NO

1
NO
(Nitric Oxide)
Jon Fukuto Department of Pharmacology UCLA School
of Medicine, CHS jfukuto_at_mednet.ucla.edu
2
EDRF
Endothelium-derived relaxing factor
Robert Furchgott
3
Tissue Bath Studies
4
The Furchgott experiment
http//nobelprize.org/medicine/laureates/1998/illp
res/sandwich.html
5
EDRF is NO - We make NO!
6
NO/sGC/cGMP Pathway
7
Angina
http//nobelprize.org/medicine/laureates/1998/illp
res/medicine.html
8
Pharmacological NO Nitroglycerin
"It sounds like the irony of fate that I have
been prescribed nitroglycerine internally. They
have named it Trinitrin in order not to upset
pharmacists and the public. Your affectionate
friend, A. Nobel"
http//nobelprize.org/medicine/laureates/1998/illp
res/application.html
9
NO activates soluble guanylate cyclase via heme
ligation
10
Guanylate cyclases
Numerous isoforms - both soluble and particulate.
Soluble forms have basal activity but can be
activated by nitric oxide (NO).
11
NO-sGC-cGMP signaling
12
NO biosynthesis
13
NO in the vascular system
14
eNOS
http//www.sigmaaldrich.com/Area_of_Interest/Life_
Science/Cell_Signaling/Pathway_Slides_and_Charts/N
itric_Oxide_Metabolism.html
15
NO in the CNS
Certain neurons make NO via a Ca2-dependent
process. However, the function of NO in the
brain remains unestablished.
16
nNOS

http//www.sigmaaldrich.com/Area_of_Interest/Life_
Science/Cell_Signaling/Pathway_Slides_and_Charts/N
itric_Oxide_Metabolism.html
17
NO in the Immune System
There is no doubt that macrophages make NO.
However the function of NO in immune response is
not established.
18
iNOS
http//www.sigmaaldrich.com/Area_of_Interest/Life_
Science/Cell_Signaling/Pathway_Slides_and_Charts/N
itric_Oxide_Metabolism.html
19
Non-vascular Functions of NO

• NO generated in an immune response from activated
  macrophages. Presumably NO is acting as a
  specific toxin against invading pathogens.
  Mechanism unknown.
• Stimulation of glutaminergic neurons (NMDA)
  results in NO generation. The function of NO in
  this system likely involves cGMP generation. May
  contribute to excitotoxicity associated with
  activation of NMDA receptors.
• Although controversial at this time, NO appears
  to be made in mitochondria, possibly to to
  regulate respiration.
• Inadvertant or excess generation of NO thought to
  be involved in the etiology of numerous diseases
  (tyrosine nitration, thiol modification,
  peroxynitrite chemistry, metal ligation etc.).
• NO may have signaling functions via selective
  thiol modification (discussed later).

20
Nitric Oxide Chemistry
Colorless gas, b.p. -152C Max. water
solubility (0) 3 mM (similar to O2)
21
NO is a free radical
Is NO a good one-electron oxidant?
H-NO bond strength is approximately 50 kcal/mole
(weak) H-OH bond strength is 119 kcal/mole
(strong) H-OOH bond strength is 89
kcal/mole H-CR3 bond strength is 90-104
kcal/mole H-OO bond strength is 47 kcal/mole
(weak) Reaction E0(V) vs NHE
NO,H/HNO -0.5 (unfavorable) O2/O2- -0.33 HO
,H/H2O 2.31 (very favorable) RO,H/ROH 1.6
H3CH2C,H/CH3CH3 1.9 HOO,H/HOOH 1.06
Thus, NO is a lousy one electron oxidant!
22
Nitrogen oxide redox scheme
23
Biological Chemistry of NO
24
Biological Chemistry of NO II
25
Biological Chemistry of NO III
26
Biological Chemistry of NO IV
27
Biological Chemistry of NO VI
28
S-Nitrosothiols
29
Tyrosine
Acidic proton/nucleophilic oxygen (can react with
electrophiles)
Can be oxidized (phenoxyl radical relatively
stable due to resonance into phenyl ring)
Activated by electron-donating hydroxyl for
electrophilic aromatic substitution chemistry
30
Tyrosine Nitration
Peroxynitrite-mediated nitration
31
CO2-Assisted Tyrosine Nitration
32
CO2-Assisted Nitration of Tyrosine II
33
Tyrosine Nitration II
Peroxynitrite-mediated nitration (cont.)
34
Nitrotyrosine generation from NO2- and peroxidase
activity
35
DNA
Cytosine deamination to uracil
Note N2O3 is generated from oxidation of NO
(vide supra)
36
NO and mitochondria
37
NO and hemeproteins cytochrome oxidase
Kinetic constants for the interaction of NO with
ferrous iron in hemeproteins Protein On rate
(M-1s-1) Off rate (s-1) Kd (M) T1/2
(20C) Hemoglobin (R) 2 x 107 1.8 x 10-5 9
x 10-13 11 hrs. Myoglobin 1.7 x 107 1.2 x
10-4 7 x 10-12 1.6 hrs. Cytochrome oxidase 1 x
108 1.3 x 10-1 1.3 x 10-9 5 sec. sGC (GTP) 7
x 108 5 x 10-2 7 x 10-11 14 sec.
NO is reported to be made in the mitochondria by
mtNOS (controversial) and able to regulate
respiration by binding to cytochrome oxidase
(cytochrome a3). It has also bee reported that
NO can bind to the CuB site of CcO resulting in
inhibition.
38
Nitrofatty acids
39
Formation of Nitrofatty Acids
NO O2
40
Summary Biology

• NO generation is Ca2 regulated for the
  constitutive forms of NOS (eNOS, nNOS) and
  transcriptionally regulated for the inducible
  form (iNOS).
• For constitutively generated NO, the only clearly
  established function is the activation of sGC.
  Thus, the biology of low level NO is due to the
  actions of cGMP.
• 3. NO can act as an antioxidant by scavenging
  radical species.
• 4. High levels of NO from iNOS does more than
  activate sGC. Higher order chemistry is possible
  (especially in membranes) and amino acids,
  nucleic acids and lipids can be modified.
• 5. NO can disrupt metal metabolism and
  metalloprotein function by ligand modification
  (cysteine) or direct coordination to the metal
  center.

41
Summary Chemistry

• NO can bind heme proteins resulting in protein
  activation (sGC) or inhibition (P450, cyt c
  oxidase, Hb, Mb, etc.)
• Reaction of NO with O2 can lead to the generation
  of an oxidant (NO2) or an electrophilic
  nitrosating agent (N2O3) - both can lead to thiol
  modification.
• Reaction of NO with O2- leads to the generation
  of a potentially important oxidant, ONOO-.
  Peroxynitrite can generate HO/NO2 or nitrate
  tyrosine. The biological relevance of ONOO- is
  debatable.
• As a stable radical, NO can quench other radical
  species (thiyl, tyrosyl, lipids, etc.).

42
Chemical Properties of NO and NO-Derived Species