Module 0220502
Membrane Biogenesis and Transport
Lecture 10
Proton Pumping by the
Respiratory Chain
Dale Sanders
19 February 2009
Aims: By the end of the lecture you
should understand…
 That the mitochondrial complexes associated with H+ transport are
those that catalyse reactions with large changes in mid-point
potential;

The significance of H+/2e- stoichiometries, and how they are measured;

How the basic structural components of Complex I might be involved
in H+ pumping;

How the Q-cycle is involved in H+ pumping by Complex III;

How the three-dimensional structure of Complex IV (cytochrome
oxidase) gives information on the catalytic reduction of oxygen,
and how H+ might be pumped through cytochrome oxidase.
Reading
For this lecture, and for the ensuing two (which are on light-driven
H+ transport and ATP synthesis, respectively), the only specialist text
is:
Nicholls, DG & Ferguson, SJ (2002) Bioenergetics 3.
Good articles/minireviews on structural attributes of Complexes I, III
and IV are, respectively
Sazanov & Hinchliffe (2006) Science 311: 1430-1436
Iwata, S. et al. (1998) Science 281: 64-71
Ostermeier, 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
NADH
complexes
I
(Fe/S)2
(Fe/S)1
FMN
(Fe/S)3/4
–200
0
Em / mV
+200
FAD
Succ Fe/S
cytochromes
III
bL
bH
UQ
c1 c a
II
IV
a3
+400
+600
+800
ATP
ATP
ATP
O2
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 redox
reactions to generate PMF
(iii) a returning passive flow of H+ through an ATP synthase
provides the energy for ATP synthesis.
(iv) uncouplers work by
dissipating PMF
(≡ "Protonophores"):
Thus O2 consumption
increases in presence
of uncouplers because
no opposing force
cytoplasm
membrane
P
H+
N
Complexes
I, III, IV
ATP
synthase
mitochondrial
matrix
NADH, ½O2, H+
+
H+
NAD + H2O
ATP + H2O
ADP + Pi
Uncoupler
(artificial)
How do Respiratory Complexes
Pump Protons? – Loops vs Pumps
1. The redox loop – an early (1970s) idea:
Alternating e- and (e- + H+) carriers are part of the redox
chain
E.g. cytochrome  quinone  cytochrome 
2. Pump, with proteins undergoing redox-driven
conformational changes to move H+ uphill
across membrane
How many protons for each complex?
(H+ /2e- ratios)
Experimental systems
1. Intact mitochondria:
“Dissect” resp. chain with a combination of inhibitors, e- donors
and e- acceptors.
Complex
e- donor
e- acceptor
inhibitor
I
malate (
ubiquinone
rotenone
III
ubiquinol
Fe(CN)63-
antimycin A
IV
Fe(CN)64-
02
CN-
NADH)
2. Sub-mitochondrial particles: inside-out vesicles: allows
direct 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 of
excess oxidant: “mols” e- known.
2. Measure H+ appearing outside (or taken up: smp’s) with pH
electrode.
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
All mitochondrial homologues
Mr > 525,000
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 peripheral
subunits
Tightly-bound UQ:
Membrane sector
Measured H+/2e- = 4
Projected mechanism of H+ - pumping….
P
N
2H+
NADH
UQH2
+
2H
2e–
2e–
(Fe/S)
UQ
FMNH2
2e–
2e–
2H+
(Fe/S)
UQ
UQH2
FMN
2H+
2H+
[N-2]
2e–
NAD+ + H+
2H+
Cycling of UQ
in redox loop
hypothetical:
could just
pump 4H+ from
N to P side
Structure of the Hydrophilic Domain of Respiratory
Complex I from Thermus thermophilus
Sazanov & Hinchliffe (2006) Science 311:1430-1436
Complex III
All subunits membrane-integral
Polypeptide
Prosthetic Group(s)
Rieske protein
cytochrome c1
cytochrome b
[2Fe –2S] on P side
haem on P side
2 haem: bL on P side Em = - 100 mV
bH on N side Em = + 50 mV
Structure of Complex III Showing location of Prosthetic
Groups
Measured H+/2e- = 4
Mechanism of H+ pumping: THE Q CYCLE
• A 2-stage, branched oxidation of UQH2:
UQH2
P
myxothiazol
+
2H
e–
e–
UQ
bL
e–
2Fe-2S
e–
haem
e–
haem
c
N
UQ
–
UQH2
P
+
2H
e–
–
bH eb
e–
Rieske
UQ
bL
e–
2Fe-2S
e–
haem
c1
e–
haem
c
N
antimycin
UQ
–
–
bH eb
Rieske
c1
2H+
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 USEABLE
energy output doubled.
COMPLEX IV
(Cytochrome Oxidase; COX)
Subunit composition:
For Paracoccus:
Subunit Transmembrane
spans
I
12
II
Mitos: 13
Both crystallized:
Paracoccus: 4 Structures solved at
2.8 Å
Cofactors
Mitochondrial
homologue?
haem a, a3, CuB
Y
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:
–
2e
+
oxidized a 3
3
O2
2+
Cu2B+
a23+
+
CuB
a3
+
Cu B like oxy-
haemoglobin
O2
2HO
2
+–
2H,
e
N
e
a 43
+
O2–
–
3+
2+
CuB
HO
2
oxyferryl state
a3
+
2H,N
spontaneous
2
CuB+
2–
O2
peroxy state
Structure of Paracoccus COX, Subunit I Parallel to
Membrane
Periplasm
CuB
Haem a
Haem a3
Membrane
Iwata et al. (1995)
Nature 376, 660-669
Cytoplasm
H+ Translocation by COX
General organization:
P
N
cyt c
subunit I is H+ pump
II
2e–
CuA
a
I
2H+
a3
+
2H
CuB
IV
III
H2O
½O2
2H+
'chemical' protons
'pumped' protons
O2 channel?
Structure of Paracoccus COX, Subunit I from Periplasmic Side
Iwata et al. (1995)
Nature 376, 660-669
• Site-directed mutagenesis suggests separate pathways for
chemical and pumped H+
D124N mutation: H2O formation unaffected, but
H+ pumping blocked
• Folding of SU I shows 3-fold symmetry with pores accessible
from the N side
IX
XI
XII
X
I, II etc: helices
VIII
haem a
CuB
I
VI
Pore C
haem a3
VII
Pore B
II
III
IV
V
Pore A
Hydrophilic residues lining pores form pathway for H+:
Pore A:
H+ pumping?
Pore B:
H+ consumption (i.e. H2O formation)?
SUMMARY
1. H+ pumping at
Complexes I, III and IV
occurs by a variety of
mechanisms.
Including
(i) Protein conformational
changes (Complex I,
Complex IV)
(ii) Q cycle (Complex III;
[Complex I?])
3.
Summary diagram:
P
N
I
2e–
4H+
UQ
III 2e–
+
4H
2. Overall stoichiometry of
H+ translocation /2e- is
10 which comprises 4H+/
2e- (I); 4H+/ 2e- (III): 2H+/
2e- (IV)
NADH
Krebs cycle
+ + H+
NAD
4H++
2H
2H+
cyt c
2e–
2H+
IV
½O2 + 2H+
H2O
2H+