Membrane proteins

1
Structural studies of the respiratory complexes
Complex II and Fdh-N
Susanna Törnroth 041114 Chalmers University of
Technology
2
Crystallisation setup
Drop containing protein solution and
reservoir solution in 11 ratio.
Reservoir solution with precipitant
Hanging drop
The drop volume is slowly decreasing and ordered
precipitate is formed in the form of crystals.
3
Xray Crystallography
Edge 2.6 Å
Crystal
Diffraction
4
Solving a structure
Electron density
5
Membrane proteins
Peripheral
Integral
Lipid-anchored
6
Integral membrane proteins
Hydrophilic part
Hydrophobic part
7
Difficulties with membrane proteins

• More difficult to overexpress
• More difficult to obtain crystals
• More difficult to get good enough resolution

8
Solubilisation of membrane proteins
Mild detergent
9
Crystallisation of membrane proteins
crystallisation
10
Aerobic respiration
Matrix
H
e-
e-
ADP ATP
NADH NAD
Succinate Fumarate
O2 H2O
Q
Q
I
II
III
IV
V
Cyt. c
H
H
H
Inter-membrane space
11
Complex II

• Succinate dehydrogenase (SDH) or Succinate
  Quinone Oxidoreductase (SQR)
• Links the Krebs cycle with the respiratory chain
• Oxidises Succinate to Fumarate and reduces
  quinone in the membrane

12
Complex II from E. coli
succinate
fumarate
FAD
2Fe2S
4Fe4S
3Fe4S
periplasm
Q
Heme b
QH2
cytoplasm
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Overall Structure

• Physiological (non-functional) trimer (MW 360
  kDa)
• Monomer contact surface area 1242 Å2

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Monomer structure
FAD
2Fe2S
4Fe4S
3Fe4S
Heme b
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Electron transport pathway

• 50 Å long electron transport pathway
• All distances within the limit of physiological
  electron transfer
• Branched pathway at 3Fe-4S
• Redox centres in adjacent monomers are gt30 Å away

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Quinone binding site
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What is the role of heme b?
Ubiquinone binds between 3Fe4S and heme Heme is
not required for electron transfer to quinone
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Complex II and ROS formation

• Mutations in the Q-site give rise to tumour
  formation in humans and premature ageing in C.
  elegans.
• This is most likely due to Reactive Oxygen
  Species formation, ROS.
• ROS causes damage to cell components by forming
  highly reactive oxygen radicals.

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ROS and the role of heme b
Heme b can hold electrons that are released from
the oxidation of succinate before the
reduction of UQ takes place or when UQ- binding i
dysfunctional
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In conclusion

• E. coli Complex II is a physiological trimer
• Mutations that causes tumours in humans have been
  located in the Quinone binding site.
• A possible role for heme b as an ROS-formation
  preventer has been proposed.

21
Anaerobic respiration

• Many prokaryotes are able to use other terminal
  electron acceptors than oxygen, such as nitrate
  and fumarate
• Different respiratory chains are expressed
  depending on enviromental factors
• E. coli is a facultative aerobic bacterium, it
  can respire with or without oxygen as the
  electron acceptor

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The chemiosmotic model Peter Mitchell 1961
H
High protonic potential
respiratory or photo- redox chain
ATP synthase
Low protonic potential
ADP
H
ATP
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Proton motive force generation
4H
2H
2e-
periplasm
2QH2
Q
2e-
QH2
Q
cytoplasm
2H
1/2O2
H2O
2H
Proton pump cytochrome c oxidase
Q-cycle cytochrome bc1 complex
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The redox loop
2H
AH
A H
periplasm
Q
2e-
QH2
2e-
QH2
Q
cytoplasm
BH
2H
B H
oxidation
reduction
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Fdh-N / NAR system in E. coli
CO2H
HCOO-
a
2e-
2xMGD
2H
4Fe4S
Nitrate reductase
-
b
2e-
44Fe4S
periplasm
g
b
b
MK
Quinone pool
2e-
2e-
MKH2
g
b
b
cytoplasm
2e-
3Fe4S 34Fe4S
b
Formate dehydrogenase N
4Fe4S
2H
2xMGD
a
2e-
NO2- H2O
NO3- 2H
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Overall structure of Fdh-N

• -structure solved to 1.6 Å
• -physiological trimer
• (MW510 kDa)
• -a- and b-subunits are
• located on the periplasmic
• side of the membrane
• -distances between redox
• centres are within the limit
• of physiological electron
• transfer

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The a-subunit and formate oxidation site
-core structure is similar to other structurally
known MGD-containing enzymes, for example
Fdh-H (r.m.s1.9Å, 599 Ca) -one HEPES molecule
was found in the active site cleft
28
The b-subunit
-consist of an extrinsic domain containing the
four 4Fe4S and one transmembrane helix -the
extrinsic domain belong to a superfamily of
four FeS cluster binding domains found in many
membrane-bound oxidoreductases
-the core region of the extrinsic domain folds
into two subdomains related by an approximate
twofold symmetry (r.m.s 1.9Å, 56 Ca)
29
The g-subunit
-membranebound cytochrome b with four
transmembrane helices -contains two heme b and
the menaquinone reduction site -three helices
are involved in ligation of the hemes -the
g-subunit, the tmb and the cardiolipin form the
tightly packed trimer
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The menaquinone reduction site
-the menaquinone binding site was characterised
using the menasemiquinone analogue HQNO -the
heme ligand H169 is directly involved in quinone
binding -there is a proton pathway from the
cyto- plasm to the menaquinone reduction site
HQNO
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The quinone reduction mechanism
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In conclusion
-the Fdh-N/Nar system from E. coli generates pmf
through a redox loop -the enzyme is a
physiological trimer -a detailed mechanism for
menaquinone reduction and how the protons are
has been proposed.
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Complex II and Fdh-N

• Physiological trimers
• Couples substrate
• oxidation in the cytoplasm
• to quinone reduction in the
• membrane
• Has a similar overall design
• Large cytoplasmic domain
• electron transfer chain with
• FeS clusters
• a-helical transmembrane
• domain with heme b