MiPschool 2008Schröcken, July 2008
Mitochondrial Respiratory Physiology.
Erich GnaigerMedical University Innsbruck, Austria
Mitochondrial respiratory control: Electron transport system, oxidative phosphorylation and leak – ETS, OXPHOS and LEAK.
http://www.mitophysiology.org/index.php?id=mip-textbook
Path
way
flux
Enzymatic defect0.0 0.5 1.0
0
1
2
Excess Capacityand Biochemical Threshold
Threshold
Biochemical threshold:Cellular function is buffered against a specific enzymatic defect.
Excess capacityExcess capacity:Insurance against a specific enzymatic injury.
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH
Different excess capacities imply tissue-specific (in)sensitivity to enzymatic defects in:• genetic mitochondrial disorders• aging• ischemia-reperfusion injury• degenerative diseases
Excess Capacityand Biochemical Threshold
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH
KCN concentration [µM]
Rel
. inh
ibiti
on o
f CO
X
0 10 200.00
0.25
0.50
0.75
1.00
Cytochrome c Oxidase
KCN Titration
caa3
F1
O2 ADP ATPH+
H+
TMPDAscorbate
KCN
Antimycin A
Isolated step in intact isolated mitochondria:
TMPD+Ascorbate
Cyanide titration
Glu+MalTMPD+AscFl
ux/C
ompl
ex I
Rel. inhibition of COXKCN concentration [µM]
Rel
. inh
ibiti
on o
f CO
X
0 10 200.00
0.25
0.50
0.75
1.00
0.00 0.25 0.50 0.75 1.000.0
0.5
1.0
1.5
2.0
2.5
Electron Transport Chainand Cytochrome c Oxidase
Excess capacityH+
ATP
F1
O2
aa3bc1Q
ADPH+
H+ H+
DHI
cH+
NADHGlutamate+Malate
0.5 mM TMPD + 2 mM ascorbate: 2-fold relative COX capacity
Excess capacity
Electron Transport System
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
A. Definition of ETS capacity.B. Measurement in mitochondria and
permeabilized cells.C. Measurement in intact cells.
Which metabolic state represents electron transport capacity?
Conventional ProtocolDerived from Bioenergetics
Electron Transport Chain
Bioenergetic paradigm (1): Respiratory capacity in State 3, feeding electrons specifically into
complex I
H+ATP
F1
O2
aa3bc1Q
ADPH+
H+ H+
DHI
cH+
NADH
Conventional ProtocolDerived from Bioenergetics
Electron Transport Chain
Bioenergetic paradigm (1): Respiratory capacity in State 3, feeding electrons specifically into
complex I, or complex II
H+ATP
F1
O2
aa3bc1Q
ADPH+
H+ H+
cII
FADH 2
Conventional ProtocolDerived from Bioenergetics
Electron Transport Chain
Bioenergetic paradigm (1): Respiratory capacity in State 3, feeding electrons specifically into
complex I, or complex II (rotenone+succinate)
H+ATP
F1
O2
aa3bc1Q
ADPH+
H+ H+
cII
FADH 2
Then we are surprised to find ...
Intact versusPermeabilized Cells
Uncoupled
Intact
0
1
2
3
CellROUTINE uncoupl.
Res
pira
tion
/ GM
AD
P
Permeabilized
State 3GMADP
In permeabilized cells, State 3 respiration (Glutamate+Malate) is short of representing respiratory capacity of intact uncoupled cells.
ATP
F1
O2
aa3bc1Q
ADPH+
H+ H+
IIDH
NADH
I
Succinate
cH+
GM H+
FCCP
Fibroblasts NIH3T3
Coupled
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH
Contoversy on Isolated Mitochondria
• 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.
• Villani, Attardi (1997) PNAS 94: 1166.
But low COX excess in intact cells „raises the critical issue of how accurately the data obtained with isolated mitochondria reflect the in vivo situation“.
COX excess capacity is high in isolated mitochondria, with corresponding phenotypic threshold.
ControversyLiving cells vs isolated mitochondria
Bioenergetic paradigm (2) of substrate/uncoupler combinations which yield maximum flux in:
• Isolated mitochondria: Rasmussen et al (2001) AJP
• Intact cells: Villani and Attardi (1997) PNAS• Permeabilized muscle fibers: Kunz et al (2000) JBC
Oxidative Phosphorylation in Top Gear
Gold standard to assess maximum aerobic capacity in cultured cells:
→Uncoupled flux
• Villani, Attardi (1997) PNAS 94: 1166
Res
pira
tion [p
mol
·s-1·1
0-6]
0
90
180
360
270
0 20 40 60 80Time [min]
RoutineAma
Oligo-mycin
FCCPRot
But intact cells do not have uncoupled mitochondria !
Oxidative Phosphorylation in Top Gear – Mitochondrial Physiology
Gold standard to assess maximum aerobic capacity in humans:
→VO2 max
Electron Transport Coupled to ATP Synthesis
Oxidative Phosphorylation:Coupling
O2 ATPH+H+
O2
Δp
ATP
OXPHOS andRespiratory Capacity
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH
Q
Linear
O2
aa3
H+
bc1
H+
cNADH I
H+
Mitochondrial PathwaysConvergent Redox and ET System
Coupled
OXPHOS
F1
H+H+
ADP
ETFβ-Oxidation
Convergent
II
Succinate
4 : 1
SDH
II
Glycolysis
Convergent
GDH
ODH
IDH
MDH
PDH
GpDHGp
p. 24www.oroboros.at/index.php?id=mipnet-publications
Question 1
How do we measure mitochondrial electron transport capacity?
A. MitochondriaB. Intact cells
ETS O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
Succinate2-
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
MitoPathwaysSuccinate + Rotenone
Malate2-
Fumarate2-
Pi2-
Pi2-
II
S F
I
Succinate2-
II
FADH2
Oxaloacetate2-
NADH
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Succinate2-
Malate2-
Malate2-
Pyruvate-
H+
Malate2-
MitoPathwaysPyruvate+Malate+Succinate, PMS
Oxaloacetate2-
Malate2-
Fumarate2- 2-Oxoglutarate2-
NADH
NADH
Succinate2-
Acetyl-CoANADHPyruvate-
HCO3-
NADHCO2FADH2
H+
Citrate3-
H+
Pi2-
Pi2-
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH
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Malate2-
Complex II is not active in respiration on pyruvate + malate.Pi
2-
Pyruvate-
H+
Malate2-
Oxaloacetate2-
Malate2-
Fumarate2- 2-Oxoglutarate2-
Acetyl-CoANADH
NADH
NADH
NADHFADH2
HCO3-
CO2
Malate2-
Succinate2-
H+
Citrate3-
H+Pyruvate-
Pi2-
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
I
H+
NADH
MitoPathwaysPyruvate+Malate, PM
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Succinate2-
MitoPathwaysGlutamate+Malate+Succinate, GMS
Oxaloacetate2-
Malate2-
Fumarate2-
NADH
NADHFADH2
Glutamate-
Aspartate-
Glutamate-
2-Oxoglutarate2-
Glutamate-
Succinate2-Glutamate-
H+
Malate2-
H+
H+
CO2
NADH
H+
Malate2-
NH4+
Pi2-
Pi2-
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1:101:000:500:400:300:20
O2 C
once
ntra
tion 200
160
120
80
40
0 O2 F
low
per
cel
ls250
200
150
100
50
0
Time [h:min]endogen.
ROUTINE
+c
c
Cytochrome c test: Intact mitochondrial outer membrane
+uncoupler
F F
ETS
CICI
+ADP
ADP
OXPHOS
GMN
GM
+Dig
CI
LEAK
Permeab.
CI Substrates
High-Resolution Respirometryin Permeabilized Cells
1:101:000:500:400:300:20
O2 C
once
ntra
tion 200
160
120
80
40
0 O2 F
low
per
cel
ls250
200
150
100
50
0
Time [h:min]
CeR
endogen.
ROUTINE
CI
+D
D
OXPHOS
+c
c
CI+II
+S
S1
SF
CII
+Rot
Rot
+u
F F
ETS
CI
High-Resolution Respirometryin Permeabilized Cells
Am
a
+AmaGMN
GM
+Dig
CI
LEAK
PC
ETS capacity with CI+II substrates
Maximum electron transport capacity is obtained with convergent CI+II electron input.
Reference State
1
O2
aa3
H+
bc1
H+
Qc
I
H+
NADH
F1
H+H+II
S F
FADH2
CI+II:
ETS O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
With CI substrates, respiration is limited to 0.70 of ETS capacity.
Q-Junction Ratio
1
0.70
O2
aa3
H+
bc1
H+
Qc
I
H+
NADHF1
H+H+ CI+II:
CI:
ETS O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
With CII substrates, respiration is limited to 0.36 of ETS capacity.
Q-Junction Ratio
0.36
1
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S FFADH2
CI+II:
CII:
ETS O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
Convergent CI+II electron input exerts an additive effect in human fibroblasts.
Q-Junction Ratio
0.36
1
0.70
O2
aa3
H+
bc1
H+
Qc
I
H+
NADH
F1
H+H+II
S F
FADH2
CI+II:
CI:
CII:
ETS O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
O2
CIVaa3
H+
CIIIbc1
H+
c
H+
Uncoupler
Electron Transport:from Chain to System
QCI
H+
Glutamate
Pyruvate
Malate
Isocitrate
3-Hydroxyacyl CoA
NADH
• Electron Transport ChainSuccinate
CII
?
Fatty acyl CoA
?
3-Glycerol phosphate
?• Electron Transport System, ETS
Convergent Electron Flux and the Q-junction
www.oroboros.at/index.php?id=mipnet-publications
The most frequent misnomer in bioenergetics:
Electron Transport Chain
E
ETS
Question 1
How do we measure mitochondrial electron transport capacity?
A. MitochondriaB. Intact cells
ETS O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
O2 C
once
ntra
tion 200
16012080400
Res
pira
tion
[pm
ol∙s
-1∙1
0-6 c
ells
]250200150100500
Time [h:min]3:002:302:001:301:000:300:00
Uncoupling
FCCP
ETS
Olig
omyc
in
LEAKROUTINE
Cells
Fibroblasts NIH3T3
High-Resolution Respirometryin Intact Cells
Gnaiger E (2008) In: Mitochondrial Dysfunction in Drug-Induced Toxicity. (Dykens JA, Will Y, eds) John Wiley.
Mitochondrial Pathwaysand Q-Junction
0 50 100 150 200 2500
50
100
150
200
250
Perm
. cel
ls [p
mol
∙s-1∙1
0-6 c
ells
]
Intact cells [pmol∙s-1∙10-6 cells]
CI+II: Glutamate+Malate+Succinateuncoupled
ETS capacities were identical in intact and permeabilized cells, with convergent electron flow through Complexes I and II (CI+II e-input).
ETS
Identical ETS Capacity in Permeabilized and Intact Cells
EL
LEAK ROUTINE ETSR/EL/R
L/E
10.09 0.29 0.290.32
0.09CI+II combined
E
ETS
1
Coupling
B: Intact CellsA: Permeabilized Cells
Control
Culture medium
uncouplerR
Oligomycin
186 199
pmol∙s-1∙10-6 cells
O2 C
once
ntra
tion 200
16012080400
Res
pira
tion
[pm
ol∙s
-1∙1
0-6 c
ells
]250200150100500
Time [h:min]3:002:302:001:301:000:300:00
Uncoupling
FCCP
ETS
Olig
omyc
in
LEAKROUTINE
State 3?
OXPHOS Capacity ?
O2 ATP
H+H+
ADP
Cells
Fibroblasts NIH3T3
High-Resolution Respirometryin Intact Cells
Question 2
How do we measure OXPHOS capacity?
A. MitochondriaB. In intact cells ?
OXPHOS
O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
O2 C
once
ntra
tion 200
160
120
80
40
0 O2 F
low
per
cel
ls250
200
150
100
50
0
Time [h:min]1:451:301:151:000:450:300:15
ADP
Dig
itoni
n
Glu
tam
ate
+Mal
ate
CI
Rot
CII
c ADPSuccinate
CI+II
OXPHOS
Om
y
FFCCP
CI+II
ETSuncoupled
OXPHOS capacity is less than ETS
High-Resolution Respirometryin Permeabilized Cells
ADP-stimulated
P
OXPHOS
Flux Control Diagrams for Permeabilized and Intact Cells
EL
LEAK ROUTINE ETSR/EL/R
L/E
10.09 0.290.32
0.09CI+II combined
EL
LEAK OXPHOS ETS
L/E
P/E
L/P
0.50 10.100.20
0.10
Coupling
B: Intact CellsA: Permeabilized Cells
Control
Culture medium
uncouplerR
OligomycinP
uncouplerADP
Glutamate+Malate+Succinate
0.29Reserve capacity is overestimated 2-fold
R/E
0.58R/P
The phosphorylation system exerts strong control over OXPHOS in human fibroblasts.
O2 ATPH+H+
P E0.50
OXPHOS O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
Question 3
How do we express respiratory coupling ratios?
A. MitochondriaB. Intact cells
LEAK O2
aa3
H+
bc1
H+
Qc
F1
H+H+
II
S F
I
H+
NADH ADP ATP
III IV
O2 C
once
ntra
tion 200
160
120
80
40
0 O2 F
low
per
cel
ls250
200
150
100
50
0
Time [h:min]1:451:301:151:000:450:300:15
ADP
Dig
itoni
n
Glu
tam
ate
+Mal
ate
CI
Rot
CII
c ADP
Succinate
CI+II
OXPHOS
Om
y
F
FCCP
CI+II
ETSuncoupled
L/E ratio but not L/P ratio reflects the relative LEAK.
High-Resolution Respirometryin Permeabilized Cells
ADP-stimulated
CI
ETS Capacity versus OXPHOS Capacity
Permeabilized Cells, NIH3T3 Fibroblasts
Glutamate+Malate
EL
LEAK OXPHOS ETS
L/E
P/EL/P
PL 0.25 E0.55
Substrate
Coupling ADP
Control PuncouplerADP
Limitation by the phosphorylation system
4.0
Respiratory Control Ratio (State 3/State 4)is the inverse L/P ratio
ETS Capacity versus OXPHOS Capacity
Permeabilized Cells, NIH3T3 Fibroblasts
Glutamate+Malate
EL
LEAK OXPHOS ETS
L/E
P/EL/P
PL 0.25 E0.55
Substrate
Coupling ADP
Control PuncouplerADP
7.1
Respiratory Control Ratioshould be the inverse L/E ratio
L/E ratio expresses uncoupling
0.14
Mitochondrial Pathwaysand Q-Junction
0 50 100 150 200 2500
50
100
150
200
250
Perm
. cel
ls [p
mol
∙s-1∙1
0-6 c
ells
]
Intact cells [pmol∙s-1∙10-6 cells]
CI+II: GMSE
CI+II: GMSL
CI: GML
CrE
ETS capacities and LEAK respiration were identical in intact and permeabilized cells, with convergent electron flow through Complexes I and II (CI+II e-input)
LEAK
L
LEAK
1. Convergent e-input at the Q-junction corresponds to the
operation of the citric acid cycle.
2. 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
Mitochondrial Respiratory Control: The Q-Junction
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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 CI+II substrates explain apparent discrepancies between mitochondria and intact cells.
Mitochondrial Respiratory Control: The Q-Junction
p. 33www.oroboros.at/index.php?id=mipnet-publications
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.
Mitochondrial Respiratory Control: The Q-Junction
p. 33www.oroboros.at/index.php?id=mipnet-publications
5. 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 Respiratory Control: The Q-Junction
Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publ. 2007 p. 33www.oroboros.at/index.php?id=mipnet-publications
High-Resolution RespirometryState-of-the-art polarography
www.oroboros.at
OROBOROS INSTRUMENTShigh-resolution respirometryOxygraph-2k
FacultyDisclosureStatement
O2 H+ Ca2+
TPP+ NO
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.
Mitochondrial Respiratory Control: The Q-Junction
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Coupling ControlLEAK, OXPHOS, ETS
L: LEAK
P: Oxidative Phosphorylation
E: Electron Transport SystemOdra Noel
EL
LEAK Routine ETSR/EL/R
L/E
199 = 10.09 0.29 0.290.32
0.09
slow
com-bined
fast
S
GMS
GMS
GM
EL
LEAK OXPHOS ETS
L/E
P/EL/P
0.34
0.50
0.38
0.36(0.12)
186 = 10.10
0.700.090.25
0.98 0.77
0.20
0.68(1.2)(0.35)
0.55
0.14
0.50
0.70
0.10
0.360.93
(0.32)
Substrate
Coupling ADP
B: Intact CellsA: Permeabilized CellsControl
Culturemedium
uncouplerR
OligomycinP
uncouplerADP
