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.

erich.gnaiger@i-med.ac.at

http://www.mitophysiology.org/index.php?id=mip-textbook

Pat

hw

ay f

lux

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]

Re

l. in

hib

itio

n 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 ATP

H+

H+

TMPDAscorbate

KCN

Antimycin A

Isolated step in intact isolated mitochondria:

TMPD+Ascorbate

Cyanide titration

Glu+MalTMPD+AscF

lux/

Com

ple

x I

Rel. inhibition of COXKCN concentration [µM]

Re

l. in

hib

itio

n 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 capacity

H+

ATP

F1

O2

aa3bc1Q

ADPH+

H+ H+

DH

Ic

H+

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.

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+

DH

Ic

H+

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+

c

II

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+

c

II

FADH 2

Then we are surprised to find ...

Intact versusPermeabilized Cells

Uncoupled

Intact

0

1

2

3

CellROUTINE uncoupl.

Res

pir

atio

n /

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

c

H+

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

pir

atio

n [p

mo

l·s-1

·10-6

]R

esp

irat

ion

[pm

ol·

s-1·1

0-6]

0

90

180

360

270

0 20 40 60 80Time [min]Time [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 ATP

H+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-

PiPi2-2-

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-CoA

NADHPyruvate-

HCO3-HCO3-

NADHCO2CO2FADH2

H+H+

Citrate3-

H+

PiPi2-2-

PiPi2-2-

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-CoA

NADH

NADH

NADH

NADH

FADH2

HCO3-HCO3-

CO2CO2

Malate2-

Succinate2-

H+H+

Citrate3-

H+

Pyruvate-

PiPi2-2-

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+H+

Malate2-

NH4NH4++

PiPi2-2-

PiPi2-2-

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1:101:000:500:400:300:20

O2 C

on

cen

tra

tio

n 200

160

120

80

40

0 O2 F

low

pe

r ce

lls

250

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

+D

ig

CI

LEAK

Permeab.

CI Substrates

High-Resolution Respirometryin Permeabilized Cells

1:101:000:500:400:300:20

O2 C

on

cen

tra

tio

n 200

160

120

80

40

0 O2 F

low

pe

r ce

lls

250

200

150

100

50

0

Time [h:min]

CeR

endogen.

ROUTINE

CI

+D

D

OXPHOS

+c

c

CI+II

+S

S1

SF

CII

+Rot

Ro

t

+u

F F

ETS

CI

High-Resolution Respirometryin Permeabilized Cells

Am

a

+AmaGMN

GM

+D

ig

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+

NADH

F1

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

CIV

aa3

H+

CIII

bc1

H+

c

H+

Uncoupler

Electron Transport:from Chain to System

QCI

H+

Glutamate

Pyruvate

Malate

Isocitrate

3-Hydroxyacyl CoA

NADH

• Electron Transport Chain

Succinate

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

Co

nce

ntr

atio

n 200

160

120

80

40

0

Res

pir

atio

n[p

mo

l∙s-1

∙10-6

cel

ls]

250

200

150

100

50

0

Time [h:min]3:002:302:001:301:000:300:00

Uncoupling

FCCP

ETS

Olig

om

ycin

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

Per

m. c

ells

[p

mo

l∙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 ETS

R/EL/R

L/E

10.09 0.290.290.32

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

Co

nce

ntr

atio

n 200

160

120

80

40

0

Res

pir

atio

n[p

mo

l∙s-1

∙10-6

cel

ls]

250

200

150

100

50

0

Time [h:min]3:002:302:001:301:000:300:00

Uncoupling

FCCP

ETS

Olig

om

ycin

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

on

cen

tra

tio

n 200

160

120

80

40

0

O2 F

low

pe

r ce

lls

250

200

150

100

50

0

Time [h:min]1:451:301:151:000:450:300:15

ADP

Dig

ito

nin

Glu

tam

ate

+M

ala

te

CI

Ro

t

CII

c ADP

Succinate

CI+II

OXPHOS

Om

y

F

FCCP

CI+II

ETS

uncoupled

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 ETS

R/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

Oligomycin

PuncouplerADP

Glutamate+Malate+Succinate

0.29

Reserve capacity is overestimated 2-fold

R/E

0.58

R/P

The phosphorylation system exerts strong control over OXPHOS in human fibroblasts.

O2 ATP

H+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

on

cen

tra

tio

n 200

160

120

80

40

0

O2 F

low

pe

r ce

lls

250

200

150

100

50

0

Time [h:min]1:451:301:151:000:450:300:15

ADP

Dig

ito

nin

Glu

tam

ate

+M

ala

te

CI

Ro

t

CII

c ADP

Succinate

CI+II

OXPHOS

Om

y

F

FCCP

CI+II

ETS

uncoupled

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

PL0.25

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

PL0.25

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

Per

m. c

ells

[p

mo

l∙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

High-Resolution RespirometryState-of-the-art polarography

www.oroboros.at

OROBOROS INSTRUMENTShigh-resolution respirometry

Oxygraph-2k

FacultyDisclosureStatement

FacultyDisclosureStatement

O2

H+ Ca2+

TPP+ NO

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

p. 33www.oroboros.at/index.php?id=mipnet-publications

Coupling ControlLEAK, OXPHOS, ETS

L: LEAK

P: Oxidative Phosphorylation

E: Electron Transport System

Odra Noel

EL

LEAK Routine ETS

R/EL/R

L/E

199 = 10.09 0.290.290.32

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

0.36

0.93

(0.32)

Substrate

CouplingADP

B: Intact CellsA: Permeabilized CellsControl

Culturemedium

uncouplerR

Oligomycin

PuncouplerADP