Day 21 Chemiosmosis Evidence
• Monday, Oct. 5: Mitochondria – Chemiosmosis,
Ch. 14
• Exam 2 due 1 PM
• Flu clinic, 9-6, Bovee
• Wednesday, Oct. 7: Chemical signaling 1, Ch. 15
• Friday, Oct. 9: Chemical signaling 2, Ch. 15
I. Mechanism of coronavirus infection
II. The chemiosmotic theory
A. Review of in mitochondrial function
B. Evolution of chemiosmosis
C. Evidence that ETS > H+ gradient
D. Evidence that H+ gradient > ATP synthesis
Respiration rap: http://www.youtube.com/watch?feature=player_embedded&v=VCpNk92uswY
After reading the text, attending
lecture, and reviewing lecture
notes, you should be able to:
• Explain the mechanism of infection
of SARS coronaviruses.
• Describe the chemiosmotic model
of mitochondrial ATP production.
• Explain why anaerobic bacteria run
ATP synthase backwards.
• Give evidence that electron
transport causes proton pumping.
• Give evidence that the protein
gradient drives ATP synthesis.
 Review: coronavirus replication
ACE2 “receptor”

Endocytosis Budding
RDRP (-) by Zn2+,
and remdesivir? and
Replication: Exocytosis

(cis-Golgi)

https://microbenotes.com/coronavirus/
 pyr

Chemiosmosis
in mitochondrion:
4 4
4 4
transporters
Review: Mitochondria and chloroplasts compared and
contrasted
Feature Mitochondria Chloroplasts
Site of electron transport inner mito- membrane thylakoid membrane
chain, ATP synthase
Source of e- for chain NADH, FADH2 H2O
Final e- acceptor after e- O2 NADP+
transport
Proton motive force mostly from Vm, less from mostly from H+ conc.
(pmf) H+ conc. gradient gradient, less from Vm
Size of H+ gradient 1 pH unit 2.5 pH units
Direction of H+ flow From IMS to matrix From thylakoid lumen to
through ATP synthase stroma
ATP made in matrix stroma
ATP synthase (like any enzyme) can work in either direction;
working backwards it acts as a proton pump
Direction depends on Vm , H+ gradient, conc. of ATP, ADP, Pi.
Reversal of ATP synthase in facultatively anaerobic
bacteria

ATP made by fermentation:

 Selected for by
1. H+ pump acidic products
of fermentation

Possible evolution of 2. e- transport

Selected for by
reduced availability

chemiosmosis
of nutrients, using
fermentation
products as e-
source

Efficiency of H+
pumping by e-
3. Chemiosmotic transport so great
that ATP-driven
coupling
pump can be driven
backwards.
A. Chemiosmosis Evidence:
1. ETS  H+ pumping
1. Mitochondria + O2
2. Chloroplasts + light
3. Orientation of mitochondrial vesicles determines direction
of pumping
4. Asymmetrical orientation of IMM components correlates
with unidirectional pumping
1. Mitochondria + O2  pH change

O2
pH N2 pH 7
meter

time
Mitochondria w/o O2

• Mitchell and Jennifer Moyle, 1965

• Conclusion: Stimulation of electron transport is necessary for proton pumping in
mitochondria.
2. Chloroplasts + light  pH change

pH
meter pH 6

on off

Chloroplasts in the dark time

• Jagendorf and Hind, 1963

• Conclusion: Stimulation of electron transport is necessary for proton pumping
in chloroplasts.
3. Inside-out IMM vesicles now pump
protons inward
Inside-out
Right-side out H+

H+

Conclusion: Reversal of electron transport system asymmetry is sufficient to cause H+ pumping in

the reverse direction.
4. RC crystal structures show asymmetrical orientation of
ETS proteins, correlating with unidirectional H + pumping

RCIV Cyt c2e-

2H+

Q QH2

Q QH2

2e- 2H+
RCIII from
RC1
 RCIV crystal structure
2 H+
Cyt c IMS

MATRIX 2e- ½ O2
H+ H+

2 H+ H2O
Conclusion: Structure of respiratory complex structure correlates with
unidirectional proton pumping.
B. Evidence that electrochemical H+
gradient  ATP Synthesis
1. Chloroplast loading w/acid  ATP synthesis.
2. A membrane is needed.
3. Uncoupling agents abolish H+ gradient and ATP synthesis.
4. H+ pumping, ATP Synthase can be isolated and reconstituted to
make ATP.
1. Chloroplast Loading With Acid
(Jagendorf et al., 1966)

Burst of ATP
synthesis,
followed by
disappearance
of pH gradient.
2. A membrane is needed for ATP synthesis
• Control: Respiratory complexes and ATP synthase in membrane
vesicles  ATP

• Experiment: Respiratory complexes and ATP synthase w/ no

membrane  no ATP

• Conclusion: A membrane, allowing for a H+ gradient, is necessary for

ATP synthesis.
3. Uncoupling agents abolish H+ gradient and ATP
synthesis

• Dinitrophenol (DNP) is a chemical that dissipates the H + gradient, stopping ATP synthesis.
• Thermogeninin is a natural uncoupler protein in brown fat cells.
• Conclusion: A H+ gradient is necessary for ATP synthesis.
4. ATP synthesis in artificial vesicles: H+ gradient and
ATP synthase are necessary