MiPschool 2008 Schröcken, July 2008 Mitochondrial Respiratory Physiology. Erich Gnaiger Medical University Innsbruck, Mitochondrial respiratory control: Electron transport system, oxidative phosphorylation and leak – ETS, OXPHOS and LEAK. [email protected]http://www.mitophysiology.org/index.php?id=mip-textbook
MiP school 2008 Schröcken, July 2008. http://www.mitophysiology.org/index.php?id=mip-textbook. Mitochondrial Respiratory Physiology. Mitochondrial respiratory control: Electron transport system, oxidative phosphorylation and leak – ETS , OXPHOS and LEAK . Erich Gnaiger - PowerPoint PPT Presentation
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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.
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
www.oroboros.at/index.php?id=mipnet-publications
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
www.oroboros.at/index.php?id=mipnet-publications
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
www.oroboros.at/index.php?id=mipnet-publications
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-
www.oroboros.at/index.php?id=mipnet-publications
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
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
www.oroboros.at/index.php?id=mipnet-publications
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
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
p. 33www.oroboros.at/index.php?id=mipnet-publications