Biochem Lect 32: ETC, Oxidative Phos

purpose of electron transport

create proton gradient using electron movement

purpose of oxidative phosphorylation

use proton gradient to make ATP

Enzymes of each Complex

Complex I = _____________

Complex II = _____________

Complex III = _____________

Complex IV = _____________

Complex V = _____________

Enzymes of each complex

Complex I = NADH dehydrogenase

Complex II = Succinate CoQ Reductase

Complex III = UQ-Cytochrome C Reductase

Complex IV = Cytochrome C Oxidase

Complex V = ATP Synthase

Order of Complexes

Which complexes contain Fe-S clusters or electron wires?

Complex I = Fe-S clusters and electron wire

Complex II = Fe-S clusters only

Complex III = 1 Fe-S cluster (Rieske)

Complex IV = None

Complex V = None

How many protons does each complex pump?

Complex I = 4

Complex II = 0

Complex III = 4

Complex IV = 2

Complex I = ?

NADH dehydrogenase

Complex I Structure

includes a large matrix arm on matrix side

___________ (on the NuoL subunit) on Complex I helps drive _________ that couples electron transfer to proton pumping

A long alpha helix (on the NuoL subunit) on Complex I helps drive conformational changes that couples electron transfer to proton pumping

Ubiquinone = ________, _____-soluble electron carrier

Also known as: _______ (____), ____, ____

Reduced form: ____

Ubiquinone = coenzyme, lipid-soluble electron carrier

Also known as: Coenzyme Q (CoQ), UQ, Q

Reduced form: QH₂

Complex I Mechanism

1) ______ gets oxidized by matrix arm → sends ____ e-

2) ///

3) ///

Complex I Mechanism

1) NADH gets oxidized by matrix arm → sends 2 e-

2) ///

3) ///

Complex I Mechanism

1) NADH gets oxidized by matrix arm → sends 2 e-

2) ____ gets 1 electron at a time from NADH to send through ________ within matrix arm

3) ///

Complex I Mechanism

1) NADH gets oxidized by matrix arm → sends 2 e-

2) FMN gets 1 electron at a time from NADH to send through electron wire within matrix arm

3) ///

Complex I Mechanism

1) NADH gets oxidized by matrix arm → sends 2 e-

2) FMN gets 1 electron at a time from NADH to send through electron wire within matrix arm

3) ___ gets reduced (gains ___ e-) and transfers electrons to Complex ___

Complex I Mechanism

1) NADH gets oxidized by matrix arm → sends 2 e-

2) FMN gets 1 electron at a time from NADH to send through electron wire within matrix arm

3) Q gets reduced (gains 2 e-) and transfers electrons to Complex III

How many H+ are pumped across membrane in Complex I (for every 2e-)?

4 H+

Q gets reduced to QH₂ and later QH₂ will get re-oxidized. What is the phenomenon called?

redox loop

Complex II = ?

Succinate-CoQ Reductase

Alternate names for Complex II

• succinate dehydrogenase (from TCA cycle)

• flavoprotein 2 (FP₂) → contains FAD

Complex II Structure

• ___ type cytochrome

• contains ___ subunits

  1. ___ Fe-S clusters (…)

  2. contains ___

  3. contains non-covalently bound _____

Complex II Structure

• b type cytochrome

• contains 4 subunits

  1. 3 Fe-S clusters (3Fe4S, 4Fe4S, 2Fe2S)

  2. contains FAD ← flavoprotein 2

  3. contains non-covalently bound heme b (Fe protophyrin IX)

Can heme participate in redox? Is it involved in the complex II reaction?

Yes, it can transfer 1 electron.

However in this reaction, it is not involved.

How many protons are pumped across complex II?

0

Does complex II contain an electron wire?

no

Complex II Mechanism

1) ________ oxidizes to ________ ←→ ____ reduces to ____

2) ///

3) ///

Complex II Mechanism

1) succinate oxidizes to fumarate ←→ FAD reduces to FADH₂

2) ///

3) ///

Complex II Mechanism

1) succinate oxidizes to fumarate ←→ FAD reduces to FADH₂

2) FADH₂ oxidizes to FAD ←→ 2___ reduce to 2____

3) ///

Complex II Mechanism

1) succinate oxidizes to fumarate ←→ FAD reduces to FADH₂

2) FADH₂ oxidizes to FAD ←→ 2Fe³⁺ reduce to 2Fe²⁺

3) ///

Complex II Mechanism

1) succinate oxidizes to fumarate ←→ FAD reduces to FADH₂

2) FADH₂ oxidizes to FAD ←→ 2Fe³⁺ reduce to 2Fe²⁺

3) 2Fe²⁺ oxidize to 2Fe³⁺ ←→ ____ reduces to ____

Complex II Mechanism

1) succinate oxidizes to fumarate <=> FAD reduces to FADH₂

2) FADH₂ oxidizes to FAD ←→ 2Fe³⁺ reduce to 2Fe²⁺

3) 2Fe²⁺ oxidize to 2Fe³⁺ ←→ Q reduces to QH₂

As complex II reduces Q, complex ___ oxidizes QH₂

As complex II reduces Q, complex III oxidizes QH₂

electrons flow in what order for the follwing:

FAD, heme B, Fe-S clusters

Succinate creates more/less ATP than NADH (and why?)

NADH gives electrons to which complexes?

Succinate gives electrons to which complexes?

less protons = more/less ATP

Succinate creates less ATP than NADH

NADH gives electrons to complex I (4 H+) → III → IV → V

Succinate gives electrons to complex II (0 H+) → III → IV → V

less protons = less ATP

_______________ oxidize fatty acyl CoAs and give electrons to Q.

FAD/NAD takes electrons.

Fatty acyl CoA dehydrogenase oxidize fatty acyl CoAs and give electrons to Q.

FAD takes electrons.

Complex III = ?

UQ-Cytochrome C Reductase

Complex III Structure

• contains ___ cytochrome which contains hemes ___ and ___

Complex III Structure

• contains b cytochrome which contains hemes b_L and b_H

Q Cycle Phase I

• QH₂ binds to ___ site and gets reduced to ___ → releases ___H+

• ///

• ///

Q Cycle Phase I

1. QH₂ binds to Q_p site and gets reduced to Q → releases 2H+

2. ///

3. ///

Q Cycle Phase I

1. QH₂ binds to Q_p site and gets reduced to Q → releases 2H+

2. 1st electron gets transferred to ___ → ___ → ___

3. ///

Q Cycle Phase I

1. QH₂ binds to Q_p site and gets reduced to Q → releases 2H+

2. 1st electron gets transferred to Fe-S (in Rieske subunit) → cyt c₁ → cyt c

3. ///

Q Cycle Phase I

1. QH₂ binds to Q_p site and gets reduced to Q → releases 2H+

2. 1st electron gets transferred to Fe-S (in Rieske subunit) → cyt c₁ → cyt c

3. 2nd electron gets transferred to ___ → ___ → reduces Q → ___ in ___ site

Q Cycle Phase I

1. QH₂ binds to Q_p site and gets reduced to Q → releases 2H+

2. 1st electron gets transferred to Fe-S (in Rieske subunit) → cyt c₁ → cyt c

3. 2nd electron gets transferred to cyt b_L → cyt b_H → reduces Q → Q- in Q_n site

Q Cycle Phase II

1. A second QH₂ binds to Q_p site and gets reduced to Q → releases 2H+

2. At Q_n site, ___ is able to reduce to ___ (requires ___H+) → ___ is released into __________.

Q Cycle Phase II

1. A second QH₂ binds to Q_p site and gets reduced to Q → releases 2H+

2. At Q_n site, Q- is able to reduce to QH₂ (requires 2H+) → QH₂ is released into lipid bilayer.

cytochrome C is an electron tranferer (transfers ___ e-)

• it is ______ soluble, so it moves in the ______

(QH₂ is ______ soluble, so it moves in the ______)

cytochrome C is an electron tranferer (transfers 1 e-)

• it is water soluble, so it moves in the intermembrane space

(QH₂ is lipid soluble, so it moves in the bilayer)

cytochrome C passes electrons from complex ___ to ___

cytochrome C passes electrons from complex III to IV

cytochrome C structure

• total ___ sulfurs

• ____ at center, covalently linked with ___ S

• ___ S coordinates ___

cytochrome C structure

• total 3 sulfurs

• heme at center, covalently linked with 2 S

• 3rd S coordinates Fe 2+ (in heme)

Complex IV = ?

Cytochrome C Oxidase (COX)

COX oxidizes ________, reduces ________ (___ electrons)

→ ___ H₂O released

COX oxidizes cytochrome C, reduces O₂ (4 electrons)

→ 2 H₂O released

_________ is the terminal oxidoreductase, ___ is the final acceptor of electrons.

COX is the terminal oxidoreductase, O₂ is the final acceptor of electrons.

How is 2 H₂O created in Complex IV?

4H+ reduces O₂

COX Structure

How many hemes and how many coppers?

• 2 hemes (a and a₃)

• 2 copper sites (Cu_A and Cu_B)

COX Mechanism

(idk if i need details, view image)

E. coli ETC (view image)

Mitchell Hypothesis = ?

proton gradient across the inner membrane drives ATP synthesis

• ridiculed but right, won a Nobel prize

• aka chemiosmotic hypothesis

In complexes I-IV, are H+ released into intermembrane space or matrix?

intermembrane space

electrons pass through ETC and H+ is released into intermembrane space → electric potential and proton gradient = (2 names)

electrochemical proton gradient,

proton motive force

many/few protons are present in IMS → goes back to matrix with/against concentration gradient (through _________)

many protons are present in IMS (due to ETC) → goes back to matrix with concentration gradient (through ATP synthase)

Evidence of Mitchell Hypothesis

• pH increases/decreases when O₂ added to respiring mitochondria

• ATP synthesis stops if inner/outer membrane is disrupted

  • placing mitochondria in water causes swelling → ___ leak → no ATP synthesis

• ATP synthesis inhibitors work by disrupting ___________

Evidence of Mitchell Hypothesis

• pH decreases when O₂ added to respiring mitochondria (more H+)

• ATP synthesis stops if inner membrane is disrupted

  • placing mitochondria in water causes swelling → H+ leak → no ATP synthesis

• ATP synthesis inhibitors work by disrupting proton gradient

2 types of ATP synthesis ihibitors

1) …

2) …

• both types are hydrophobic/hydrophilic

1) uncouplers = equalize H+ concentration

2) ionophores = poke pores into membrane → dissipate H+ gradient

• both types are hydrophobic

Uncouplers

• uncouples _____________ and _____________

• hydrophobic molecules with ____________

• (how do they equalize gradient?)

Uncouplers

• uncouples electron transport and proton gradient

  • hydrophobic molecules with dissociable proton

• shuttle across membrane while carrying proton → equalizes gradient

Chemiosmotic coupling

electron transfer coupled with H+ gradient

It is thermodynamically favorable for protons to move up or down gradient?

down!!

Approximate number of protons pumped per 2 electrons

1. succinate = ____

2. NADH = ____

Approximate number of protons pumped per 2 electrons

1. succinate = 6

2. NADH = 10

ATP Synthase

-

ATP Synthase has 2 main components:

1. F₁ (subunits = ________)

2. F₀ (subunits = ________)

ATP Synthase has 2 main components:

1. F₁ (subunits = ɑ, β, ɣ, ε, σ)

2. F₀ (subunits = a, b, c)

ATP Synthase subunits

____, ____ → make ATP

____ → make up top of stalk

____, ____ → middle portion (like umbrella handle)

____ → site of H+ entry (part of _____)

____ → makes up stalk (part of _____)

____ → make up channel (_____)

ATP Synthase subunits

ɑ, β, → make ATP

σ → make up top of stalk

ɣ, ε → middle portion (like umbrella handle)

a → site of H+ entry (part of stator)

b → makes up stalk (also part of stator)

c → make up channel (rotor)

ATP Synthase Mechanism

1. H+ flows from ___ through ___ and back to ___ which turns ________ → turns ɣ

2. This causes conformational changes in ___ and ___

3. ATP Synthesis

ATP Synthase Mechanism

1. H+ flows from a through c and back to a which turns rotor → turns ɣ

2. This causes conformational changes in ɑ and β

   1. ATP Synthesis

F1 structure of ATP Synthase

John Walker discovered that F1 contains 3 β subunits:

1. contains ____ and a honhydrolyzable ___ bond

2. contains ____

3. empty

F1 structure of ATP Synthase

John Walker discovered that F1 contains 3 β subunits:

1. contains AMP-PNP and a honhydrolyzable β-ɣ bond

2. contains ADP

3. empty

Paul Boyer’s Binding Change Mechanism

The 3 β subunits of F1 have different conformations:

1. tight = contains ___ = high/low/no affinity for ADP

2. loose = contains ___= high/low/no affinity for ADP

3. open = contains ___= high/low/no affinity for ADP

Which one makes ATP? _____

Paul Boyer’s Binding Change Mechanism

The 3 β subunits of F1 have different conformations:

1. tight = contains AMP-PNP = high affinity for ADP

2. loose = contains ADP = low affinity for ADP

3. open = contains nothing = no affinity for ADP

Which one makes ATP? tight

Overall order of conformation switch in Paul Boyer’s mechanism

O → L → T → O …

Paul Boyer’s Binding Change Mechanism

1. O → L

   • ____ presses against β subunit, kind of closing it

   • ATP/ADP does what?

2. L → T

   • ____ rotates and presses more against β subunit, completely closing it

   • ATP/ADP does what?

3. T → O

   • ____ no longer presses against β, relaxes

   • ATP/ADP does what?

Paul Boyer’s Binding Change Mechanism

1. O → L

   • ɣ presses against β subunit, kind of closing it

   • ADP/Pi enter

2. L → T

   • ɣ rotates and presses more against β subunit, completely closing it

   • ADP/Pi are forced together → ATP

3. T → O

   • ɣ no longer presses against β, relaxes

   • ATP leaves

In ATP Synthase, H+ enter the a subunit _____ half channel → c → a _____ half channel.

In ATP Synthase, H+ enter the a subunit inlet half channel → c → a outlet half channel.

One complete rotation of rotor produces ____ ATP molecules

3

A and C Subunit Residues

1) ____ (___) on a subunit (end of inlet half channel)

2) ____ (___) on a subunit (between half channels) moves H+ to c subunit

• long side chain to pick up H+ easily

• cannot be replaced with ___

3) ____ (___) on c subunit

• can be replaced with ___

4) ____ (___) on a subunit (end of outlet half channel)

A and C Subunit Residues

1) Asn²¹⁴ (N) on a subunit (end of inlet half channel)

2) Arg²¹⁰ (R) on a subunit (between half channels) moves H+ to c subunit

• long side chain to pick up H+ easily

• cannot be replaced with K

3) Asp⁶¹ (D) on c subunit

• can be replaced with E

4) Ser²⁰⁶ (S) on a subunit (end of outlet half channel)

-

**NeRDS

A and C Subunit Mechanism

1. H+ enters ___ at the __________

2. H+ hops onto ___

3. H+ goes to ___ on __________

4. H+ rides around rotor

5. H+ exits on ___ at the __________

A and C Subunit Mechanism

1. H+ enters N at the a inlet half channel

2. H+ hops onto R

3. H+ goes to D on c subunit

4. H+ rides around rotor

5. H+ exits on S at the a outlet half channel

ATP-ADP Translocase in Mitochondria

• ATP is only transported in/out

• ADP is only transported in/out

ATP-ADP Translocase in Mitochondria

• ATP is only transported out

• ADP is only transported in

Cytosol (outside) is more positive/negative, so ATP movement out is spontaneous.

ATP movement is equivalent to 1 electron leaving/entering or 1 H+ leaving/entering.

-

Thus, the total cost of making and exporting ATP is ___ H+ (in).

Cytosol (outside) is more positive, so ATP movement out is spontaneous.

ATP movement is equivalent to 1 electron leaving or 1 H+ entering.

-

Thus, the total cost of making and exporting ATP is 4 H+ (in).

P/O =

# ATP / electron pair

ATPase with 10c subunits yields ____ ATP,

ATPase with 8c subunits yields ____ ATP

ATPase with 10c subunits yields 3.33 ATP,

ATPase with 8c subunits yields 2.7 ATP

Bacteria have no mitochondria, how does this affect ATP yield?

higher ATP yield (lose no H+ to export ATP)