same experiment done

the other way:

• As described, ATP synthesis requires electron transport.

• But electron transport does not require ATP synthesis.

Phosphorylation
How Does Oxidative Phosphorylation Form ATP?

• Before the 1970’s, it was thought that the ET chain

used the energy of redox to make a ”high-energy”
intermediate for “substrate-level phosphorylation, as
occurs in glycolysis and TCA cycle.
• It was clear that one of the most abundant proteins
on the inner-mitochondrial membrane had a curious
mushroom-shaped structure.
• It was purified, along with the other Complexes I-IV,
initially called Complex V.

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 Phosphorylation
How Does Oxidative Phosphorylation Form ATP?
• Complex V, when purified
separately from the F1-ATPase
membrane is an effective
ATPase enzyme, hence it
was initially called the F1-
ATPase.
• The F1 part had 9 subunits F0
(a3b3gde)
• When more careful
purifications were
performed, it was clear that
F1
its activity was closely
coupled to an intact inner
membrane with little activity.
• When membrane proteins
were isolated, the F0 part • Inhibited by
had 15 subunits of 3 venturicidin or
different proteins (a, b2, c10). oligomycin

Phosphorylation
How Does Oxidative Phosphorylation Form ATP?
• Complex V, when purified
separately from the
membrane is an effective
ATPase enzyme, hence it
was initially called the F1-
ATPase.
• The F1 part had 9 subunits
(a3b3gde)
• When more careful
purifications were
performed, it was clear that
its activity was closely
coupled to an intact inner
membrane with little activity.
• When membrane proteins
were isolated, the F0 part • Inhibited by
had 15 subunits of 3 venturicidin or
different proteins (a, b2, c10). oligomycin

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 Phosphorylation

Contains two functional units:

• F1
• soluble complex in the matrix
• individually catalyzes the
hydrolysis of ATP
• F0
• integral membrane complex
• transports protons from IMS
to matrix, dissipating the
proton gradient
• energy transferred to F1 to
catalyze phosphorylation of
ADP.
• This mechanism was only
fully accepted in late 1970s.

Phosphorylation
Hypothesis: The Respiratory Chain and Hypothesis: The Respiratory Chain
ATP Synthase Produce ATP by a Produce ATP by a substrate-level-
Chemiosmotic Mechanism VS. phosphorylation mechanism

Peter Mitchell
1920-1992

The search was for a compound, like 1,3

bisphosphoglycerate, that would be used
by the ATP Synthase to make ATP.

Peter Mitchell, 1920-1992

1978 Nobel Prize

for Chemistry

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 Phosphorylation
Chemiosmotic Theory
Ø ADP + Pi à ATP is highly thermodynamically unfavorable.
• How do we make it possible?
• Phosphorylation of ADP is not a result of a direct reaction
between ADP and some high-energy phosphate carrier.
• The energy released by the exergonic flow of electrons to
oxygen in electron transport is used to transport protons
against the electrochemical gradient. Secondary active transport
principles are at work.
• Energy needed to phosphorylate ADP is provided by the flow
of protons down this electrochemical gradient. This can be
calculated.
• If all that was needed was a proton gradient, could
one be established without the ET chain and still
drive ATP biosynthesis?

Phosphorylation
Chemiosmotic Theory • The proteins in the electron-transport chain
created the electrochemical proton gradient
(proton-motive force (PMF)) by one of three
means:
– actively transporting (pumping) protons
across the membrane
• Complex I and Complex IV
– releasing protons into the
intermembrane space
• oxidation of QH2 at Complex III
– chemically removing protons from the
matrix
• reduction of CoQ in Q-cycle (Complex I,
II, & III)
• reduction of oxygen (Complex IV)
DG’ = 1.0 + 1.4 = 2.4 kcal/mol

As a consequence, it will take

~3 protons per ATP. But, how
many precisely?

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 Phosphorylation
Chemiosmotic Theory Mitchell’s experiment:

Nobel Prize in Chemistry

1978 For 3 protons:
DG’ = 3.0 + 4.2 = 7.2 kcal/mol

Phosphorylation
Chemiosmotic Theory for
ATP Synthesis:
• Electron transport sets up a proton-
motive force.
• Energy of proton-motive force drives
synthesis of ATP.

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 Phosphorylation
Chemiosmotic Theory
Chemiosmotic Energy Coupling
Requires Membranes
• The proton gradient needed for ATP synthesis can be stably
established across a membrane that is impermeable to ions.
– plasma membrane in bacteria
– inner membrane in mitochondria
– thylakoid membrane in chloroplasts

• The membrane must contain proteins that couple the

“downhill” flow of electrons in the electron-transfer chain
with the “uphill” pumping of protons across the membrane.
• The membrane must contain a protein that couples the
“downhill” flow of protons to the phosphorylation of ADP.

Phosphorylation
The F1 catalyzes ADP + Pi ATP + H2O
Mechanism:
BINDING – O from water is quickly
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incorporated into Pi
–Kinetic studies
–Isotope studies

– Compare to >105 for in

–Structural studies solution

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 Phosphorylation
–MORE Structural
Mechanism: studies
RELEASE • Hexamer arranged in three αβ dimers
• Dimers can exist in three different
conformations:
– open: empty
– loose: binding ADP and Pi
– tight: catalyzes ATP formation and binds
product

T=tight
L O=open
L=loose

T
WHERE does the Called the Binding-
energy come from Change Model
for the release?
– This is where
ALL the energy
(in Dconf. from tight to
of PMF is used open;
O to release ATP!
thus, releasing ATP)

Phosphorylation
Mechanism
Binding-Change Model

WHAT drives the

g-subunits
motion?

John E. W alker

Nobel Prize in
ATP cannot be released from
Chemistry 1997 one site unless and until ADP
and Pi are bound at the
other…………without
substrates IT ALL GRINDS TO
A HALT!

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