Electron Transport and Oxidative Phosphorylation

The biochemical fate of NADH and FADH2

These are energy rich molecules coming from the degradation of sugars, FA, and AA.

Overview: The Three Stages of Cellular Respiration Stage 1: Acetyl-CoA Production from
Glucose Fatty Acids Amino Acids

Stage 2: Acetyl-CoA Oxidation (TCA cycle Stage !: "lectron Transfer # Oxidati$e P%os&%orylation

Whats chieved!

Oxygen is t%e ultimate acce&tor of electrons from 'A() and FA()2* T%us* oxygen is reduced to )2O+ A large amount of free energy is li,erated in t%e &rocess and it is used to dri$e t%e &%os&%orylation of A(P - ATP+

Oxidative Phosphorylation

Oxidati$e P%os&%orylation is t%e &rocess in .%ic% ATP is formed as a result of t%e transfer of electrons from 'A() and FA()2 to O2 ,y a series of electron carriers+

/emem,er: 0ost of t%e ATP is &roduced in t%e mitoc%ondria+

"itochondria: Power Plants of the Cell

T%e inner mem,rane is
an essential ,arrier t%at carries essential mac%inery for ,ot% lateral and trans$erse translocation of su,strates

T%e cristae* ,ounded ,y t%e inner mem,rane* in$aginate dee& into t%e matrix 1 why? All fuel oxidation &at%.ays* except for glycolysis* occur in t%e mitoc%ondrial matrix

Concept: Oxidative Phosphorylation

2Oxidati$e &%os&%orylation3 is t%e term used to descri,e t%e synt%esis of ATP* .%ic% is energetically cou&led to t%e oxidation of reducing e4ui$alents to .ater* in t%e mitoc%ondria+ T%e mec%anism of t%e cou&ling of t%ese t.o &%enomena .as elusi$e for many years ,ut is no. acce&ted to ,e $ia t%e 2c%emiosmotic model3 of &roton moti$e force+

What is substrate-level phosphorylation? How is it different?

Oxidative Phosphorylation:

"lectrons are &assed from 'A() and FA()2 to molecular oxygen (oxidation) Carried ,y redox intermediates suc% as 4uinones* cytoc%romes* and Fe-S &roteins T%is exergonic electron flo. is cou&led to )5 trans&ort to create a mem,rane &otential 6%ic% allo.s 2do.n%ill3 flo. of )5 t%roug% s&ecific c%annels to dri$e ATP synt%esis (&%os&%orylation

As the electrons flow through a series of protein complexes, protons are pumped into the intermembrane space. A pH gradient is formed that creates a protonmotive force.

Energy Transfor#ations

T%e &rocess:

"lectron1moti$e force ('A() # FA()2 Protonmoti$e force (&roton gradient P%os&%oryl-transfer &otential in t%e form of ATP.

Three protein co#plexes do the pu#ping$

1+

2+

!+

'A()-7 oxidoreductase 7-cytoc%rome c oxidoreductase Cytoc%rome c oxidase

T%ese are transmem,rane &rotein com&lexes containing 4uinones* fla$ins* sulfur clusters* %emes* and co&&er ions+

Complex I, III, IV pump protons.

NADH and FADH2 reduce

2

to water.

The reducing potential of NADH!than FADH2, so that more AT" is produced from NADH2. 2.# AT"$NADH %s &.#AT"$FADH2.

NADH is a stronger reducing agent than FADH2.

xidati%e "hosphorylation

'emember( The mitochondrial synthesis of AT" )oxidati%e phosphorylation* is not stoichiometric.

AT" is not synthesi+ed in exact proportions as in a typical chemical reaction for example, substrate le%el phosphorylation.

The Respiratory Chain: Overall Electron %low

"m,edded in t%e inner mem,rane* it em&loys integral mem,rane &roteins %a$ing &rost%etic grou&s t%at can carry 1 or 2 electrons 6%ic% are &assed laterally and se4uentially from com&lexes 8 to 89* .it% t.o mo,ile carriers along t%e .ay (7 and cyt c 8n concert .it% electron transfer (laterally is &roton transfer (trans$ersely from t%e matrix to t%e intermem,rane s&ace

Electron Carriers and Prosthetic &roups

'A(5 and 'A(P5 use &yridine nucleotides

6it% a fixed reduction &otential to carry %ydride ions (2 electrons* 1 &roton after transfer of 2 %ydrogen atoms in 'A(-lin:ed de%ydrogenase reactions T%e remaining &roton ()5 is released to sol$ent T%ese carriers are .ater solu,le and only .ea:ly en;yme-associated* %ence can carry electrons ,y diffusion to t%eir targets in t%e res&iratory c%ain T%oug% t%ey t%emsel$es cannot cross t%e inner mem,rane

F0' and FA( use fla$in nucleotides

T%ey %a$e a $aria,le reduction &otential de&endent on t%e &rotein .it% .%ic% t%ey are tig%tly (co$alently associated And can &artici&ate in ,ot% 1 and 2-electron transfers

"ore Electron Carriers and Prosthetic &roups

<,i4uinone (7 : can acce&t 1 or 2 electrons* is small* %ydro&%o,ic* and freely diffusi,le in t%e li&id ,ilayer Cytoc%romes (a* ,* and c : can carry single electrons in t%eir nonco$alently ,ound (exce&t cyt c %eme (Fe55 &rost%etic grou&s= cyt c also is mo,ile on t%e outer surface of t%e inner mem,rane (it>s &eri&%eral* not integral 8ron-sulfur &roteins: carry 1 electron using Fe55* coordinated to inorganic S and?or sulf%ydryl grou&s of t%e &rotein

'sing ( to Carry Electrons

<,i4uinone can ,e &artially reduced* to semi4uinone* or Fully reduced to u,i-4uinol (7)2 * .%ic% carries 2 electrons* and 2 &rotons as .ell* and T%us can cou&le electron flo. to &roton mo$ement

'sing to Carry Electrons

)) %e

Cytochromes

Iron-sulfur proteins

*ow is the Pathway of Electron Transfer Ordered!

T%e carriers c%ange t%eir s&ectral &ro&erties .%en oxidi;ed* so .%en Oxygen is suddenly added to a fully reduced &at%.ay* t%e rate at .%ic% eac% carrier gets oxidi;ed (measured s&ectro&%otometrically re$eals its &lace in t%e c%ain

The Se+uence of Electron Carriers

Can ,e &redicted ,ased on reduction &otentials (see Ta,le 1@-2 * ,ecause electrons should flow fro# carriers of lower Eo to higher Eo 6%ic% agrees .it% s&ectral determinations* and 6it% measurements using in%i,itors:

Conceptually Si#ple, Structurally Co#plex

Fragments of mitoc%ondrial inner mem,rane* dissol$ed in detergent* yield se&ara,le s&ecies of large mem,rane-em,edded su&ramolecular com&lexes (see a,o$e "ac% ca&a,le of carrying out alone one ste& in t%e res&iratory c%ain

The Se+uence of Electron Carriers -s:

NADH

cytb

cytc

cyt(a+a3)

FADH2

O2

Matrix
2H+ + O2 H2O

H+ + NADH NAD+ + 2H+

III IV

2 e Q

++
4H
+

4H

cyt c

2H+

Intermembrane Space

Co#plex - .'A() (e%ydrogenase trans&orts /*) out of t%e mitoc%ondrial matrix &er 0e transferred from 'A() to Co7+

Co#plex - Cataly1es Two Coupled Processes

)ydride transfer from 'A() to F0' t%roug% FeS centers to 7 (ri$es t%e ex&ulsion of A &rotons &er electron &air* T%us creating an electroc%emical &otential across t%e gradient* <sed (e$entually to dri$e ATP synt%esis

Other Carriers %unnel Electrons to (

Com&lex 88 is t%e only mem,rane,ound en;yme in t%e TCA cycle T%e acyl-CoA de%ydrogenase iso;ymes acce&t electrons in t%e first ste& of -oxidation* also &assing t%em along to 7 Glycerol!P is oxidi;ed on t%e cytosolic face of t%e inner mem,rane ,y t%e en;yme G!Pd) 'ote: t%ese are all fla$o&roteinsB
Electron-Transferring Flavoprotein

Structure of Co#plex --: Succinate 2ehydrogenase

2Sim&ler3 t%an Com&lex 8+++ Four &rotein su,units 1 t.o integral mem,rane and t.o in matrix (mammals C &rost%etic grou&s (FA(* t%ree Fe-S* and Coen;yme 7 AD angstroms from su,strate to t%e 7-,inding site (.it% C %andoffs of electrons 1 ,lue cur$ed arro.s And a cardioli&in anc%orE
E. coli enzyme

( ccepts Electrons fro# %our Sources in Respiration

And carries t%em in t%e ,ilayer to Com&lex 888F

The ( Cycle in Co#plex --The other 11-protein monomer of the dimer is not shown

6%ic% accommodates t%e s.itc% ,et.een 2-electron and 1electron carriers and results in A translocated &rotons &er &air of electrons transferred To t%e second mo,ile carrier* cyt c* on t%e cytosolic face of t%e inner mem,rane* .%ic% ta:es t%em toF

Matrix
2H+ + O2 H2O

H+ + NADH NAD+ + 2H+

III IV

2 e Q

++
4H
+

4H

cyt c

2H+

Intermembrane Space

Co#plex --- (,c1 com&lex :

)5 trans&ort in com&lex 888 in$ol$es coen1y#e ( (Co7 +

O CH3O
CH3

CH3O

CH3

CH3 CH3

e
CH3O (CH2 CH O C CH2)nH

CH3O C CH2)nH

(CH2 CH

coenzyme Q e + 2 H+
OH CH3O CH3

coenzyme Q

CH3 CH3O OH (CH2 CH C CH2)nH

coenzyme QH2

T%e 2( cycle3 de&ends on #o3ility of coen;yme 7 .it%in t%e li&id ,ilayer+ T%ere is e$idence for one4electron transfers* .it% an intermediate se#i+uinone radical+

matrix 2 H+ Q. QH2
Complex III

Q cyt bH e

QH2

cyt bL e Q Q 2 H+ intermembrane space

Fe-S

cyt c1 cyt c

One version of Q Cycle:

"lectrons enter com&lex 888 $ia coen1y#e (*0* .%ic% 3inds at a site on t%e &ositi$e side of t%e inner mitoc%ondrial mem,rane* adGacent to t%e intermem,rane s&ace+

matrix 2 H+ Q. QH2
Complex III

Q cyt bH

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

QH2

cyt c1 cyt c

Fe-S is reoxidized cyt bL e e by transfer of the Q Fe-S Q e to cyt c1, which + 2 H passes it out of the intermembrane space complex to cyt c.

T%e loss of one electron from 7)2 .ould generate a se#i+uinone radical* s%o.n %ere as 7H* t%oug% t%e semi4uinone mig%t initially retain a &roton as 7)H+

A 2nd is transferred from Q Q. QH2 QH2 the semiquinone to cyt bH cyt bL (heme bL) Complex III which passes it via cyt bH across the cyt b e L e membrane to Q Q Fe-S cyt c1 another CoQ bound at a site on 2 H+ cyt c intermembrane space the matrix side.

matrix

2 H+

T%e fully oxidi1ed Co(* generated as t%e 2nd e is &assed to t%e , cytoc%romes* may t%en dissociate from its ,inding site adGacent to t%e intermem,rane s&ace+

Accom&anying t%e t.o-electron oxidation of ,ound 7)2* 0*) are released to t%e intermem,rane s&ace+

matrix 2 H+ Q. QH2
Complex III

Q cyt bH e

QH2

cyt bL e Fe-S

cyt c1 cyt c

Q 2 H+ intermembrane space

8t ta:es 0 cycles for Co7 ,ound at t%e site %ear t%e matrix to ,e reduced to 7)2* as it acce&ts 2e from t%e , %emes* and 2)5 are extracted from t%e matrix com&artment+

8n 2 cycles* 0(*0 enter t%e &at%.ay # one is regenerated+

matrix 2 H+ Q. QH2
Complex III

Q cyt bH e

QH2

cyt bL e Q Q 2 H+ intermembrane space

O$erall reaction cataly;ed ,y com&lex 888* including net in&uts # out&uts of t%e 7 cycle :
Fe-S

cyt c1 cyt c

(*0 ) 0*).#atrix5 ) 0 cyt c .%e6)5 ( ) /*).outside5 ) 0 cyt c .%e0)5

Per 0e transferred t%roug% t%e com&lex to cyt c* /*) are released to t%e intermem,rane s&ace+

Co#plex ---:
PDB 1BE3

)alf of t%e %omodimeric structure is s%o.n+

Complex III (bc1 Complex)

A&&roximate location of t%e mem,rane ,ilayer is indicated+

heme bH heme bL membrane Fe-S heme c1

'ot s%o.n are t%e Co7 ,inding sites near %eme ,) and near %eme ,I+

T%e 3 he#es are &ositioned to &ro$ide a &at%.ay for electrons across t%e mem,rane+

T%e domain .it% attac%ed /ies:e %e4S %as a flexi3le lin7 to t%e rest of t%e com&lex+ (Fe-S &rotein in green+

PDB 1BE3

Complex III (bc1 Complex)

Fe-S changes position during e transfer+

membrane

heme bH heme bL

After %e4S extracts an e from 7)2* it mo$es closer to he#e c8* to .%ic% it transfers t%e e+
Fe-S

heme c1

After t%e 1st e transfer from 7)2 to Fe-S* t%e Co7 se#i+uinone is &ostulated to s%ift &osition .it%in t%e 7-,inding site* mo$ing closer to its e acce&tor* he#e 39+
membrane

PDB 1BE3

Complex III (bc1 Complex)

heme bH heme bL

T%is .ould %el& to prevent transfer of t%e 0nd electron from t%e semi4uinone to %e4S+
Fe-S

heme c1

Com&lex 888 is an o,ligate ho#o4di#er+

PDB-1BGY

Complex III homo-dimer

Fe-S in one %alf of t%e dimer may interact .it% ,ound Co7 # %eme c1 in t%e ot%er %alf of t%e dimer+

Fe-S heme c1

Arro.s &oint at: J %e4S in t%e %alf of com&lex colored .%ite?grey J he#e c8 in t%e %alf of com&lex .it% &roteins colored ,lue or green+

Matrix
H+ + NADH NAD+ + 2H+ 2H+ + O2 H2O

III IV

2 e Q

++
4H+ 4H+

Co#plex -: (Cytoc%rome Oxidase :

cyt c Intermembrane Space

2H+

Electrons are donated to com&lex 89* one at a time* ,y cytoc%rome c* .%ic% ,inds from t%e intermem,rane s&ace+

"ac% e &asses $ia CuA # %eme a to t%e 3inuclear center* ,uried .it%in t%e com&lex* t%at cataly;es O2 reduction: Ae 5 A)5 5 O2 - 2)2O+ Protons utili;ed in t%is reaction are ta:en u& from t%e matrix com&artment+

Matrix
2H+ + O2 H2O

H+ + NADH NAD+ + 2H+

III IV

2 e Q

++
4H+ 4H+

cyt c

2H+

Intermembrane Space

*) pu#ping ,y com&lex 89:

8n addition to &rotons utili;ed in reduction of O2* t%ere is electron transfer-lin:ed transport of 0*) per 0e (A)5 &er Ae from t%e matrix to t%e intermem,rane s&ace+

Co#plex -:: Cytochro#e Oxidase

! of t%e 1! su,units are critical for electron transfer and &roton &um&ing For e$ery A electrons from mo,ile cyt c* A &rotons are &um&ed out* and A more are com,ined .it% molecular oxygen to form 2 molecules of .ater <sing 1-electron redox centers (.it% 2 %emes and ! co&&er atoms

Electron Transfer Through the Respiratory Chain 9eads to a "e#3rane Potential

Created ,y concomitant &roton &um&ing in Com&lexes 8* 888* and 89 0a:ing t%e inside t%e 2'side3* t%us creating a differential in ,ot% C%emical concentration (&) and C%arge distri,ution ()

Concept: Proton "otive %orce

T%e c%emiosmotic model of oxidati$e &%os&%orylation %olds t%at: a gradient of &rotons is created across t%e inner mitoc%ondrial mem,rane (,y t%e electron trans&ort c%ain and it is t%e energy stored in t%is gradient t%at is lin:ed to t%e synt%esis of ATP from A(P and Pi+ T%is conce&t .as re$olutionary in its time (1@KD>s B

Electron %low Thus Captures Energy as Proton4"otive %orce

= 0.15-0.2 V

0.75 pH units higher than P-side

For t%e o$erall transfer of 2 electrons from 'A() to molecular oxygen* G>o L -22D :M?mol* and 1D &rotons are &um&ed out* .%ic% conser$es a,out 2DD :M?mol (of 'A() in t%e electroc%emical &otential 6%en &rotons are allo.ed to flo. ,ac: in* .or: can ,e &erformed* suc% as t%e synt%esis of ATP

Oxidation of &lucose: Where re We ;ow!

CK)12OK 5 KO2

KCO2 5 K)2O
-2840 kJ/mole

6e>re (O'"B

,ummary of the electron flow

The pH gradient pro%ides a proton moti%e force that is used to con%ert AD" to AT". The en+yme complex that cataly+es the reaction is ATP syntase ) complex -.*.

Peter "itchell

"lectron trans&ort and ATP synt%esis are cou&led ,y a &roton gradient across t%e inner mitoc%ondrial mem,rane+ C%emiosmotic )y&ot%esis Oxidation and &%os&%orylation are cou&led t%roug% a &roton 1 moti$e force+

"eter /itchell Nobel "ri+e in 0hemistry, &123

"for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory"

-t ain4t a theory, it4s a fact.

Matrix
2H+ + O2 H2O

ADP + Pi ATP

H+ + NADH NAD+ + 2H+

F1 Fo

III IV

2 e Q

++
4H+

4H+

cyt c
2H+

3H+

Intermembrane Space

T%e Che#ios#otic Theory of oxidati$e &%os&%orylation* for .%ic% Peter 0itc%ell recei$ed t%e 'o,el &ri;e:

Coupling of ATP synt%esis to res&iration is indirect* $ia a )5 electroc%emical gradient+

Matrix
2H+ + O2 H2O

ADP + Pi ATP

H+ + NADH NAD+ + 2H+

F1 Fo

III IV

2 e Q

++
4H+

4H+

cyt c
2H+

3H+

Intermembrane Space

Che#ios#otic theory 4 respiration:

S&ontaneous e transfer t%roug% com&lexes 8* 888* # 89 is cou&led to non-s&ontaneous )5 eGection from t%e matrix+

)5 eGection creates a #e#3rane potential (* negati$e in matrix and a p* gradient (&)* al:aline in matrix +

Matrix
2H+ + O2 H2O

ADP + Pi ATP

H+ + NADH NAD+ + 2H+

F1 Fo

III IV

2 e Q

++
4H+

4H+

cyt c
2H+

3H+

Intermembrane Space

Che#ios#otic theory 4 %8%o TP synthase:

'on-s&ontaneous ATP synt%esis is cou&led to s&ontaneous )5 trans&ort into t%e matrix+ T%e &) # electrical gradients created ,y res&iration are t%e dri$ing force for )5 u&ta:e+

)5 return to t%e matrix $ia Fo Nuses u&N &) # electrical gradients+

ADP + Pi F1 3 H+ matrix Fo intermembrane space

ATP

TP synthase* em,edded in cristae of t%e inner mitoc%ondrial mem,rane* includes:

%8 catalytic su,unit* made of C &oly&e&tides .it% stoic%iometry 33+

%o com&lex of integral mem,rane &roteins t%at mediates proton transport+

ADP + Pi F1 3 H+ matrix Fo intermembrane space

ATP

%8%o cou&les ATP synt%esis to )5 trans&ort into t%e mitoc%ondrial matrix+ Trans&ort of least 6 *) &er ATP is re4uired* as estimated from com&arison of: & for TP synthesis under cellular conditions (free energy re4uired & for transfer of each *) into t%e matrix* gi$en t%e electroc%emical )5 gradient (energy a$aila,le &er )5 +

F: 5 The proton channel &:;&< c subunits. F& 5 6787( 6 and 8 alternate in a ring and both bind nucleotides, only the 8 is catalytic. 9ach 8 subunit is distinct because it interacts with a different face of the subunit. There are two functional components( The mo%ing rotor )the c ring, , and *. The stationary stator )the rest of the subunits*.
ligomycin )F:* inhibits AT" synthesis.

TP Synthesis
ATP-A 5 )2O

A(P-! 5 )POA-2 5 )5

8soto&ic exc%ange ex&eriments s%o.ed t%at ATP forms in t%e acti$e site of t%e en;yme .it%out &roton-moti$e force+ ATP and A(P are in e4uili,rium in t%e acti$e site+ Out* ATP can>t lea$e t%e acti$e site unless &rotons flo. t%roug% t%e en;yme+

/emem,er* t%e ! P su,units interact .it% t%ree different faces of t%e su,unit+ T%us* t%e P su,unit can %a$e t%ree different conformations+ T (tig%t conformation * strongly ,inds ATP+ 8t s%ifts t%e e4uili,rium of A(P 5 Pi - ATP+ )o.e$er t%e T conformation can not release t%e ATP+ 9 (loose conformation * ,inds A(P and Pi and can not release nucleotides+

O (o&en conformation can ,ind nucleotide and is similar to t%e T and I forms ,ut it can s%ift to an o&en conformation t%at releases ,ond nucleotide+ T%e intercon$ersion of t%e t%ree forms is dri$en ,y t%e rotation of t%e su,unit+

Nobel "ri+e in 0hemistry, &112

Afor their elucidation of the en+ymatic mechanism underlying the synthesis of adenosine triphosphate )AT"*A

"aul D. =oyer

>ohn 9. ?al@er

Actin filament fluorescently labeled. AT" is added causing a unidirectional countercloc@wise rotation.

The hydrolysis of AT" dri%es the rotation of the subunit.

0ountercloc@wise rotation, since obser%ation was from below with a fluorescence microscope.

The Blycerol 7;"hosphate ,huttle

Remember - glyceraldehyde 3-phosphate DH reaction from glycolysis.

G = 1.5 kcal mol-1

?hat is the fate of NADH produced from glycolysis under aerobic conditionsC

Dihydroxyacetone;" is reduced to glycerol 7;". Blycerol 7;" is then reoxidi+ed to dihydroxyacetone;" howe%er, the mitochondrial DH ids an FAD en+yme.

Blycolysis

What is achieved fro# the &lycerol 64Phosphate Shuttle!

'A() is reoxidi;ed to 'A(5+ )o.e$er* t%e cost of aero,ically reoxidi;ing t%e 'A() is less ATP t%an ex&ected+ 'A() is con$erted to FA()2 in t%e s%uttle+ T%e ex&ected 2*C ATP ('A() yield is actually a,out 1+C ATP (FA()2 + This shuttle is very active in #uscle to ena3le a high rate of O<4Phos$

AT";AD" translocase )adenine nucleotide translocase or ANT* transports these nucleotides from the mitochondria to the cytosol.

For e%ery AD" that enters the mitochondrial matrix and AT" exists.