Proton Pumping by theRespiratory Chain

Dale Sanders

19 February 2009

Module 0220502

Membrane Biogenesis and Transport

Lecture 10

That the mitochondrial complexes associated with H+ transport are

those that catalyse reactions with large changes in mid-pointpotential;

The significance of H+/2e- stoichiometries, and how they are measured;

How the basic structural components of Complex I might be involvedin H+ pumping;

How the Q-cycle is involved in H+ pumping by Complex III;

How the three-dimensional structure of Complex IV (cytochromeoxidase) gives information on the catalytic reduction of oxygen,and how H+ might be pumped through cytochrome oxidase.

Aims: By the end of the lecture youshould understand…

ReadingFor this lecture, and for the ensuing two (which are on light-drivenH+ transport and ATP synthesis, respectively), the only specialist textis:

Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3.

Good articles/minireviews on structural attributes of Complexes I, IIIand IV are, respectively

Sazanov & Hinchliffe (2006) Science 311: 1430-1436

Iwata, S. et al. (1998) Science 281: 64-71Ostermeier, C. et al. (1996) Curr. Opin. Struct. Biol. 6: 460-466

H+ Translocation by the Respiratory Chain

The mito. resp. chain, arranged according to mid-point potentials

(Fe/S)2

(Fe/S)1

FMN

FAD

(Fe/S)3/4

Fe/SSuccUQ

bL bH

c1 c a

a3

I

III

IV

II

NADH

ATP ATP ATP

O2

complexes

cytochromes

E / mVm

–200

0

+200

+400

+600

+800

ATP production coupled to e- transport at Complexes I, III, IV

These are Complexes with a large change in mid-point potential

Chemiosmotic Coupling

(i) respiratory chain is a proton pump

(ii) low intrinsic membrane permeability to H+ allows redoxreactions to generate PMF

(iii) a returning passive flow of H+ through an ATP synthaseprovides the energy for ATP synthesis.

H+H+Complexes

I, III, IV

ATPsynthase

Uncoupler(artificial)

cytoplasm membrane mitochondrialmatrix

NADH, ½O2, H+

NAD+ + H2O

ATP + H2O

ADP + Pi

P N

(iv) uncouplers work bydissipating PMF

(≡ "Protonophores"):

Thus O2 consumptionincreases in presenceof uncouplers becauseno opposing force

How do Respiratory ComplexesPump Protons? – Loops vs Pumps1. The redox loop – an early (1970s) idea:

Alternating e- and (e- + H+) carriers are part of the redoxchain

E.g. cytochrome quinone cytochrome

2. Pump, with proteins undergoing redox-drivenconformational changes to move H+ uphillacross membrane

How many protons for each complex?(H+ /2e- ratios)

Experimental systems

1. Intact mitochondria:

“Dissect” resp. chain with a combination of inhibitors, e- donorsand e- acceptors.

Complex e- donor e- acceptor inhibitor

I malate ( NADH) ubiquinone rotenone

III ubiquinol Fe(CN)63- antimycin A

IV Fe(CN)64- 02 CN-

2. Sub-mitochondrial particles: inside-out vesicles: allowsdirect access of substrate to matrix side.

3. Reconstituted complexes:

“dissect” resp. chain physically

[detergent, centrifugation]

incorporate complexes into lipid vesicles

EXPERIMENTAL PROTOCOL

1. Initiate e- flow with known amount of reductant in presence ofexcess oxidant: “mols” e- known.

2. Measure H+ appearing outside (or taken up: smp’s) with pHelectrode.

Stoichiometries and Mechanisms

COMPLEX I

In mitos > 41 subunit types Mr > 850,000

7 integral membrane 34 peripheral

encoded on mito genome nuclear genome

In E. coli 14 subunits Mr > 525,000

All mitochondrial homologues

Cofactors and subunits of Complex I

NAD+, FMN, [4Fe-4S] centre: 51 kDa peripheral subunit

3 more [4Fe-4S], + 1 [2Fe-2S]: each on separate peripheralsubunits

Tightly-bound UQ: Membrane sector

Measured H+/2e- = 4

Projected mechanism of H+ - pumping….

UQH2

UQ

(Fe/S)FMNH2

2e–

2e–

2e–

FMN

NADH

NAD+ + H+

2H+

2H+

2H+

2H+

[N-2]

UQ

UQH2

2H+

2H+(Fe/S)

2e–

2e–

P N

Cycling of UQin redox loophypothetical:could justpump 4H+ fromN to P side

Structure of the Hydrophilic Domain of RespiratoryComplex I from Thermus thermophilus

Sazanov & Hinchliffe (2006) Science 311:1430-1436

Complex III All subunits membrane-integral

Polypeptide Prosthetic Group(s)

Rieske protein [2Fe –2S] on P side

cytochrome c1 haem on P side

cytochrome b 2 haem: bL on P side Em = - 100 mV

bH on N side Em = + 50 mV

Structure of Complex III Showing location of ProstheticGroups

Measured H+/2e- = 4

Mechanism of H+ pumping: THE Q CYCLE

• A 2-stage, branched oxidation of UQH2:

UQH2

UQ

2Fe-2S

2H+

e–

P N

UQ–

bL bHe–

e–

b

Rieske

c1

e–

haem

e–

haem

c

myxothiazol

UQH2

UQ

2Fe-2S

2H+

e–

P N

UQ–

bL bHe–

e–

b

Rieske

c1

e–

haem

e–

haem

c

2H+

e– e–

antimycin

Net result of Q Cycle: oxidation of 1 UQH2

with 2e- passed to cyt c and 4H+ pumped

BUT: 2 UQH2 oxidized (1 regenerated)

1 e- each to bL + [2Fe-2S]

Significance: By recirculating ½ of e-,maximise H+ translocation ie USEABLEenergy output doubled.

COMPLEX IV (Cytochrome Oxidase; COX)

Subunit composition: Mitos: 13 Both crystallized:

Paracoccus: 4 Structures solved at

2.8 ÅFor Paracoccus:

Subunit Transmembrane Cofactors Mitochondrialspans homologue?

I 12 haem a, a3, CuB Y

II 2 CuA Y

III 7 None Y

IV 1 None N

Measured H+/2e- = 2

[plus 2H+ consumed on N side in ½ 02 reduction]

Haem a3 + CuB: binuclear centre

Redox reactions during 02 reduction:

a3

3

+

a a4

3

3 3

++

a2

3

+ a2

3

+

Cu2

B

+

Cu2

B

+Cu

2

B

+

CuB

+ CuB

+

2e–

e–

O2

O2

oxidized

spontaneous

like oxy-haemoglobin

2HO

2H, e-

2

+–

N

2H,+

NHO

2O2– O

2–

oxyferryl state peroxy state

2

Structure of Paracoccus COX, Subunit I Parallel toMembrane

Haem a

CuB

Haem a3

Membrane

Cytoplasm

Periplasm

Iwata et al. (1995)

Nature 376, 660-669

H+ Translocation by COX

General organization:

cyt c

CuA

2e–

½O2

2H+

2H+

2H+

H2O

CuB

a3

a

II

I

IV

III

P N

O2 channel?

'chemical' protons

'pumped' protons

subunit I is H+ pump

Structure of Paracoccus COX, Subunit I from Periplasmic Side

Iwata et al. (1995)

Nature 376, 660-669

• Site-directed mutagenesis suggests separate pathways forchemical and pumped H+

D124N mutation: H2O formation unaffected, but

H+ pumping blocked

• Folding of SU I shows 3-fold symmetry with pores accessiblefrom the N side

haem a3

CuB

Pore B

Pore A

Pore C

haem a

XI

XII

I

II

X

IX

VIII

VIIVI

VIV

III

I, II etc: helices

Hydrophilic residues lining pores form pathway for H+:

Pore A: H+ pumping?

Pore B: H+ consumption (i.e. H2O formation)?

SUMMARY

1. H+ pumping atComplexes I, III and IVoccurs by a variety ofmechanisms.Including(i) Protein conformational

changes (Complex I,Complex IV)

(ii) Q cycle (Complex III;[Complex I?])

2. Overall stoichiometry ofH+ translocation /2e- is10 which comprises 4H+/2e- (I); 4H+/ 2e- (III): 2H+/2e- (IV)

3. Summary diagram:

2e–

½O2 + 2H+

2H+

2H+ H2O

cyt c

2H+

2H+4H+

4H+

4H+

P N

NADH

2e–

I

IV

NAD+ + H+

UQ

2e–III

Krebs cycle