Exam 2: Lecture 9 Ch 5- Mitochondrial Membranes and Mitochondrial Function

Describe how the proton gradient created across the mitochondrial inner membrane leads to ATP synthesis in cellular respiration

• In an actively respiring mitochondrion, the intermembrane-space side of the inner membrane has more H+, lower pH, and a relatively positive charge.

• The matrix has fewer H+, a higher pH, and a relatively negative charge. The ETC- created electrochemical gradient stores energy, so H+ returns from the intermembrane space to the matrix through the embedded F0 portion of ATP synthase (or Complex V)

• H+ movement also drives the rotation of the c ring on the F0 portion of ATP synthase, which then turns the gamma stalk and changes the conformations of the catalytic beta subunits in the matrix-facing G1 head. They bind ADP and Pi, synthesize ATP, and release ATP.

In terms of the F1 head of ATP and the catalytic beta subunits rotating via the gamma stalk, what conformations (O, L, or T) do what?

1. O for open state= ATP can leave and ADP + Pi can bind

2. L for loose state = ADP and Pi are associated with the beta subunit

3. T for tight state = ATP is synthesized from ADP and Pi.

• Loose → Tight = drives catalysis of ATP

• Tight → Open = releases ATP

Offer a molecular description of one of the components of ATP synthase: F0

The membrane proton-conducting portion:

• The hydrophobic portion embedded in the inner mitochondrial membrane.

• Contains the rotating ring of c subunits and interacts with the stationary subunit

• Proton movement through this machinery drives c-ring rotation (contains 10-14 subunits)

Offer a molecular description of one of the components of ATP synthase: F1

The matrix-facing catalytic head:

• Contains three alpha and three beta subunits arranged around a central gamma stalk

• The three beta subunits contain the catalytic sites; each occupies a different binding conformation at a given instant (L, T, or O)

• ATP is synthesized and released on the matrix side

Offer a molecular description of one of the components of ATP synthase: Gamma and epsilon stalk

• The central stalk that connects the membrane rotor to the F1 head.

• The c ring is tightly associated with this component and is the reason this component rotates, which drives the changes in conformations of the beta subunits

• The mechanical connection between proton movement and catalytic-site cycling

Offer a molecular description of one of the components of ATP synthase: A subunit and stationary components

• The component that contacts the c ring and contains TWO physically separate half-channels: one opens toward the intermembrane-space side and the other toward the matrix.

• Neither half-channel alone spans the entire membrane.

• Slide 16 describes an immobile exterior column containing one a, two b, and one delta (δ) subunit.

• The stationary portion allows the ring/stalk to rotate relative to the other parts of the enzyme.

Offer a molecular description of one of the components of ATP synthase: Proton-binding residues

• Each c subunit has this component that picks up and releases H+- aspartic acid (Asp) or glutamic acid (Glu)

• Protonation neutralizes its negatively charged carboxylate; deprotonation restores the negative charge.

• The annotated diagram also identifies positively charged Arg in the a subunit interacting with a negatively charged acidic residue.

Describe the sequence of events in ATP synthase function whereby the energy of the proton gradient is coupled to the synthesis of ATP

1 — A proton enters the first half-channel. A proton enters the a-subunit half-channel that opens to the H⁺-rich intermembrane-space side. Slides that label this the “cytoplasmic (+)” side are referring to the side opposite the matrix in this mechanism.

2 — Proton binding advances the ring. The proton binds the Asp/Glu residue of a c subunit. Protonation and the associated changes/interactions permit the c ring to advance. Sequential proton binding and ring movement carry protons around with the c subunits. In a 10-subunit ring, one proton corresponds to one-tenth of a rotation; the fraction depends on ring size.

3 — The proton exits into the matrix. After being carried around, the proton-binding residue reaches the second a-subunit half-channel, which opens to the higher-pH matrix. The residue releases H⁺, becomes deprotonated, and can participate in another cycle. Net H⁺ movement is from intermembrane space to matrix.

4 — Rotation changes catalytic binding sites. Because the c ring is attached to the stalk, ring movement rotates gamma. Successive 120° changes of gamma alter the three beta-subunit conformations. The beta sites cycle through loose, tight, and open states, coordinating substrate binding, ATP formation, and ATP release. One full 360° rotation yields approximately 3 ATP in the lecture model.

Which sequence of events in ATP synthase function whereby the energy of the proton gradient is coupled to the synthesis of ATP is being described? Options: 1 — A proton enters the first half-channel. 2 — Proton binding advances the ring. 3 — The proton exits into the matrix. 4 — Rotation changes catalytic binding sites.

Because the c ring is attached to the stalk, ring movement rotates gamma. Successive 120° changes of gamma alter the three beta-subunit conformations. The beta sites cycle through loose, tight, and open states, coordinating substrate binding, ATP formation, and ATP release. One full 360° rotation yields approximately 3 ATP in the lecture model.

4 — Rotation changes catalytic binding sites.

Which sequence of events in ATP synthase function whereby the energy of the proton gradient is coupled to the synthesis of ATP is being described? Options: 1 — A proton enters the first half-channel. 2 — Proton binding advances the ring. 3 — The proton exits into the matrix. 4 — Rotation changes catalytic binding sites. :

The proton binds the Asp/Glu residue of a c subunit. Protonation and the associated changes/interactions permit the c ring to advance. Sequential proton binding and ring movement carry protons around with the c subunits. In a 10-subunit ring, one proton corresponds to one-tenth of a rotation; the fraction depends on ring size.

2 — Proton binding advances the ring.

Which sequence of events in ATP synthase function whereby the energy of the proton gradient is coupled to the synthesis of ATP is being described? Options: 1 — A proton enters the first half-channel. 2 — Proton binding advances the ring. 3 — The proton exits into the matrix. 4 — Rotation changes catalytic binding sites.

A proton enters the a-subunit half-channel that opens to the H⁺-rich intermembrane-space side. Slides that label this the “cytoplasmic (+)” side are referring to the side opposite the matrix in this mechanism.

1 — A proton enters the first half-channel

Which sequence of events in ATP synthase function whereby the energy of the proton gradient is coupled to the synthesis of ATP is being described? Options: 1 — A proton enters the first half-channel. 2 — Proton binding advances the ring. 3 — The proton exits into the matrix. 4 — Rotation changes catalytic binding sites.

After being carried around, the proton-binding residue reaches the second a-subunit half-channel, which opens to the higher-pH matrix. The residue releases H⁺, becomes deprotonated, and can participate in another cycle. Net H⁺ movement is from intermembrane space to matrix.

3 — The proton exits into the matrix.

Predict the impact of mutating Asp/Glu in the C ring of the Fo complex.

ATP synthesis would be reduced significantly:

• If a mutation removes the acidic side chain’s ability to bind and release H⁺, the c ring cannot carry out normal proton-coupled rotation.

• Gamma rotation and beta-site conformational cycling would slow or stop, reducing ATP synthesis and release.

• The immediate defect is in USING the proton gradient; it does not directly remove an ETC electron-transfer cofactor.

• The prediction applies to mutations that impair the required residue function, rather than every possible substitution.

Explain why NADH results in ~3 ATP and whereas FADH₂ results in only ~2 ATP

1. Different electron-entry points:

• NADH supplies the Complex I pathway, whereas the succinate/FADH₂ route enters through Complex II.

• Complex I transfers electrons from NADH to Q, and Complex II transfers succinate-linked electrons to Q.

• Both routes then feed the downstream Q-to-cytochrome-c and cytochrome-c-to-O₂ steps.

• Complex II does NOT PUMP PROTONS

2. Different proton supply for ATP synthesis:

• The NADH route supports more proton pumping because it includes the Complex I contribution that the Complex II entry route bypasses.

• More H⁺ can then return through ATP synthase, supporting more rotation and ATP release.

• Thus approximately 3 ATP per NADH and approximately 2 per FADH₂.

• The larger medium-pH decrease for NADH than succinate in slide 27 is consistent with more proton pumping in the NADH condition.

Explain how liposomes/vesicles containing ETC Complexes can be used to demonstrate that electron flow is coupled to proton pumping

• ETC components are isolated by detergent solubilization and chromatography, then reconstituted into synthetic lipid vesicles.

• A vesicle creates separate inside and outside aqueous compartments, and there is an appropriate reduced electron donor and an available electron acceptor for the inserted complex, and a sensitive pH electrode that monitors the surrounding solution.

• Electron transfer accompanied by a directional pH change demonstrates coupling of electron flow to H⁺ transport

EX: A liposome contains cytochrome oxidase, Complex IV. Reduced cytochrome c supplies electrons, and O2 is the terminal acceptor, forming water. H+ is pumped from the vesicle interior into the surrounding solution. Outside H+ increases so the pH falls, and the interior loses H+ so the pH rises.

Describe what happens during each conformational change of the beta subunits: Loose state L

ADP and Pi are bound at the beta-subunit active site. Compared with the open state, substrates are held more tightly. This conversion traps/binds substrates for the next step

Describe what happens during each conformational change of the beta subunits: Tight state T

The active site converts ADP and Pi to ATP, and the ATP remains tightly bound. This transition therefore corresponds to ATP formation

Describe what happens during each conformational change of the beta subunits: Open state O

This transition lowers the active site’s affinity for ATP and releases it. The open site allows ADP to enter or leave; further rotation returns it to L. The slides identify energy-driven ATP release as the principal energetic requirement of the binding-change mechanism.

What happens during conformational changes? What drives the conformational change in the Beta subunits?

• At a given instant, one beta subunit is L, one is T, and one is O.

• As gamma rotates, all advance to their next states; each individual site follows L → T → O → L.

• One 120° step advances all three sites, with one ATP-release event across the head; a complete rotation lets each site complete a cycle, yielding approximately 3 ATP total.

• The beta subunits change shape because gamma rotates, and gamma rotates because H⁺ flow drives the c ring.

Relate the number of protons translocated during ETC flow to the rotation of the C ring

• The number of H+ translocated through ATP synthase per complete rotation equals the number of c subunits.

EX: a ring with 10 c subunits requires 10 H+ for a full turn

• One proton advances a 10-subunit ring by 36 degrees and a 14 unit subring by approx. 25.7 degrees

Relate the number of protons translocated during ETC flow to the synthesis of ATP

• A full turn produces approximately 3 ATP

• Dividing the number of c subunits by 3 therefore gives the APPROXIMATE H+ requirement per ATP at ATP synthase

Relate the number of protons translocated during ETC flow to actual electron transport

• ETC electron flow builds the H⁺ gradient, while ATP synthase uses H⁺ returning through its rotor.

• More ETC-driven proton translocation can support more rotary cycles and ATP production.

• Do not equate the c-ring subunit count with the number of protons pumped by a particular ETC complex: the count specifies the H⁺ used per ATP synthase revolution

Predict the impact that increasing the number of c ring subunits would have on the ratio of NADH molecules required to produce 1 ATP

More c subunits require more H⁺ per revolution, while ATP output remains approximately 3 per revolution.

• Therefore MORE NADH molecules are required per ATP, and fewer ATP are produced per NADH

Predict the impact that decreasing the number of c ring subunits would have on the ratio of NADH molecules required to produce 1 ATP

Fewer c subunits require fewer H⁺ per revolution.

• Therefore FEWER NADH molecules are required per ATP, and more ATP are produced per NADH.

Predict how the pH of the solution containing vesicle with Complex I would/would not change if incubated with NADH, FADH2, QH2, or reduced cytochrome c

• NADH is the matching donor; oxidized Q is needed as acceptor. With electron transfer and the specified orientation, H⁺ is pumped outward and external pH decreases.

• FADH₂, QH₂, or reduced cytochrome c do not replace NADH as the entry donor for isolated I in the pathway shown.

Predict how the pH of the solution containing vesicle with Complex II would/would not change if incubated with NADH, FADH2, QH2, or reduced cytochrome c

• Complex II does not pump protons; thus, no change in outward pH occurs when incubated with NADH, FADH₂, QH₂, or reduced cytochrome c.

• Complex II transfers electrons but does not pump H⁺, so there is no pump-driven decrease in external pH from isolated II, even when electron transfer occurs.

Predict how the pH of the solution containing vesicle with Complex III would/would not change if incubated with NADH, FADH2, QH2, or reduced cytochrome c

• Reduced ubiquinone, QH₂ or ubiquinol, is the matching donor; oxidized cytochrome c is needed as acceptor.

• Electron flow supports outward H⁺ translocation and external pH decreases.

• NADH or FADH₂ cannot supply an isolated III vesicle through I or II because those complexes are absent; reduced cytochrome c is downstream of III rather than its forward electron donor.

Predict how the pH of the solution containing vesicle with Complex IV would/would not change if incubated with NADH, FADH2, QH2, or reduced cytochrome c

• Reduced cytochrome c is the matching donor and O₂ is the acceptor.

• External pH decreases.

• NADH, FADH₂, and QH₂ do not directly supply IV in this isolated-complex arrangement.

Explain how liposome/ vesicles containing rhodopsin and ATP synthase can be used to demonstrate the chemiosmotic theory (protein gradient is coupled to ATP synthesis)

1. Bacteriorhodopsin pumps H+ INTO the vesicle, producing a higher H+ concentration inside. ADP and Pi are available on the side facing ATP synthase’s catalytic head, which faces outward towards the matrix.

2. H+ then moves OUT of the vesicle through ATP synthase, down the gradient created by the light-driven pump. This powers c-ring/ stalk rotation and ATP production outside. The sequence is light energy → H+ gradient → ATP synthase rotation and binding changes → ATP.

3. This demonstrates that a proton gradient generated by a light-powered protein can support ATP synthesis, linking the gradient itself to ATP production without requiring an ETC to create it in this setup. The pump uses light to establish the gradient. ATP synthase uses this gradient. This supports chemiosmotic coupling.

Predict the impact of disrupted flow through the ETC on ATP synthase

• An ETC block reduces or stops electron-flow-driven H+ pumping along the affected route.

• ATP synthase may initially use a pre-existing gradient, but continued H+ return without equivalent replenishment weakens that gradient.

• Proton-driven c-ring rotation, gamma rotation, and productive beta-site cycling then slow or stop, decreasing oxidative ATP synthesis and release.

• An ETC inhibitor can therefore inhibit ATP production indirectly even if it does not bind ATP synthase.

If a toxin blocks electron flow at COMPLEX I in the ETC, would the cofactors in each complex (upstream or downstream) be found in the reduced or oxidized state?

• If Complex I is blocked (ex: Fe-S centers and ubiquinone), NADH and the upstream Complex I cofactors remain reduced, while Q and later carriers get oxidized

  • Upstream (NADH, FMN, Fe-S clusters in Complex I): Stuck holding electrons = reduced

  • Downstream (Ubiquinone, Complex III, Cytochrome c Complex IV): Depleted of electrons = oxidized

If a toxin blocks electron flow at COMPLEX II in the ETC, would the cofactors in each complex (upstream or downstream) be found in the reduced or oxidized state?

• If Complex II is blocked (e.g., QH2 into Complex II), FADH2 and the cofactors in Complex II remain reduced, while Q and downstream carriers become oxidized.

• Upstream (FADH2, succinate in Complex II): Stuck holding electrons = reduced

• Downstream (Ubiquinone, Complex III, Cytochrome c, Complex IV): Depleted of electrons = oxidized.

How would the toxin impact oxygen consumption and ATP synthesis?