module 0220502 membrane biogenesis and transport lecture … · 2009-02-19 · proton pumping by...
TRANSCRIPT
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