12857_OXIDATIVE PHOS

8/3/2019 12857_OXIDATIVE PHOS

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Oxidative Phosphorylation


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Respiration-linked H+pumping out of the matrix conservessome of the free energy of spontaneous e transfers as

potential energy of an electrochemical H+ gradient.

matrix

innermembrane outermembrane

inter-membrane

space

mitochondrion

cristae

Conventional view ofmitochondrial

structure is at right.

Respiratory chain isin cristae of the innermembrane.

Spontaneous electrontransfer through

respiratory chain complexes I, III & IV is coupled toH+ ejection from the matrix to the intermembrane space.

Because the outer membrane contains large channels,these protons may equilibrate with the cytosol.


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3-D reconstructionsbased on electron micrographs of

isolated mitochondria taken with a large depth of field, atdifferent tilt angles have indicated that the infoldings of

the inner mitochondrial membrane are variable in shape

and are connected to the periphery and to each other by

narrow tubular regions.

matrix

innermembrane outermembrane

inter-membrane

space

mitochondrion

cristae


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between the lumen ofcristae & the intermembrane space.There is evidence also that protonspumped out of thematrix spread along the anionic membrane surface andonly slowly equilibrate with the surrounding bulk phase,

maximizing the effective H+ gradient.

Electron micrograph by Dr.C.

Mannella of a Neurosporamitochondrion in a frozen sample

in the absence of fixatives orstains that might alter appearance

of internal structures.

Wadsworth Centerwebsite.

Tubular cristae connect to theinner membrane via narrowpassageways that may limitthe rate ofH+ equilibration


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A total of10 H+ are ejected from the mitochondrial matrix

per 2 e transferred from NADH to oxygen via therespiratory chain.

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

Matrix

H++NADHNAD++2H+ 2H++O2 H2O

2e I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc

Spontaneous

electron flow

through eachof complexesI, III, & IV is

coupled to H

+

ejection fromthe matrix.


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Complex I (NADH Dehydrogenase) transports4H+ out of the mitochondrial matrix per2e

transferred from NADH to CoQ.

Matrix

H+

+NADHNAD++2H+ 2H++O2 H2O

2e I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc


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Lack of high-resolution structural information for themembrane domain ofcomplex I has hindered elucidationof the mechanism of H+ transport.

Direct coupling of transmembrane H+

flux & e

transfer isunlikely, 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 mitochondrialmembrane

matrix

NAD+

NADH

Complex I

FMN peripheraldomain

membrane domain

n FMN

A B

n FMN

Peripheral domain of a bacterial Complex I

membranedomain

qPDB 2FUG

n N2


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Complex III (bc1 complex):

H+ transport in complex III involves coenzyme Q (CoQ).

Matrix


2e I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc


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The Q cycle depends on mobility of coenzyme Q withinthe lipid bilayer.

There is evidence forone-electron transfers, with an

intermediate semiquinone radical.

O

O

CH3O

CH3CH3O

(CH2 CH C CH2)nH

CH3

OH

OH

CH3O

CH3CH3O

(CH2 CH C CH2)nH

CH3

e + 2H+

coenzyme Q

coenzyme QH2

O

O

CH3O

CH3CH3O

(CH2 CH C CH2)nH

CH3e

coenzyme Q


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

space.

One version

ofQ Cycle:

2H+

Q Q QH2 QH2

cyt bH

cyt bLQ Q Fe-S cyt c1

2H+

matrix

Complex III

e

intermembrane space

.

cyt c

e


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

QH2 gives up 1e

to the Rieskeiron-sulfur center,Fe-S.

Fe-S is reoxidized

by transfer of thee to cyt c

1, which

passes it out of thecomplex to cyt c.

2H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2H+

matrix

Complex III

e

intermembrane space

.

cyt c

e


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The fully oxidizedCoQ, generated as the 2nd

e

is passedto the b cytochromes, may then dissociate from its bindingsite adjacent to the intermembrane space.

Accompanying the two-electron oxidation of bound QH2,

2H+ are released to the intermembrane space.

A2nd e istransferred from

the semiquinone tocyt bL (heme bL)which passes it viacyt bH across themembrane toanotherCoQbound at a site onthe matrix side.

2H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2H+

matrix

Complex III

e

intermembrane space

.

cyt c

e


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In an alternative mechanism that has been proposed, the2 e transfers, from QH2 to Fe-S & cyt bL, may beessentially simultaneous, eliminating the semiquinone

intermediate.

2H+

Q Q QH2 QH2

cyt bH

cyt bLQ Q Fe-S cyt c1

2H+

matrix

Complex III

e

intermembrane space

.

cyt c

e


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It takes 2 cycles forCoQ bound at the site hear the matrixto be reduced to QH2, as it accepts 2e

from the b hemes,and 2H+ are extracted from the matrix compartment.

In 2 cycles, 2QH2

enter the pathway & one is regenerated.

2H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2H+

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 (Fe

2+)

Per 2e transferred through the complex to cyt c, 4H+ are

released to the intermembrane space.

Animation

Overall reactioncatalyzed by

complex III,including netinputs & outputsof the Q cycle :

2H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2H+

matrix

Complex III

e

intermembrane space

.

cyt c

e


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While 4H+ appear outside per net 2e transferred in 2cycles, only 2H+ are taken up on the matrix side.

In complex IV, there is a similarly uncompensated protonuptake from the matrix side (4H+per O

2or 2 per 2e).

2H+

Q Q QH2 QH2

cyt bH

cyt bL

Q Q Fe-S cyt c1

2H+

matrix

Complex III

e

intermembrane space

.

cyt c

e


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T

hus there are 2H

+

per 2e

that are effectively transportedby a combination of complexes III & IV.

They are listed with complex III in diagrams depictingH+/e stoichiometry.

Matrix


2e I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc


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Complex III:

Half of the homodimeric

structure is shown.Approximate location ofthe membrane bilayer isindicated.

Not shown are the CoQbinding sites near hemebH and near heme bL.

The b hemes arepositioned to provide a

pathway for electronsacross the membrane.

heme bL

heme c1

Fe-S

PDB

1BE3Complex III

(bc1Complex)

membrane

heme bH


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The domain with

attached Rieske Fe-S hasa flexible linkto the restof the complex.(Fe-S protein in green.)

Fe-Schanges positionduring e transfer.

AfterFe-S extracts an e

from QH2, it movescloser to heme c1, to

which it transfers the e.

View an animation.

heme bL

heme c1

Fe-S

PDB

1BE3Complex III

(bc1Complex)

membrane

heme bH


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After the 1st e transfer

from QH2 to Fe-S, theCoQ semiquinone ispostulated to shift positionwithin the Q-binding site,

moving closer to its eacceptor, heme bL.

This would help toprevent transfer of the2nd electron from thesemiquinone to Fe-S.

heme bL

heme c1

Fe-S

PDB

1BE3Complex III

(bc1Complex)

membrane

heme bH


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Complex III is anobligate homo-dimer.

Fe-S in one half of thedimer may interact withbound CoQ & heme c1in the other half of the

dimer.

Arrows point at:

Fe-S in the half ofcomplex coloredwhite/grey

heme c1

in the half ofcomplex with proteins

colored blue or green.

PDB-1BGY Complex IIIhomo-dimer

Fe-Sheme c1


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Electrons are donated to complex IV, one at a time, bycytochrome 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


2e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc

Complex IV

(Cytochrome

Oxidase):


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H+ pumpingby complex IV:

In addition to protons utilized in reduction of O2, thereis electron transfer-linked transport of 2H+ per 2e

(4H+per 4e) from the matrix to the intermembranespace.

Matrix

H+


2e I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc


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Structural & mutational studies indicate that protons passthrough complex IV via chains of groups subject to

protonation/deprotonation, called "protonw

ires."

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 themembrane to the buried binuclear center where O2reduction 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 centerdeep within complex IV.

Images in web pages of: IBI, & Crofts.


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Aswitch mechanism controlled by the reaction cycle isproposed to effect transfer of a proton from one half-wire (half-channel) to the other.

There cannot be an open pathway for H+

completelythrough the membrane, or oxidative phosphorylationwould be uncoupled.(Pumped protons would leak back.)

Switching may involve conformational changes, and

oxidation/reduction-linked changes in pKa of groupsassociated with the catalytic metal centers.

Detailed mechanisms have been proposed.


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Ejection of a total of20H+ from the matrix per 4e

transferred from 2 NADH to O2 (10H+per O2).

Not shown is OH that would accumulate in the matrixas protons, generated by dissociation of water(H2O m H

+ + OH), are pumped out.

Also not depicted is the effect of buffering.

Matrix


2e

I Q III IV

+ +

4H

+

4H

+

2H

+

Intermembrane Space

cytcSimplified

animationdepicting:


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ATP synthase, embedded in cristae of the innermitochondrial membrane, includes:

F1 catalytic subunit, made of 5 polypeptideswith stoichiometry EFKHI.

Fo complex of integral membrane proteins thatmediates proton transport.

ADP + Pi ATP

F1

Fo

3 H+matrix

intermembrane

space


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F1F

o couples ATP synthesis to H+ transport into the

mitochondrial matrix.Transport of least 3 H+perATP isrequired, as estimated from comparison of:

(GforATP synthesis under cellular conditions (freeenergy required)

(G fortransfer of each H+ into the matrix, given the

electrochemical H+

gradient (energy available per H+

).

ADP + Pi ATP

F1

Fo

3 H+matrix

ntermem rane

space


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TheC

hemiosmotic Theory of oxidative phosphorylation,for which Peter Mitchell received the Nobel prize:

Coupling ofATP synthesis to respiration is indirect,via a H+ electrochemical gradient.

Matrix

H+


2e I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc 3H+

F

Fo

ADP+Pi ATP


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Chemiosmotic theory - respiration:

Spontaneous e transfer through complexes I, III, & IV iscoupled to non-spontaneous H+ ejection from the matrix.

H+ ejection creates a membrane potential ((=, negativein matrix) and a pH gradient ((pH, alkaline in matrix).

Matrix

H+


2e I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc 3H+

F

Fo

ADP+Pi ATP


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Chemiosmotic theory - F1Fo ATP synthase:

Non-spontaneous AT

P synthesis is coupled to spontaneousH+ transport into the matrix.The pH & electrical gradientscreated by respiration are the driving force for H+ uptake.

H+ return to the matrix via Fo "uses up" pH & electrical

gradients.

Matrix


2e

I Q III IV

+ +

4H+

4H+

2H+

Intermembrane Space

cytc 3H+

F

Fo

ADP+Pi ATP


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Transport ofATP, ADP, & Pi

ATPproduced in the mitochondrial matrix must exit tothe cytosol to be used by transport pumps, kinases, etc.

AD

P & Pi arising from AT

P hydrolysis in the cytosolmust reenter the matrix to be converted again to ATP.

Two carrier proteins in the inner mitochondrialmembrane are required.

The outer membrane is considered not a permeabilitybarrier.Large outer membrane VDAC channels areassumed to allow passage of adenine nucleotides and Pi.


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Adenine nucleotide translocase (ADP/ATP carrier) is an

antiporter that catalyzes exchange ofADP forAT

Pacross the inner mitochondrial membrane.

At cell pH, ATP has 4 () charges, ADP 3 () charges.

ADP3/ATP4 exchange is driven by, and uses up,

membrane potential (one charge perATP).

ADP+Pi ATP matrix lower [H+]

_ _

3H+ ATP

4 ADP

3H2PO4

H

+

higher [H+] ADP + Pi cytosol

energy

requiring

reactions

ATP4

+ +


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Phosphate re-enters the matrix with H+by an electroneutral

symport mechanism.Pi entry is driven by, & uses up, thepH gradient (equivalent to one mol H+per mol ATP).

Thus the equivalent of one mol H+ enters the matrix withADP/ATP exchange & Pi uptake.Assuming 3H

+ transported

by F1Fo, 4H+

total enter the matrix per ATP synthesized.

ADP+Pi ATP matrix lower [H+]

_ _

3H+ ATP

4 ADP

3H2PO4

H

+

higher [H+] ADP + Pi cytosol

energy

requiring

reactions

ATP4

+ +

Animation


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Questions: Based on the assumed number of H+pumpedout per site shown above, and assuming 4H+ aretransferred back to the matrix per ATP synthesized:

What would be the predicted P/O ratio, the # ofATPsynthesized per 2e transferred from NADH to O2?

What would be the predicted P/O ratio, if the e source is

succinate rather than NADH?

Matrix

H+


2

e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc


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2.5 ~P bonds synthesized during oxidation ofNADHproduced via Pyruvate Dehydrogenase & Krebs Cycle(10 H+pumped; 4 H+ used up perATP).

1.5 ~P bonds synthesized per

NAD

Hproduced in thecytosol in Glycolysis (electron transfer via FAD to CoQ).

1.5 ~P bonds synthesized during oxidation ofQH2produced in Krebs Cycle (Succinate Dehydrogenase

electrons transferred via FAD & Fe-S to coenzyme Q).

For, summing upsynthesis of~Pbonds via oxphos, assume:

Matrix


2e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc


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PathwayNADH

produced

QH2produced

(via

FADH2)

~P bondsATP or

GTP direct

~P bonds1.5 or 2.5

per NADH

in oxphos

~P bonds1.5 per

QH2 in

oxphos

Total ~P

bonds

Glycolysis

Pathway

Pyruvate

Dehydrogenase

Krebs Cycle

Sum ofPathways

All Quantities Per Glucose


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Above is represented an O2 electrode recording while

mitochondria respire in the presence of Pi and an e

donor(succinate or a substrate of a reaction to generate NADH).

The dependence of respiration rate on availability ofADP,the ATP Synthase substrate, is called respiratory control.

[O2]

time

ADP added

ADP allconvertedto ATP

a

bc

An oxygen electrode

may be used to record[O2] in a closed vessel.

Electron transfer, e.g.,NADH O2, is

monitored by the rateof O2 disappearance.


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Respiratory control ratio is the ratio of slopes after and

before ADP addition (b/a).

P/O ratio is the moles ofADP divided by the moles of O

consumed (based on c) while phosphorylating the ADP.

[O2]

time

ADP added

ADP allconvertedto ATP

a

bc


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Chemiosmotic explanation of respiratory control:

Electron transfer is obligatorily coupled to H+ ejection

from the matrix. Whether this coupled reaction isspontaneous depends on pH and electrical gradients.

Reaction (G

e

transfer(NADH

O2) negative value*

H+ ejection from matrix positive; depends on H+

gradient**

e- transfer with H+ ejection algebraic sum of above

*(Go' = nF(Eo' = 218 kJ/mol for 2eNADHO2.

**For ejection of 1 H+ from the matrix:

(G = RT ln ([H+]cytosol/[H+]matrix) + F(=

(G = 2.3 RT(pHmatrix

pHcytosol

) + F(=


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With no ADP, H+ cannot flow through Fo.(pH & (= aremaximal.As respiration/H+pumping proceed, (G for H+

ejection increases, approaching that for e transfer.

When the coupled reaction is non-spontaneous,respiration stops.This is referred to as a static head.

In fact there is usually a low rate of respiration in the

absence ofADP, attributed to H

+

leaks.

Matrix

H+


2e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc 3H+

F1

Fo

ADP+Pi ATP


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When ADP is added, H+ enters the matrix via Fo, as ATPis synthesized.This reduces (pH & (=.

(G of H+

ejection decreases.The coupled reaction of electron transfer with H+ ejectionbecomes spontaneous .

Respiration resumes or is stimulated.

Matrix

H+


2

e

I Q III IV

+ +

4H+ 4H+ 2H+

Intermembrane Space

cytc 3H+

F

Fo

ADP+Pi ATP


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Uncoupling reagents (uncouplers) are lipid-soluble

weak acids.E.g., H

+

can dissociate from the OH groupof the uncouplerdinitrophenol.

Uncouplers dissolve in the membrane and function as

carriers for H+.

OH

NO2

NO2

2,4-dinitrophenol


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Uncouplersblock oxidative phosphorylation bydissipating the H+ electrochemical gradient.

Protons pumped out leak back into the mitochondrial

matrix, preventing development of(pH or(=.

Matrix


2e

I Q III IV

4H+ 4H+ 2H+ H+

Intermembrane Space

cytc

uncoupler


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With uncouplerpresent, there is no (pH or (=.

(G for H+ ejection is zero

(G for e transfer coupled to H+ ejection is maximal(spontaneous).

Respiration proceeds in the presence of an uncoupler,

whether or not ADP is present.

Matrix


2e

I Q III IV

4H+

4H+

2H+

H+

Intermembrane Space

cytc

uncoupler


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(G for H+

flux is zero in the absence of a H+

gradient. Hydrolysis ofATP is spontaneous.

The ATP Synthase reaction runs backward in presence

of an uncoupler.

ADP+Pi ATP

F1

Fo

3H+

ATPase with H+ gradient dissipated

matrix

intermembranespace


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Uncoupling Protein

An uncoupling protein (thermogenin) is produced inbrown adipose tissue of newborn mammals andhibernating mammals.

This protein of the inner mitochondrial membranefunctions as a H+carrier.

The uncoupling protein blocks development of a H+

electrochemical gradient, thereby stimulating

respiration.(G of respiration is dissipated as heat.

This "non-shivering thermogenesis" is costly in termsof respiratory energy unavailable forATP synthesis,but provides valuable warming of the organism.

12857_OXIDATIVE PHOS

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