The respiratory chain
Citric acid cycle
Respiratory Chain
P. Frederix
Electron carriers
Nicotinamide Adenine Dinucleotide
(Ubi)Quinone
Prosthetic groups
Non-protein chemical compounds that are tightly bound to a
protein and are required for the protein's biological activity
flavins
FMN (Flavin MonoNucleotide) FAD (Flavin Adenine Dinucleotide)
Carbon as electron donor
1
Prosthetic groups: Hemes
Prosthetic groups: Iron-Sulfur clusters
17-hydroxyethylfarnesyl
1
1
Heme b
histidine
cystein
Hemes a and b are generally coordinated by conserved
amino acid side chains (XY): e.g. Heme b inside complex
II, kept in place between 2 histidines
Prosthetic groups
Spectroscopy for studying respiration
chain versus catalytic site
Hemes have characteristic absorption spectra
Hemes are the prothetic groups of
Cytochromes
α-peak was used
to categorize heme
a, b and c types
SMP = submitochondrial particle
Lower temperature: narrower peaks
Absorption spectrum cyt c
(Lehninger, Biochemistry)
Page et al. 2003,
2
Spectroscopy for studying respiration
Spectroscopy for studying respiration
Hemes have characteristic absorption spectra
Hemes have characteristic absorption spectra
Detection of redox centers
Redox potentiometry
Iron-sulfur centers
EPR spectroscopy
∆E = hν = geµBB
Signal is from unpaired electrons in: Fe3+, Mn2+, Cu2+
Biological redox couple plus secondary redox mediator
(1-100 µM) that has similar mid-point potential, e.g.
ascorbate (Em,7≈ +60 mV). For this experiment the
complex must be kept in solution with detergent or a
bilayer permeant mediator must be used.
To titrate a redox process, either an electron donor (reducing agent) like dithionite (Em,7≈ -660
mV) or an electron acceptor (oxidizing agent) like ferricyanide (Em,7 ≈ +420 mV) is added.
Titration with small aliquots of dithionite makes the potential more negative, with ferricyanide
more positive.
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Redox potentiometry
Electron transfer: Marcus’ theory
Example: Mixture of complex II & III in detergent
If wavefunctions
overlap, transfer is
possible.
To titrate the redox process the electron acceptor ferricyanide is
added, and spectra are recorded at different potentials.
Here 4 Spectra were recorded (-100mV, -10mV, 145mV, 180mV)
and difference spectra are calculated, by substracted the next
oxidized stage (giving a reduced-oxidized difference spectrum):
i. Abs(145mV) – Abs(180mV)
ii. Abs(-10mV) – Abs(145mV)
iii. Abs(-100mV) – Abs(-10mV)
Overlap is maximal
when ∆G=-λ
(product curve crosses
reactant curve at
minimum)
Question: Which prosthetic groups do we look at and in which
range are their mid-point potentials under the experimental
conditions?
The reorganization energy λ is the energy required for
all structural adjustments (in the reactants and in the
surrounding solvent molecules) which are needed in
order that A and D assume the configuration required
for the transfer of the electron.
Marcus, J Chem Phys 1956, Moser et al. Nature, 1992
Practical tunneling expression
Frank-Condon factor
λ
Reorganization
energy λ (eV)
k ET ∝ 10−3.1(∆G +λ )
2
Dutton’s plots
Tunneling versus distance
/λ
Driving force
∆G (eV)
∆G can be adjusted
using mutants with
shifted mid-potentials
Haffa, A.L., Lin, Su, Katilius, E., Williams, J.C., Taguchi, A.K. J. Phys. Chem B. 106:7376-7384 (2002)
Gunner, M. R and Dutton P.L. J.A.C.S. 111:3400-3412 (1989)
Moser, C. C. et al. Nature 355:796-802 (1992).
Page, C. C. et al. Nature 402:47-52 (1999).
Moser, C. C. et al. In Enzyme-Catalyzed Electron Radical Transfer (Scrutton, N. S.), pp 1-30 (Plenum, 2000)
Page, C. C. et al Current Opinion 7:1-6 (2003)
Moser et al., Nature 1992
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Dutton’s Equations
Electron tunneling in oxidation-reduction
If the structure of the protein is not known, electron transfer rates
are calculated based on the following equations:
For exothermic (downhill) reactions (∆G < 0):
Log10 ket = 13 - 0.6 (R - 3.6) - 3.1 (∆G + λ )2 / λ
For endothermic (uphill) reactions (∆G > 0):
Log10 ket = 13 - 0.6 (R - 3.6) - 3.1 (- ∆G + λ )2 / λ - ∆G/0.06
The initial constant 13 is the rate at van der Waals contact distance (R = 3.6 Å).
The second term describes an approximately exponential fall-off in electron
tunneling rate with distance through the insulating barrier. R is the edge-to-edge
distance.
The third term is the quantized Frank-Condon factor at room temperature. ∆G is
free energy change and λ is reorganization energy, both in units of eV.
The last term in the uphill reactions incorporates thermal excitation (kT≈0.025eV).
Experimental rate data compared with edge-toedge distances lie close to the ∆G=-λ line
Theoretical curves for exo/endothermic with
previous equations
Page et al, Nature 1999
Electron tunneling in oxidation-reduction
Electron tunneling in oxidation-reduction
at the catalytic site
Proximity between redox centers in catalytic clusters allows
rapid tunneling to endergonic radical intermediate states.
Heme a3 and CuB
In complex IV
Insertion of “high energy” intermediates is faster than no intermediate
Page et al, Nature 1999
Xanthine
dehydrogenase
Moving radical
states into the
thermally
accessible regime
Page et al, Nature 1999
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Summary I
• Enzymes can be studied by redox potentiometry, optical spectroscopy,
EPR spectroscopy
The respiratory chain: Cofactors
Mitochondrial:
• Electrons tunnel from one to the next prosthetic group
• The mid-point potential of prosthetic groups depends on their direct
environment
• Electron tunneling requires prosthetic groups to be precisely arranged
(<14Å)
• Electrons are shuttled by electron carriers from one to the next complex
• Electron carriers (NADH, quinone, cytochrome c, oxygen) must have
precise docking sites to accept/deliver electrons from/into the tunneling
pathway.
Note:
The counterpart of Complex I in bacteria has generally only 14 subunits (13 in E. coli, where
two are fused as one)
Other names:
Complex I: NADH-UQ oxidoreductase
Complex II: Succinate-UQ oxidoreductase, Succinate dehydrogenase, SQR
Complex III: UQ-cyt c oxidoreductase, cyt bc1 complex
Complex IV: ferrocytochrome c-O2 oxidoreductase, cytochrome c oxidase, cytochrome aa3
oxidase
• Uphill intermediate prosthetic groups are possible
Respiratory energy conversion (By courtesy of Leslie Dutton)
Respiratory energy conversion (By courtesy of Leslie Dutton)
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Respiratory energy conversion (By courtesy of Leslie Dutton)
Respiratory energy conversion (By courtesy of Leslie Dutton)
Respiratory energy conversion (By courtesy of Leslie Dutton)
Respiratory energy conversion (By courtesy of Leslie Dutton)
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Respiratory energy conversion (By courtesy of Leslie Dutton)
Respiratory energy conversion (By courtesy of Leslie Dutton)
Respiratory energy conversion (By courtesy of Leslie Dutton)
Respiratory energy conversion (By courtesy of Leslie Dutton)
8
Respiratory energy conversion (By courtesy of Leslie Dutton)
Respiratory energy conversion (By courtesy of Leslie Dutton)
Respiratory energy conversion (By courtesy of Leslie Dutton)
Redox potential ladder drives
electronflow
Em,7, in non-respiring mammalian
mitochondria
Eh,7, in state 4, depends on ratio of
components
• There are four “isopotential” groups
• Potential gaps correspond to regions where
proton translocation occurs
∆µH+
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Complex II
Complex I
Succinate Dehydrogenase from E. coli is
Complex I catalyzes oxidation of NADH, with reduction of coenzyme Q. Complex I is the point of
entry for the major fraction of electrons that traverse the respiratory chain, is L-shaped and includes
(in mammals) ~ 45 unlike proteins (Mr about 2 MDa). Coupled to the electron transfer, protons are
pumped from the matrix side to the inter-membrane space of mitochondria.
NADH + Q + H+ + nH+in → NAD+ + QH2 + nH+out (n=3-5)
This enzyme complex contains a noncovalently-bound
FMN molecule and 2 binuclear and 6 tetranuclear
iron-sulfur clusters. The initial electron transfers are:
NADH + H+ + FMN → NAD+ + FMNH2
FMNH2 + (Fe-S)ox → FMNH· + (Fe-S)red + H+
Thereafter Fe-S is reoxidized by transfer of the electron to the next iron-sulfur center in the pathway:
FMNH· + (Fe-S)ox → FMN + (Fe-S)red + H+
packed as a trimer. The complex II prosthetic
group FAD is reduced to FADH2 during oxidation
of succinate to fumarate. FADH2 is reoxidized by
transfer of electrons through a series of three Fe-S
centers to Coenzyme Q, yielding QH2.
X-ray crystallographic analysis of related bacterial
electron transfer complexes indicates a linear
arrangement of these electron carriers across the
membrane consistent with the predicted sequence
of electron transfers:
FAD → FeScenter 1 → FeScenter 2 → FeScenter 3
→ CoQ
There are other non-membrane bound complexes
that fulfill similar tasks as complex II
Electrons pass through a series of iron-sulfur centers
in complex I, and are eventually transferred to
coenzyme Q. Coenzyme Q accepts 2 e− and picks
M. Lazarou et al. / Biochimica et Biophysica Acta
up 2 H+ to yield the fully reduced QH2.
1793 (2009) 78–88
Research on complex I has recently taken on greater significance since the finding that many human
mitochondrial diseases involve structural and functional defects at the level of this enzyme complex.
“Isolated complex I deficiency is the most common cause of respiratory chain dysfunction”
(1st sentence in abstract Lazarou et al.)
Bacterial complex II analogue: (QFR)
Menaquinol oxidation by Wolinella succinogenes QFR. The prosthetic groups of the W.
succinogenes QFR dimer are displayed (PDB entry 1QLA). Distances between prosthetic
groups are edge-to-edge distances in Å. Drawn in red is the side chain of Glu C66.
Menaquinol binding (drawn in green) is modelled. The position of bound fumarate (Fum)
is taken from PDB entry 1QLB.
E. coli
W. succinogenes
Yankoskaya et al., science 2003 According to Lancaster, FEBS 2001
Calculate transfer rates using Duttons rule.
( MK = Menaquinone)
E. Coli complex II
Yankovskaya et al., science 2003
Complex II
Why is heme b present?
E. Coli complex II
Yankovskaya et al., science 2003
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Complex III (bc1 complex)
Complex III (bc1 complex)
Complex III accepts electrons from the coenzyme QH2 that is generated by
electron transfer in complexes I and II. The pathway is called the "Q cycle” and
depends on:
•the mobility of CoQ within the lipid bilayer
•the existence of binding sites for CoQ within bc1 that stabilize the
semiquinone radical, Q·−.
matrix
Q cycle: Electrons enter complex III via QH2, which
binds at a site near the membrane surface facing the
intermembrane space. QH2 gives up an e− to the Rieske
iron-sulfur center (Fe-S), which makes a conformational
change, so Fe-S can be reoxidized by electron transfer to
cytochrome c1, which passes the electron out of the
complex to cytochrome c. Loss of one e− to the Fe-S
complex, and release of 2 H+ to the intermembrane space,
generates a Q·− radical. Q·− becomes Q as it gives up the
second electron that is transferred to the other side of the
membrane via hemes bL and bH.
It takes 2 cycles for CoQ, bound at a site near the matrix
side of the membrane, to be reduced to QH2, as 2
electrons are transferred from the b hemes and 2 H+ are
extracted from the matrix compartment. In 2 cycles, 2
QH2 enter the pathway, and one is regenerated.
intermembrane
space
Complex III (bc1 complex)
-77±11mV
51±7mV
Complex III (bc1 complex)
257±4mV
1)
2)
3)
4)
5)
Control
+ myxothiazol
+ antimycin
+ myxothiazol + antimycin
+ antimycin + ascorbate
Berry et al., 1991, JBC, 266, 9064-9077
Reducing titration (open symbols) and re-oxidation
titration (closed symbols) give the mid-potential of the
complex III heme groups.
Berry et al., 1991, JBC, 266, 9064-9077
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http://www.life.uiuc.edu/crofts/bc-complex_site/
Complex III
Three positions of the Rieske Fe/S protein in beef mytochondrial
cyt. bc1 complex
Fe/S protein in its cyt c1
positional state (1BE3)
Fe/S protein in
intermediate location
(1BCC)
From Ed Berry
Complex III
The net Q cycle reaction: QH2 +2H+(matrix)+2 cyt c (Fe3+) Q + 4 H+(out) + 2 cyt c (Fe2+)
Per 2e- transferred through the complex to cytochrome c, 4H+ are released to the intermembrane space.
Fe/S protein in its b
positional state, close to
QP-site (1BGY)
Bovine Complex IV (cytochrome c oxidase)
Subunits I and II of 13 subunits in cytochrome oxidase (complex IV)
contain the metal centers:
Bovine Complex IV (cytochrome c oxidase)
Structure is solved for mitochondrial and bacterial complex:
Intramembrane domains of cytochrome oxidase (complex IV) consist mainly of
transmembrane α-helices.
Cua (2 adjacent Cu-atoms)
heme a
heme a3
Cub
Tsukihara et al. (1995)
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Bacterial Complex IV
Cytochrome oxidase (complex IV) carries out the following irreversible reaction:
H+
O2 + 4
+4
cytochrome c.
e−
→ 2 H2O. The four electrons are transferred into the complex one at a time from
Complex IV
O2 reacts at a binuclear center, consisting of heme a3 and CuB.
For cytochrome c oxidase, the overall
reaction is:
4 ferrocyt c + 4H+N + 4H+N + O2 4
ferricyt c+ + 2H2O + 4H+P
Cytochrome oxidase viewed
from the side.
(Paracoccus denitrificans)
Since cytochrome c is in the P-phase, 8
charges are transfered from N- to P-phase
per oxygen consumed.
Subunit I is yellow
Subunit II is magenta
Subunit III is blue
Subunit IV is in green
The cyan subunit is from the
antibody used to aid
crystallization of the complex.
Iwata et al. (1995)
Complex IV
Summary II
• The respiratory chain is a nanoscale redox machine built of complex I to
IV, which are embedded in a lipid bilayer. These enzymes convert the
redox energy obtained from oxidation of carbohydrates into a proton
gradient
•The energy liberated drives protons uphill against a concentration
gradient and a potential.
• Whereas the structure of complex I is still missing, those of complex IIIV are now resolved to high resolution
CO-bound heme a3-CuB unit
Azide-bound heme a3-CuB unit
(Sodium azide, NaN3 is often
used to keep buffers free of
bacteria)
Yoshikawa et al., 1996, science 280, 1723-1729
• Structures from different states of the reaction cycles of complex II-IV
help to understand the function
• Nanometer scale movements are associated with functional cycles
• Proton pathways are lined with water molecules, but mechanism in
complex I and IV are still not proven
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