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Nitric oxide and other gas
interactions with mitochondria
Guy Brown
Department of Biochemistry,
University of Cambridge
Gases bind to the oxygen binding site of mitochondrial complex IV,
blocking energy production, in the same way as hypoxia.
This may be one way in which our cells regulate their energy production.
It may also be one way in which our body kills pathogens.
However, it may also kill our cells in disease.
NO
CO
HCN
H2 S
I
Mitochondrial
inner
membrane
III
QH2
c
out
IV
O2
H2O
in
NADH
Path of electrons through
Complex IV
• Electrons pass from
c > CuA > a > a3/CuB.
• Consumes 90% of our
oxygen.
• Major generator of
proton motive force.
Figure from Lehninger: Principles of Biochemistry
Gas binding to complex IV
Binuclear centre consists
of heme a3 and CuB.
O2 binds only when both
Fe & Cu reduced.
NO
NO & CO bind when Fe
reduced (Fe2+).
HCN & H2S bind when
Fe oxidised (Fe3+).
CO – CARBON MONOXIDE
O2 haem
haem oxygenase
biliverdin
Fe2+
CO
sGC or
Cyt Ox?
low sensitivity
Vascular relaxation
Synaptic modulation
Anti-inflammatory
Induction of HO-1 causes
small inhibition of cellular
respiration at low O2.
Probably insignificant in
vivo due to CO binding to
haemoglobin.
HCN – HYDROGEN CYANIDE
H2O2
?
Peroxidase in
neutrophils &
neurons?
HCN
catalase
Cyt Ox?
Synaptic modulation
Estimated 5 mM in brain &
1 mM in blood, but may be
bound (e.g. to metHb).
Cellular respiration is half
inhibited by about 10-50
mM cyanide
Probably insignificant in
vivo.
H2S – HYDROGEN SULPHIDE
cysteine
cystathionine beta-synthase
(CBS) or cystathionine
gamma lyase (CSE)
H2S
KATP channels
Cyt Ox?
Vascular relaxation
Synaptic modulation
Heart contractility
Estimated 1-10 mM in aorta.
Cellular respiration is half
inhibited by 10-30 mM.
Lower concentrations are
rapidly oxidised by
mitochondria.
High concentration induce
suspended animation state.
Inhibitor
Mechanism
Ki
kon(M-1 s-1)
koff (s-1)
HCN
Noncompetitive
200 nM
2 x 103
5 x 10-4
H2S
Noncompetitive
200 nM
104
10-3
CO
Competitive
with O2
200 nM
105
2 x 10-2
NO
Competitive
with O2
0.2 nM
108
2 x 10-2
Cooper C.E. & Brown G.C. (2008) The inhibition of mitochondrial cytochrome oxidase by the gases carbon
monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: J. Bioenergetics & Biomembranes
Inhibitor
Mechanism
Ki
Light
sensitive
In vivo
nM
HCN
Noncompetitive
200 nM
No
< 1000
H2S
Noncompetitive
200 nM
No
< 1000
CO
Competitive
with O2
200 nM
Yes
< 1000
NO
Competitive
with O2
0.2 nM
Yes
< 100
Summary
NO & CO inhibition is rapidly reversed by O2, light or Hb.
HCN & H2S inhibition is slow & insensitive to O2, light or Hb.
It is unclear whether gas inhibition is significant in vivo.
HCN
H2 S
H2 S
I
Mitochondrial
inner
membrane
III
QH2
c
NO
CO
out
IV
O2
H2O
in
NADH
Nitric oxide (NO) and its derivatives have 3 major effects on mitochondria:
1. NO inhibition of cytochrome oxidase
2. SNO inactivation of complex I
3. ONOO- activation of permeability transition.
NO
cGMP
ONOO-
NO
SNO
c
eI
PTP
NADH
QH2
out
e-
O2
H2O
III
IV
Mitochondrial inner membrane
in
Sources of nitric oxide (NO)
Ca2+ ˜P
eNOS
Ca2+
nNOS
Cytokines,
pathogens
NO
iNOS
mtNOS?
Targets/reactions of nitric oxide (NO)
Fe2+-NO
Iron nitrosyl
ONOO-
Peroxynitrite
Fe2+
O2O2
NO
RS
.
nitration
NO2
R-NO2+
N2O3
nitrosation
RSNO
S-nitrosothiol
Function of NO:
“The double-edged sword”
Ca2+/Calmodulin
Phosphorylation
eNOS
nNOS
Smooth muscle relaxation
Neuromodulation
Platelet aggregation
Soluble guanylate
Low,
cyclase
transient
levels
Cytokines
Pathogens
Inflammation
iNOS
High,
sustained
levels
?
Pathogen & Host:
Cell Death/Cytostasis
Function of NO:
“The double-edged sword”
Ca2+/Calmodulin
Phosphorylation
eNOS
nNOS
Smooth muscle relaxation
Neuromodulation
Platelet aggregation
Soluble guanylate
Low,
cyclase
transient
levels
Cytokines
Pathogens
Inflammation
iNOS
High,
sustained
levels
Mitochondria?
Pathogen & Host:
Cell Death/Cytostasis
Function of NO:
“The double-edged sword”
Ca2+/Calmodulin
Phosphorylation
eNOS
nNOS
Smooth muscle relaxation
Neuromodulation
Platelet aggregation
Soluble guanylate
Low,
cyclase
transient
levels
Biogenesis
Protection
Cytokines
Pathogens
Inflammation
iNOS
High,
sustained
levels
Mitochondria?
Pathogen & Host:
Cell Death/Cytostasis
Exercise, Cold, Calorie restriction
NO
AMP cGMP cAMP
PGC-1
NRF-1
mtTFA
Nuclear mt genes
mtDNA transcription
& replication
More mitochondria
Brown, G. C. (2007) Nitric oxide and mitochondria. Front. Biosci. 12, 1024-33.
NO effects on mitochondria:
• Inhibition of respiration
• O2-, H2O2 & ONOO- production
• Mitochondrial permeability transition
Cytochrome oxidase is
inhibited by NO
•Inhibition is rapid.
•Inhibition is potent.
•Inhibition is reversible when
NO gone.
•Inhibition reversible by light.
•Inhibition at oxygen binding
site in competition with O2.
Brown, G. C. & Cooper, C. E. (1994) Nanomolar concentrations of
nitric oxide reversibly inhibit synaptosomal respiration by competing
with oxygen at cytochrome oxidase. FEBS Lett. 356, 295-298.
NO inhibition of
cytochrome oxidase is
competitive with O2,
raising the KM of
respiration into the
physiological range.
Brown, G. C. & Cooper, C. E. (1994) Nanomolar concentrations of
nitric oxide reversibly inhibit synaptosomal respiration by competing
with oxygen at cytochrome oxidase. FEBS Lett. 356, 295-298.
eNOS regulates cellular
respiration and its
sensitivity to oxygen in
cultured endothelial cells
NO may be a
physiological regulator of
respiration and its affinity
for oxygen
Clementi, E., Brown, G. C., Foxwell, N. & Moncada, S. (1999) On the
mechanism by which vascular endothelial cells regulate their oxygen
consumption. Proc. Natl. Acad. Sci. USA 96, 1559-1562.
Aortic endothelial cells activated with LPS+IFNg produce
NO from iNOS that continuously inhibits respiration until
reversed by oxyhaemoglobin.
Cells
Borutaite, V., Matthias, A., Harris, H., Moncada, S. & Brown, G. C. (2001) Reversible inhibition of
cellular respiration by nitric oxide in vascular inflammation. Am. J. Physiol. 281, H2256-H2260.
Oxyhemoglobin
50 mM O2
Oxygen trace
0.4 mM NO
NO trace
2 min
Respiratory rate, nmol O 2/min/106 cells
The oxygen consumption of aortic endothelial cells is
inhibited by LPS/IFNg-induced iNOS induction, and reversed
by the NO scavenger haemoglobin
5
4
#
3
2
1
0
*
Control
LPS/interferon-g
+HbO2
Borutaite, V., Matthias, A., Harris, H., Moncada, S. & Brown, G. C. (2001) Reversible inhibition of
cellular respiration by nitric oxide in vascular inflammation. Am. J. Physiol. 281, H2256-H2260.
Activated astrocytes reversibly inhibit cellular respiration via NO
Brown, G. C., Bolanos, J. P., Heales, S. J. R. & Clark, J. B. (1995) Nitric oxide produced
by activated astrocytes rapidly and reversibly inhibits cellular respiration. Neuroscience
Lett. 193, 201-204.
Cytokine-activated
macrophages express
iNOS and reversibly
inhibit the respiration
of co-incubated cells.
Brown, G. C., Foxwell, N. & Moncada, S.
(1998) Transcellular regulation of cell
respiration by nitric oxide generated by
activated macrophages. FEBS Lett.439,
321-324.
NO
iNOS
MACROPHAGE
O2
TUMOUR
CELL
Outer
membrane
I
Inner
II
III
IV
ATPase
membrane
mitochondria
Death?
Apoptosis or
Necrosis?
NO sensitizes isolated aorta to
hypoxia-induced necrosis
LDH activity, mmol/min/mg wet weight
8
Control
DETA/NO
Hypoxia
Hypoxia + DETA/NO
6
4
2
0
0
1
2
3
4
Time, hours
Borutaite, V., Moncada, S. & Brown, G. C. (2005) Nitric oxide from inducible nitric oxide synthase
sensitizes the inflamed aorta to hypoxic damage via respiratory inhibition. Shock 23, 319-323.
The oxygen consumption of aortic rings is inhibited and
oxygen-dependent after iNOS induction by LPS+IFNg
a
b c
d
1 mM NO
1 mM NO
25 mM O2
5 min
LPS/IFNg
+1400W
Control
+LPS/IFNg
Control
Borutaite, V., Moncada, S. & Brown, G. C. (2005) Nitric oxide from inducible nitric oxide synthase
sensitizes the inflamed aorta to hypoxic damage via respiratory inhibition. Shock 23, 319-323.
LDH, mmol/min/mg ww
NO produced by iNOS sensitizes aorta
to hypoxia
Control
Hypoxic
Activated
Activated-Hypoxic
Act-hypoxic+1400W
1
0
2
3
4
Time, hours
Borutaite, V., Moncada, S. & Brown, G. C. (2005) Nitric oxide from inducible nitric oxide synthase
sensitizes the inflamed aorta to hypoxic damage via respiratory inhibition. Shock 23, 319-323.
•
NO reversibly inhibits mitochondrial respiration
at cytochrome oxidase.
•
NO inhibition is competitive with O2, raising
the Km of respiration into physiological range.
•
NO from iNOS may sensitise tissues to hypoxia.
Hypoxia
Glycolysis
IMMUNE CELL
iNOS
iNOS
TISSUE CELL
CELL
DEATH
NO
Mitochondrial
Cytochrome oxidase
N
ADAPTION
/REACTION
Relevant to: inflammation, sepsis, ischaemia, cancer,
atheroschlerosis, neurodegeneration?
Do NO and hypoxia synergise to kill neurons?
NO donor DETA/NO synergises with hypoxia (2% O2)
to induce ‘apoptosis’ in CGC neurons
90
***
80
CHROMATIN CONDENSATION (%)
NORMOXIA
NORMOXIA
HYPOXIA
HYPOXIA
70
60
50
Mander, P., Borutaite, V., Moncada,
S. & Brown G. C. (2005) Nitric
oxide from glial iNOS sensitizes
neurons to hypoxic death via
mitochondrial respiratory inhibition.
J. Neurosci. Res. 79, 208-215.
40
30
20
10
###
*
0
CONTROL
CONTROL
100uM
NOC-18
MK801100mM
+ 100uM NOC-18
100mM
DETA/NO
DETA/NOD-OG + NOC-18
+MK801
10mM D
NO/HYPOXIA INDUCE NEURONAL DEATH
HYPOXIA ALONE
HYPOXIA + DETA/NO
HYPOXIA + DETA/NO + DEOXYGLUCOSE
NO donors lower cellular ATP in presence of glucose,
but completely deplete ATP in absence of glucose.
NO donor DETA/NO synergises with hypoxia (2% O2)
and deoxyglucose to induce necrosis in CGC neurons
100
***
90
NORMOXIA
HYPOXIA
PI POSITIVE NEURONS (%)
80
70
60
50
*
40
***
30
20
###
10
0
CONTROL
CONTROL
100uM NOC-18
MK801 + 100uM NOC-18
D-OG + NOC-18
100mM
DETA/NO 100mM
DETA/NO 100mM
DETA/NO
+MK801
+DeoxyGlucose
10mM D-OG
DeoxyGlucose
NO from iNOS (induced by LPS/IFNg) synergises with
hypoxia (2% O2) to induce necrosis in CGC neurons
40
***
35
NORMOXIA
HYPOXIA
PI POSITIVE NEURONS (%)
30
25
20
15
#
10
#
5
0
CONTROL
CONTROL
LPS + IFN
LPS/IFNg
1400W + LPS + IFN
LPS/IFNg
+1400W
MK801 + LPS + IFN
LPS/IFNg
+MK801
NO completely but reversibly
inhibits neuronal respiration
at cytochrome oxidase
NO from activated astrocytes
reversibly inhibits neuronal
respiration
Bal-Price, A. & Brown, G. C. (2001) Inflammatory neurodegeneration mediated by nitric oxide from activated glia, inhibiting
neuronal respiration, causing glutamate release and excitoxicity. J. Neuroscience 21, 6480-6491.
Hypoxia induces neuronal death via inhibiting
cytochrome oxidase resulting in excitotoxity
NO
HYPOXIA
NEURON
mito
N
NMDAR
GluT
2+
Ca
Glutamate
Important in stroke and dementia?
NO causes rapid depletion of ATP in
neuronal but not in astrocytic cultures
astrocyte culture
neuronal cultures
100
ATP level (% of control)
ATP level (% of control)
100
80
60
40
20
80
60
40
20
0
0
5
10
(min)
20
4
(hrs)
24
5
30
(min)
1
4
16
(hrs)
Time of exposure to NO donor DETA/NO (500 mM)
24
Rapid release of glutamate from neuronal cultures induced by an NO
donor DETA/NO and respiratory inhibitor myxothiazol
20
(mM)
(mM)
20
16
Glutamate
Glutamate
12
8
4
0
0
1
5 10 30
min
4
15
10
5
0
24
hrs
0
1
5
10
30
min
Time of exposure to DETA/NO (500 mM)
24
hrs
Time of exposure to myxothiazol (2 mM)
•Release is rapid. Over concentration range as inhibits respiration.
•Other respiratory inhibitors (e.g. cyanide) cause release.
•Release greater at low O2.
4
Calcium and cGMP independent.
nNOS is activated by NMDA receptor and might
contribute to hypoxic death by sensitising cytochrome
oxidase
HYPOXIA
mito
N
NO
nNOS
NMDAR
GluT
Glutamate
2+
Ca
Summary:
•
NO reversibly inhibits mitochondrial respiration
at cytochrome oxidase.
•
NO inhibition is competitive with O2, raising
the Km of respiration into physiological range.
•
NO from iNOS may sensitise cells to
hypoxic/ischaemic death.
•
Glycolytic capacity determines sensitivity and
form of cell death.
•
Relevant in ischaemic, infectious, inflammatory
and neurodegerative diseases.
Outer
membrane
Inner
I
O2
-
II
III
IV
ATPase
membrane
peroxynitrite
GS.
S-nitroso-glutathione
mitochondria
NO inactivates complex I
DETA/NO
+DTT,
GSH
or light
+BSO
-Hb
Incubation of cells with an NO
donor (0.5mM DETA/NO) for
hours results in inactivation of
complex I and respiration.
The inactivation is speeded by
depleting cellular GSH with BSO.
The inactivation is reversed by
DTT, GSH methylester or light.
The inactivation may be due to
nitrosation of complex I thiol.
Clementi, E., Brown, G. C., Feelisch, M. & Moncada, S. (1998)
Persistent inhibition of cell respiration by nitric oxide: Crucial role
of S-nitrosylation of mitochondrial complex I and protective action
of glutathione. Proc. Natl. Acad. Sci. 95, 7631-7636.
NO + Ca2+, peroxynitrite or S-nitrosothiols
cause inhibition of complex I and this
inhibition is reversed by light and thiols
Complex I activity, % of the control
160
140
120
NO+Ca
100
2+
SNAP
ONOO -
80
60
40
20
0
Control
+GSH +DTT +light
NO+Ca or NO donor
Borutaite, V., Budriunaite, A. & Brown, G.
C. (2000) Reversal of nitric oxide-,
peroxynitrite- and S-nitrosothiol-induced
inhibition of mitochondrial respiration or
complex I activity by light and thiols.
Biochim. Biophys. Acta 1459,405-412.
S-nitrosothiol inactivation of complex I reversibly
increases H2O2 production by mitochondria
Rate of H2O2 production, RFU/min/mg
25
20
15
10
5
0
Control
SNAP
SNAP
+light
Rotenone
Borutaite, V. & Brown, G. C. (2006) S-nitrosothiol inhibition of mitochondrial complex I causes a reversible increase in
mitochondrial hydrogen peroxide production. Biochim. Biophys. Acta 1757, 562-6.
ONOORSNO
O2 NO
NO
RSH
I
NO2
NO
NO NOIII
NO
c
out
IV
O2
QH2
N2 O 3
RSNO NADH
in
-
-
O2 NO O2
ONOOe- aconitase
H2O
O2
-
O2
ONOO-
H2O2
Mn-SOD
Brown, G. C. (2007) Nitric oxide and mitochondria. Front. Biosci. 12, 1024-33.
NO causes H2O2 production in isolated
mitochondria
H2O2, nmol/min/mg
0.05
0.04
0.03
0.02
0.01
0.00
Control
+1 mM NO
+4 mM NO
Borutaite, V. & Brown, G. C. (2003) Nitric oxide induces apoptosis via hydrogen peroxide,
but necrosis via energy and thiol depletion. Free Rad. Biol. Med. 35, 1457-68.
• NO increases oxidative
stress in cells (DCF).
• NO can induce apoptosis
via H2O2.
• Cells subsequently die by
necrosis preceded by
energy depletion.
Caspase DEVDase activity
NO-induced apoptosis is
mediated by H2O2
0.3
0.2
*
0.1
*
*
0.0
Control
+Ascorbate +NAC +Catalase
DETA/NO donor
Borutaite, V. & Brown, G. C. (2003) Nitric oxide induces apoptosis via hydrogen peroxide,
but necrosis via energy and thiol depletion. Free Rad. Biol. Med. 35, 1457-68.
APOPTOSIS
APAF1
CASPASE-3
CASPASE-9
BAX
Outer
cytosol
VDAC
membrane
Cyt.c
Inner
membrane
I
O2
-
II
III
IV
CrK
Permeability
ANT
transition
ATPase
pore
CP
peroxynitrite
GS.
S-nitroso-glutathione
mitochondria
Cytochrome c, nmol/mg protein
Nitrosothiols and peroxynitrite induce opening
of permeability transition pore and release of
cytochrome c in isolated mitochondria
0.08
0.06
0.04
0.02
0.00
+CsA
Control
NOC-18
GSNO
+CsA
ONOO-
Nitrosothiols-induced activation of caspases
is blocked by cyclosporin A
DEVDase, nmol/min/mg
0.30
NO donor
+CsA
0.25
0.20
0.15
*
0.10
0.05
0.00
Control
GSNO
NOC-18
Borutaite, V. & Brown, G. C. (2003) Nitric oxide induces apoptosis via hydrogen peroxide,
but necrosis via energy and thiol depletion. Free Rad. Biol. Med. 35, 1457-68.
NO actions on mitochondria
relevant to cell death
Outer
membrane
Inner
membrane
I
MnSOD
• Respiratory inhibition at
complexes I & IV.
BH3
VDAC
• Stimulation of oxidant
CrK PTP
Cyt.c
production.
• Induction of permeability
III IV
II
ATPase ANT
transition.
CP
• NO can induce cell death
by each of these means.
Aconitase
Collaborations:
• Salvador Moncada
Vilma Borutaite
Aiste Jekabsone
• Emilio Clementi
• Aviva Tolkovsky
Palwinder
Mander
Anna Price