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MiPschool 2008 Schröcken, July 2008
http//www.mitophysiology.org/index.php?idmip-tex
tbook
Mitochondrial Respiratory Physiology.
Mitochondrial respiratory control Electron
transport system, oxidative phosphorylation and
leak ETS, OXPHOS and LEAK.
Erich Gnaiger Medical University Innsbruck,
Austria
erich.gnaiger_at_i-med.ac.at
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Excess Capacity and Biochemical Threshold
Pathway flux
Enzymatic defect
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Excess Capacity and Biochemical Threshold
Different excess capacities imply tissue-specific
(in)sensitivity to enzymatic defects in

• genetic mitochondrial disorders
• aging
• ischemia-reperfusion injury
• degenerative diseases

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H
Antimycin A
Cytochrome c Oxidase KCN Titration
aa3
F1
TMPD Ascorbate
O2
ADP
ATP
H
Rel. inhibition of COX
KCN concentration µM
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Electron Transport Chain and Cytochrome c
Oxidase Excess capacity
H
H
H
I
aa3
bc1
Q
F
DH
1
O2
ATP
ADP
NADH GlutamateMalate
H
H
Rel. inhibition of COX
Flux/Complex I
TMPDAsc
GluMal
KCN concentration µM
Rel. inhibition of COX
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Electron Transport System
Which metabolic state represents electron
transport capacity?
A. Definition of ETS capacity. B. Measurement in
mitochondria and permeabilized cells. C.
Measurement in intact cells.
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Conventional Protocol Derived from
Bioenergetics Electron Transport Chain
H
H
H
I
aa3
bc1
Q
F
DH
1
O2
ATP
ADP
NADH
H
H
Bioenergetic paradigm (1) Respiratory capacity
in State 3, feeding electrons specifically into
complex I
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Conventional Protocol Derived from
Bioenergetics Electron Transport Chain
H
H
aa3
bc1
Q
F
1
O2
ATP
ADP
H
H
Bioenergetic paradigm (1) Respiratory capacity
in State 3, feeding electrons specifically into
complex I, or complex II
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Conventional Protocol Derived from
Bioenergetics Electron Transport Chain
H
H
aa3
bc1
Q
F
1
O2
ATP
ADP
H
H
Bioenergetic paradigm (1) Respiratory capacity
in State 3, feeding electrons specifically into
complex I, or complex II (rotenonesuccinate)
Then we are surprised to find ...
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Intact versus Permeabilized Cells
H
H
H
aa3
bc1
Q
II
F
1
Uncoupled
O2
Succinate
Coupled
ATP
ADP
GM
H
Permeabilized
Intact
In permeabilized cells, State 3 respiration
(GlutamateMalate) is short of representing
respiratory capacity of intact uncoupled cells.
Respiration / GMADP
Fibroblasts NIH3T3
GMADP
ROUTINE
uncoupl.
State 3
Cell
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Contoversy on Isolated Mitochondria
COX excess capacity is high in isolated
mitochondria, with corresponding phenotypic
threshold.

• Letellier et al (1994) Biochem. J. 302 171.
• Gnaiger et al (1998) BBA 1365 249
• Rossignol et al (2003) Biochem. J. 370 751.
• Antunes et al (2004) PNAS 101 16774.

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Controversy Living cells vs isolated mitochondria
Bioenergetic paradigm (2) of substrate/uncoupler
combinations which yield maximum flux in

• Intact cells
• Villani and Attardi (1997) PNAS

• Permeabilized muscle fibers
• Kunz et al (2000) JBC

• Isolated mitochondria Rasmussen et al (2001) AJP

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Oxidative Phosphorylation in Top Gear

• Gold standard to assess maximum aerobic capacity
  in cultured cells
• Uncoupled flux

• Villani, Attardi (1997) PNAS 94 1166

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Oxidative Phosphorylation in Top Gear
Mitochondrial Physiology

• Gold standard to assess maximum aerobic capacity
  in humans
• VO2 max
• Electron Transport Coupled to ATP Synthesis

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OXPHOS and Respiratory Capacity
Oxidative Phosphorylation Coupling
O2
ATP
ATP
H
H
O2
?p
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Mitochondrial Pathways Convergent Redox and ET
System
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Question 1
ETS
How do we measure mitochondrial electron
transport capacity?

1. Mitochondria
2. Intact cells

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MitoPathways Succinate Rotenone
Oxaloacetate2-
NADH
Malate2-
Fumarate2-
FADH2
Succinate2-
Succinate2-
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MitoPathways PyruvateMalateSuccinate, PMS
Pyruvate- H
Pyruvate-
Malate2-
NADH
H
Acetyl-CoA
Oxaloacetate2-
H
Citrate3-
NADH
Malate2-
Malate2-
HCO3-
NADH
2-Oxoglutarate2-
Fumarate2-
NADH
Malate2-
CO2
FADH2
Succinate2-
Succinate2-
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MitoPathways PyruvateMalate, PM
Pyruvate- H
Pyruvate-
NADH
Malate2-
H
Acetyl-CoA
Oxaloacetate2-
H
Citrate3-
NADH
Malate2-
Malate2-
HCO3-
NADH
2-Oxoglutarate2-
Fumarate2-
Complex II is not active in respiration on
pyruvate malate.
NADH
FADH2
Succinate2-
CO2
Malate2-
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MitoPathways GlutamateMalateSuccinate, GMS
Glutamate-
H
Malate2-
Glutamate-
H
Oxaloacetate2-
Aspartate-
NADH
Malate2-
2-Oxoglutarate2-
NADH
Fumarate2-
Malate2-
NADH
CO2
FADH2
Glutamate-
Glutamate- H
Succinate2-
H
Succinate2-
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High-Resolution Respirometry in Permeabilized
Cells
Cytochrome c test Intact mitochondrial outer
membrane
O2 Flow per cells
O2 Concentration
Time hmin
endogen.
ROUTINE
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High-Resolution Respirometry in Permeabilized
Cells
ETS capacity with CIII substrates
CeR
O2 Flow per cells
O2 Concentration
Time hmin
endogen.
ROUTINE
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Reference State
ETS
Maximum electron transport capacity is obtained
with convergent CIII electron input.
1
CIII
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Q-Junction Ratio
ETS
With CI substrates, respiration is limited to
0.70 of ETS capacity.
0.70
CI
1
CIII
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Q-Junction Ratio
ETS
With CII substrates, respiration is limited to
0.36 of ETS capacity.
1
CIII
0.36
CII
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Q-Junction Ratio
ETS
Convergent CIII electron input exerts an
additive effect in human fibroblasts.
0.70
CI
1
CIII
0.36
CII
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Electron Transport from Chain to System
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• Electron Transport Chain

• Electron Transport System, ETS

Uncoupler
H
H
c
CIV aa3
CIII bc1
Q
NADH
O2
H
Convergent Electron Flux and the Q-junction
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The most frequent misnomer in bioenergetics
Electron Transport Chain
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ETS
E
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Question 1
ETS
How do we measure mitochondrial electron
transport capacity?

1. Mitochondria
2. Intact cells

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High-Resolution Respirometry in Intact Cells
Fibroblasts NIH3T3
Respiration pmols-110-6 cells
O2 Concentration
Cells
Time hmin
ROUTINE
Gnaiger E (2008) In Mitochondrial Dysfunction in
Drug-Induced Toxicity. (Dykens JA, Will Y, eds)
John Wiley.
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Mitochondrial Pathways and Q-Junction
ETS
CIII GlutamateMalateSuccinate uncoupled
ETS capacities were identical in intact and
permeabilized cells, with convergent electron
flow through Complexes I and II (CIII e-input).
Perm. cells pmols-110-6 cells
Intact cells pmols-110-6 cells
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Identical ETS Capacity in Permeabilized and
Intact Cells
B Intact Cells
A Permeabilized Cells
Control
Coupling
ROUTINE
LEAK
ETS
ETS
R
E
L
E
uncoupler
Oligomycin
Culture medium
CIII combined
0.29
0.32
0.29
1
0.09
1
0.09
186
199
pmols-110-6 cells
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High-Resolution Respirometry in Intact Cells
Fibroblasts NIH3T3
Oligomycin
Respiration pmols-110-6 cells
O2 Concentration
Cells
Time hmin
LEAK
ROUTINE
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Question 2
OXPHOS
How do we measure OXPHOS capacity?

1. Mitochondria
2. In intact cells ?

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High-Resolution Respirometry in Permeabilized
Cells
OXPHOS capacity is less than ETS
uncoupled
ADP-stimulated
OXPHOS
ETS
Glutamate Malate
Omy
O2 Concentration
ADP
O2 Flow per cells
Digitonin
c
ADP
F
Succinate
FCCP
Rot
Time hmin
CI
CII
CIII
CIII
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OXPHOS
P
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Flux Control Diagrams for Permeabilized and
Intact Cells
B Intact Cells
A Permeabilized Cells
Control
Coupling
ROUTINE
LEAK
ETS
LEAK
OXPHOS
ETS
P
R
E
L
E
L
ADP
uncoupler
uncoupler
Oligomycin
GlutamateMalateSuccinate
Culture medium
CIII combined
0.32
0.20
0.29
1
0.09
0.50
1
0.10
0.09
0.10
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OXPHOS
The phosphorylation system exerts strong control
over OXPHOS in human fibroblasts.
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Question 3
LEAK
How do we express respiratory coupling ratios?

1. Mitochondria
2. Intact cells

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High-Resolution Respirometry in Permeabilized
Cells
L/E ratio but not L/P ratio reflects the relative
LEAK.
uncoupled
ADP-stimulated
OXPHOS
ETS
Glutamate Malate
Omy
O2 Concentration
ADP
O2 Flow per cells
Digitonin
c
ADP
F
Rot
Succinate
FCCP
Time hmin
CI
CII
CIII
CIII
CI
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ETS Capacity versus OXPHOS Capacity
Coupling
LEAK
OXPHOS
ETS
P
Control
L
E
ADP
uncoupler
Substrate
P
L
Glutamate Malate
Permeabilized Cells, NIH3T3 Fibroblasts
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ETS Capacity versus OXPHOS Capacity
Coupling
LEAK
OXPHOS
ETS
P
Control
L
E
ADP
uncoupler
Substrate
P
L
Glutamate Malate
Permeabilized Cells, NIH3T3 Fibroblasts
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Mitochondrial Pathways and Q-Junction
LEAK
ETS capacities and LEAK respiration were
identical in intact and permeabilized cells, with
convergent electron flow through Complexes I and
II (CIII e-input)
CIII GMSE
CIII GMSL
CI GML
Perm. cells pmols-110-6 cells
CrE
Intact cells pmols-110-6 cells
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LEAK
L
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Mitochondrial Respiratory Control The Q-Junction

• Convergent e-input at the
• Q-junction corresponds to the operation of the
  citric acid cycle.
• The additive Q-junction effect and
  phosphorylation limitation of OXPHOS reveal an
  unexpected diversity of mitochondrial function.
• Q-junction ratios 0.97 to 0.5

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Mitochondrial Respiratory Control The Q-Junction
3. Interpretation of apparent excess capacities
of ET complexes and of flux control coefficients
is largely dependent on the metabolic reference
state. Higher capacities with CIII substrates
explain apparent discrepancies between
mitochondria and intact cells.
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49
Mitochondrial Respiratory Control The Q-Junction
4. Interpretation of excess capacities of various
components of the respiratory chain and of flux
control coefficients is largely dependent on the
metabolic reference state. Appreciation of the
concept of the Q-junction will provide new
insights into the functional design of the
respiratory chain.
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50
Mitochondrial Respiratory Control The Q-Junction

• The relation between membrane potential and flux
  is reversed when an increase in flux is effected
  by a change in substrate supply.
• MultiSensor O2k TPMP

Mitochondrial Pathways and Respiratory Control.
OROBOROS MiPNet Publ. 2007
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OROBOROS INSTRUMENTS high-resolution
respirometry Oxygraph-2k
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Faculty Disclosure Statement
High-Resolution Respirometry State-of-the-art
polarography
O2 H Ca2 TPP NO
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Mitochondrial Respiratory Control The Q-Junction
6. ROS production and reversed electron flow from
Complex II to Complex I Multiple substrate
supply plays a key role (Capel et al 2005 Garait
et al 2005). The dependence of ROS production on
membrane potential and metabolic state will have
to be investigated further based on the concept
of the Q-junction.
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Coupling Control LEAK, OXPHOS, ETS
P Oxidative Phosphorylation
L LEAK
E Electron Transport System
Odra Noel