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Oxidative Phoshorylation

Oxidative phosphorylation is the process in which ATP is formedas a result of the transfer of electrons from NADH or FADH2 toO2 by a series of electron carriersGenerates 26 of the 30 ATP molecules from complete oxidation ofglucose to CO2 and H2O

Outline

Oxidative phosphorylation in eucaryotes takes place in mitochondriaIt depends on electron transfer

The respiratory chain consists of 4 complexes: 3 proton pumps & a physical link to the citric acid cycle

A proton gradient powers the synthesis of ATP

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Overview• Oxidative phosphorylation is the culmination of a series of energy transformations, called cellular respiration• 1st, carbon fuels are oxidized in the citric acid cycle to yield electrons with high transfer potential• Then, this electron-motive force is converted into a proton-motive force and,• Finally, the proton-motive force is converted into phosphoryl transfer potential• Conversion of electron- to proton-motive force is carried out by 3 electron-driven proton pumps: NADH-Q oxidoreductase, Q-cytochrome c oxidoreductase, and cytochrome c oxidase• They contain oxidation-reduction centers: quinones, flavins, iron-sulfur clusters, hemes, & copper ions.• The final phase is carried out by ATP synthase, driven by the flow of protons back into the mitochondrial matrix

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Mitochondrion

Double membrane

Outer membrane ispermeable

Inner membrane hasinvaginations calledcristae

The electron transport chain is located in the innermembrane

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Oxidative PhosphorylatlionOxidation & ATP synthesis are coupled by transmembrane proton flux

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Diagram of a mitochondrion

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Redox Potential

The redox potential is a measure (in volts) of the affinity of a substance for electrons — its electronegativity — compared with hydrogen (which is set at 0). Substances more strongly electronegative than (i.e., capable of oxidizing) hydrogen have positive redox potentials. Substances less electronegative than (i.e., capable of reducing) hydrogen have negative redox potentials.

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Measurement of redox potentialX(oxidized) + X-(reduced) =redox couple

Samplehalf-cell

Standard referencehalf-cell

Electrons, but notX or X-, can flowthrough the agar bridge

If the reaction is,X- + H+ X + 1/2 H2

In half-cells:X- X + e- & H+ + e- 1/2 H2

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Reduction potentials of biological redox couples

E’o = standard reduction potential

‘prime’ denotes pH 7‘zero’ denotes ‘standard’

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Distance dependence of electron-transfer rate

Rate decreases as donor & acceptor move apart

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Sequence of electron carriers in the respiratory chain

Complex Iproton pump

Complex II, does notpump protons

Complex IIIproton pump

Complex IVproton pump

Coenzyme Qelectron shuttle

Cytochrome celectron shuttle

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A total of 10 H+ are ejected from the mitochondrial matrix per 2 e transferred from NADH to oxygen via the respiratory chain.

The H+/e ratio for each respiratory chain complex will be discussed separately.

Matrix H+

+ NADH NAD+ + 2H+ 2H+ + ½ O2 H2O 2 e

– – I Q III IV

+ + 4H+ 4H+ 2H+ Intermembrane Space

cyt c

Spontaneous electron flow through each of complexes I, III, & IV is coupled to H+ ejection from the matrix. 

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Summary of electron-transport chain

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Complex I (NADH-Q Oxidoreductase) transports 4H+ out of the mitochondrial matrix per 2e transferred from NADH to CoQ.

Matrix H+

+ NADH NAD+ + 2H+ 2H+ + ½ O2 H2O 2 e

– – I Q III IV

+ + 4H+ 4H+ 2H+ Intermembrane Space

cyt c

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Structure of NADH-Q oxidoreductaseEM at moderate resolutionMatrix

side Consists of at least34 polypeptide chains

NADH is oxidized in the arm,& electrons are transferred toreduce Q in the membrane

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Lack of high-resolution structural information for the membrane domain of complex I has hindered elucidation of the mechanism of H+ transport. Direct coupling of transmembrane H+ flux & e transfer is unlikely, because the electron-tranferring prosthetic groups, FMN & Fe-S, are all in the peripheral domain of complex I.Thus is assumed that protein conformational changes are involved in H+ transport, as with an ion pump.

inner mitochondrial membrane

matrix

NAD+ NADH

Complex I

FMN peripheral domain

membrane domain

FMN

A B

FMN

Peripheral domain of a bacterial Complex I

membrane domain

PDB 2FUG

N2

[Fe-S]

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Oxidation states of flavins

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Oxidation states of quinonesCoenzyme Q (Q) is a quinone derivativewith a long isoprenoid tail, also known as ubiquinone

Reduction proceeds through a semiquinone anion intermiediate (Q.-)

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Coupled electron-proton transfer reactions

Reduction of Q can result in the uptake of 2 protons

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Complex III Q-cytochrome c oxidoreductase (bc1 complex): H+ transport in complex III involves coenzyme Q (CoQ).

Matrix H+

+ NADH NAD+ + 2H+ 2H+ + ½ O2 H2O 2 e

– – I Q III IV

+ + 4H+ 4H+ 2H+ Intermembrane Space

cyt c

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Q-cytochrome c oxidoreductase

Homodimer with 11distinct polypeptides

Major prosthetic guoups:3 hemes, & a 2Fe-2Scluster,they mediate electron-transfer between quinones in the membrane & cytochrome c in theintermembrane space

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Iron-sulfur clustersUndergo oxidation-reduction reactions

Single Fe ion+ 4 Cys cluster

2Fe-2S cluster,bridge of sulfide ions4Fe-4S cluster

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Attachment of heme group in c-type cytochromesA cytochrome is an electron-transferring

protein that contains a hemeprosthetic group

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The “Q cycle” depends on mobility of coenzyme Q within the lipid bilayer.There is evidence for one-electron transfers, with an intermediate semiquinone radical.

O

OC H 3 O

CH 3C H 3 O

(CH 2 CH C CH 2 )n H

CH 3

O H

O HC H 3 O

CH 3C H 3 O

(CH 2 CH C CH 2 )n H

CH 3

e + 2 H +

c o e n z y m e Q

c o e n z y m e Q H 2

O

OC H 3 O

CH 3C H 3 O

(CH 2 CH C CH 2 )n H

CH 3e

c o e n zy m e Q •

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Electrons enter complex III via coenzyme QH2, which binds at a site on the positive side of the inner mitochondrial membrane, adjacent to the intermembrane space.

One version of Q Cycle:

2 H+

Q Q QH2 QH2

cyt bH

cyt bL Q Q·

Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

cyt c

e

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The loss of one electron from QH2 would generate a semiquinone radical, shown here as Q·, though the semiquinone might initially retain a proton as QH·.

QH2 gives up 1e to the Rieske iron-sulfur center, Fe-S.

Fe-S is reoxidized by transfer of the eto cyt c1, which passes it out of the complex to cyt c.

2 H+

Q Q QH2 QH2

cyt bH

cyt bL Q Q·

Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

cyt c

e

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The fully oxidized CoQ, generated as the 2nd e is passed to the b cytochromes, may then dissociate from its binding site adjacent to the intermembrane space.Accompanying the two-electron oxidation of bound QH2, 2H+ are released to the intermembrane space.

A 2nd e is transferred from the semiquinone to cyt bL (heme bL) which passes it via cyt bH across the membrane to another CoQ bound at a site on the matrix side.

2 H+

Q Q QH2 QH2

cyt bH

cyt bL Q Q·

Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

cyt c

e

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It takes 2 cycles for CoQ bound at the site hear the matrix to be reduced to QH2, as it accepts 2e from the b hemes, and 2H+ are extracted from the matrix compartment.

In 2 cycles, 2 QH2 enter the pathway & one is regenerated.

2 H+

Q Q QH2 QH2

cyt bH

cyt bL Q Q·

Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

cyt c

e

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QH2 + 2H+(matrix) + 2 cyt c (Fe3+)

Q + 4H+(outside) + 2 cyt c (Fe2+)

Per 2e transferred through the complex to cyt c, 4H+ are released to the intermembrane space.

Overall reaction catalyzed by complex III, including net inputs & outputs of the Q cycle :

2 H+

Q Q QH2 QH2

cyt bH

cyt bL Q Q·

Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

cyt c

e

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While 4H+ appear outside per net 2e transferred in 2 cycles, only 2H+ are taken up on the matrix side.In complex IV, there is a similarly uncompensated proton uptake from the matrix side (4H+ per O2 or 2 per 2e).

2 H+

Q Q QH2 QH2

cyt bH

cyt bL Q Q·

Fe-S cyt c1

2 H+

matrix

Complex III

e

intermembrane space

.

cyt c

e

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Electrons are donated to complex IV, one at a time, by cytochrome c, which binds from the intermembrane space.Each e passes via CuA & heme a to the binuclear center, buried within the complex, that catalyzes O2 reduction: 4e + 4H+ + O2 → 2H2O. Protons utilized in this reaction are taken up from the matrix compartment.

Matrix H+

+ NADH NAD+ + 2H+ 2H+ + ½ O2 H2O 2 e

– – I Q III IV

+ + 4H+ 4H+ 2H+ Intermembrane Space

cyt c Complex IV (Cytochrome Oxidase):

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Cytochrome c oxidaseConsists of 13polypeptides

3 major prosthetic groups

Site of O2 reduction

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Peroxidase bridgeO2 held tightly between Fe & Cuprevents release of harmful oxides

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H+ pumping by complex IV:In addition to protons utilized in reduction of O2, there is electron transfer-linked transport of 2H+ per 2e (4H+ per 4e) from the matrix to the intermembrane space.

Matrix H+

+ NADH NAD+ + 2H+ 2H+ + ½ O2 H2O 2 e

– – I Q III IV

+ + 4H+ 4H+ 2H+ Intermembrane Space

cyt c

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Proton transport by cytochrome c oxidase

4 “chemical” protons from matrixside to reduce one O2 to two H2O4 additional protons pumpted tocytosolic side

The pumped protons double theefficiency of free-energy storagein the form of a proton gradientfor this final step in the electrontransport chain

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Structural & mutational studies indicate that protons pass through complex IV via chains of groups subject to protonation/deprotonation, called "proton wires."

These consist mainly of chains of buried water molecules, along with amino acid side-chains, & propionate side-chains of hemes.

Separate H+-conducting pathways link each side of the membrane to the buried binuclear center where O2 reduction takes place.

These include 2 proton pathways, designated "D" & "K" (named after constituent Asp & Lys residues) extending from the mitochondrial matrix to near the binuclear center deep within complex IV.

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Ejection of a total of 20 H+ from the matrix per 4 e transferred from 2 NADH to O2 (10 H+ per ½O2).

Not shown is OH that would accumulate in the matrix as protons, generated by dissociation of water (H2O H+ + OH), are pumped out. Also not depicted is the effect of buffering.

Matrix H+

+ NADH NAD+ + 2H+ 2H+ + ½ O2 H2O 2 e

– – I Q III IV

+ + 4H+ 4H+ 2H+ Intermembrane Space

cyt c

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Need for protection from free radicals