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The Virtual Free Radical School. Nitric Oxide Dioxygenase (NOD): A • NO Detoxification Enzyme. Paul R. Gardner, Ph.D. Children’s Hospital Research Foundation 3333 Burnet Ave., MLC7006, Cincinnati, OH 45229 USA Tel.: 513-636-4885 e-mail: [email protected]

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Nitric oxide dioxygenase nod a no detoxification enzyme

The Virtual Free Radical School

Nitric Oxide Dioxygenase (NOD):A •NO Detoxification Enzyme

Paul R. Gardner, Ph.D.

Children’s Hospital Research Foundation

3333 Burnet Ave., MLC7006, Cincinnati, OH 45229 USA

Tel.: 513-636-4885

e-mail: [email protected]

NODSFRBM EducationProgram Gardner 1


No is ubiquitous it can be a lethal poison
NO is ubiquitous. It can be a lethal poison.

Various life forms have evolved strategies for NO detoxification

NODSFRBM EducationProgram Gardner 2


No is ubiquitous
NO is ubiquitous

Common Biological Sources:

1. Oxidation-oxygenation of amines

•NO synthases (L-arginine)

/ immune defense (animals + plants)

organic combustions/ cigarette smoke (lung)

2. Reduction of nitrogen oxides (nitrate and nitrite)

microbial denitrification pathways (soil)

nitrite reduction by oxidoreductases (gut)

NODSFRBM EducationProgram Gardner 3


No can be a lethal poison
NO Can Be a Lethal Poison

•NO Can Poison Cell Energy Production

Sensitive Targets are:

Aconitase and

Cytochrome Oxidase that affect

Respiration.

NODSFRBM EducationProgram Gardner 4


Aconitase, a citric acid cycle enzyme, is a sensitive and critical target of •NO

•NO

Solvent accessible iron attacked by NO

+

S

[•NO] >100 nM

Fe

S

Fe

cluster dissolution +

aconitase inactivation

Cys

S

S

S

Cys

Fe

S

Fe

Iron

liberation

S

Cys

NODSFRBM EducationProgram Gardner 5


No rapidly inhibits cytochrome oxidase and thereby inhibits mitochondrial respiration

100 critical target of

50

0

1.0

0

0.5

[•NO] (µM)

•NO rapidly inhibits cytochrome oxidase and thereby inhibits mitochondrial respiration

Human A549 lung cells respiring with a physiological level of O2 (5 µM) are

poisoned by submicromolar •NO levels. Gardner et al. 2001 FRBM 31, 191.

NODSFRBM EducationProgram Gardner 6


Multiple secondary mechanisms for no toxicity
Multiple Secondary Mechanisms for critical target of •NO Toxicity

Fe-S center Iron liberation

H2O2 toxicity

&

Dinitrosyl-iron

complexes

Catalase inhibition

•NO

-e–

O2•—

+NO

+e–

O2

ONOO–

RSNO

NO–

ONO•

•OH

ONO•

NODSFRBM EducationProgram Gardner 7


Cellular strategies for no detoxification metabolism

critical target of NO + O2 + e– ONO– (nitrate)

O

Cellular Strategies for •NO Detoxification-Metabolism

1. Non-enzymatic and enzymatic ‘Oxidations’ (•NO oxidases)

2. Reduction (•NO reductases)

3. Dioxygenation (•NO dioxygenases)

•NO + O2•-/O2/H2O2 {ONOO–, ONO•, or ONO–}

Toxic products?

NOR

2 •NO + 2 e– N2O (nitrous oxide)

NOD

NODSFRBM EducationProgram Gardner 8


critical target of NO dioxygenase (NOD)

NO3-

•NO

FAD

active O2

NAD(P)H

binding

site

(flavohemoglobin,

structure by U. Ermler et al. 1995)

NODSFRBM EducationProgram Gardner 9


Proposed no dioxygenase rxn mechanism

2 critical target of •NO + 2 O2 + NAD(P)H 2 NO3– + NAD(P)+ + H+

Proposed •NO Dioxygenase Rxn Mechanism

NAD(P)H + FAD + H+ NAD(P)+ + FADH2

FADH2 + Fe3+ Fe2+ + FADH• + H+

Fe2+ + O2 Fe3+(O2•-)

Fe3+(O2•-) + •NO Fe3+ + NO3–

FADH• + Fe3+ FAD + Fe2+ + H+

FAD reduction

(-) CN—

iron reduction

(-) •NO, CO

O2 binding

•NO dioxygenation

(-) CN—

iron reduction

NODSFRBM EducationProgram Gardner 10


100 critical target of

control

80

Rate of

+ 960 ppm •NO

(<2 µM), 60 min

•NO Consumption

60

(nanomol/ min/ 108 cells)

40

0.08 ± 0.07

0.06 ± 0.03

20

0

+ hmp

plasmid

Dhmp

parent

JM109

Flavohemoglobin (hmp) catalyzes constitutive and inducible aerobic •NO consumption in Escherichia coli

E. coli lacking flavohemoglobin (Dhmp) lack constitutive and inducible aerobic •NO consumption activity. A multi-copy plasmid bearing hmp increases the •NO consumption activity in host JM109. Gardner et al. 1998 PNAS 95, 10378.

NODSFRBM EducationProgram Gardner 11


Flavohb hmp protects aconitase in aerobic escherichia coli
FlavoHb ( critical target of hmp) protects aconitase inaerobic Escherichia coli

Aconitase israpidly inactivated in E. coli lacking NOD (hmp) when exposed to an aerobic atmosphere containing 960 ppm •NO

(≤ 2 µM in solution). NOD (hmp) protects aconitase. Gardner et al. 2002 JBC 277, 8166.

NODSFRBM EducationProgram Gardner 12


5.0 critical target of

4.0

parent

3.0

Growth

(A550 nm)

2.0

hmp

D

•NO

parent + 960 ppm •NO

1.0

hmp

+ 960 ppm •NO

D

0.0

0

120

240

360

480

Minutes

FlavoHb protects aerobic E. coli against •NO-mediated growth inhibition

E. coli lacking NOD (Dhmp) that are exposed to an aerobic atmosphere containing

•NO do not grow well. Gardner et al. 1998 PNAS 95, 10378; 2002 JBC 277,

8166, 8172.

NODSFRBM EducationProgram Gardner 13


critical target of NO dioxygenation is a primal (1.8 billion year old) functionof hemoglobin/myoglobinNOD O2 storage/transport

The first hemoglobin/myoglobin most likely functioned as an

enzyme utilizing bound ‘activated’ O2 to dioxygenate NO, or

other substrates in microbes. Multicellular organisms that benefit

from the O2 storage-transport functions of hemoglobin/myoglobin

appeared much later. Gardner et al. 1998 PNAS 95, 10378.

NODSFRBM EducationProgram Gardner 14


His(E7) critical target of

Muscle Myoglobins &

RBC Hemoglobins

Microbial flavohemoglobin (Hb domain)

Tyr(B10)

His(E7)

NODs,

SODs,

etc.

Elevated O2 =

O2•-, H2O2

•NO, CO, etc.

O2 transport/storage

function

NOD function

~ 3.6 Billion years of life on earth

today

beginning

3.0 By

1.8 By

NODSFRBM EducationProgram Gardner 15


flavoHbs* critical target of Sperm Whale Mb

Vmax NOD 112-670 s-1 --

kon O2 1.7-5.0 x 107 M-1 s-1 1.7 x 107 M-1 s-1

koff O2 0.2-0.6 s-1 15 s-1

Kd O2 4-36 nM 800 nM

kox•NO 0.9-2.9 x 109 M-1 s-1 3.4 x 107 M-1 s-1

kon•NO 1.0-2.6 x 107 M-1 s-1 2.2 x 107 M-1 s-1

koff•NO 0.0002 s-1 0.0001 s-1

KM (O2) 60-90 µM --

KM (•NO) 100-250 nM --

Structure and Kinetics Control Diverse

Hemoglobin and Myoglobin Functions

*E. coli, S.cerevisiae and A. eutrophus;

Gardner et al. 2000 JBC 275, 12581, 31581

NODSFRBM EducationProgram Gardner 16


Mammalian cells produce a flavohb like nod activity for no metabolism detoxification

2 critical target of •NO + 2 O2 + NADPH 2 NO3– + NADP+ + H+

Mammalian Cells Produce a flavoHb-like NOD Activity for •NO Metabolism-Detoxification

Human Intestinal Epithelial Cells (CaCo-2)

20-30 nmol •NO/min/107 cells

Apparent Km (O2) = 17 µM

Apparent Km (•NO) = 0.2 µM

CO sensitive Ki (CO) = 3 µM (heme-dependent)

Cyanide sensitive Ki (CN-)  20 µM (heme-dependent)

Diphenylene iodonium sensitive (flavin-dependent)

Gardner et al. 2001 FRBM 31, 191

NODSFRBM EducationProgram Gardner 17


Key Points critical target of :1) •NO can be a potent toxin;2)•NO dioxygenase (NOD) is one enzyme that efficiently detoxifies •NO in bacteria, fungi, and mammals; and3)•NO dioxygenation is an ancient function for the hemoglobin/myoglobin family.

NODSFRBM EducationProgram Gardner 18


References
References critical target of

Gardner, P. R. et al., Nitric oxide sensitivity of the aconitases (1997) JBC 272, 25071.

Gardner, P. R. et al.,Constitutive and adaptive detoxification of nitric oxide inEscherichia coli Role of nitric oxide dioxygenase in the protection of aconitase (1998) JBC 273, 26528.

Gardner, P. R. et al., Nitric oxide dioxygenase: an enzymic function for flavohemoglobin (1998) PNAS 95, 10378.

Gardner, A. M. et al., Flavohemoglobin detoxifies nitric oxide in aerobic, but not anaerobic, Escherischia coli. Evidence for a novel inducible anaerobic nitric oxide scavenging activity (2002) JBC 277, 8166.

Gardner, A. M. et al., Flavorubredoxin, an inducible catalyst for nitric oxide reduction and detoxification in Escherichia coli (2002) JBC 277, 8172.

Gardner, P. R. et al., Nitric oxide dioxygenase activity and function of flavohemoglobins. Sensitivity to nitric oxide and carbon monoxide inhibition (2000) JBC 275, 31581.

Gardner, A. M. et al., Steady-state and transient kinetics of the Escherichia coli nitric oxide dioxygenase (flavohemoglobin). The tyrosine B10 hydroxyl is essential for dioxygen binding and catalysis (2000) JBC 275, 12581.

Frauenfelder, H. et al. The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin (2001) PNAS 98, 2370.

Gardner, P. R. et al., Dioxygen-dependent metabolism of nitric oxide in mammalian cells (2001) FRBM 31, 191-204.

NODSFRBM EducationProgram Gardner 19


Acknowledgements
Acknowledgements critical target of

Drs. John Olson and Yi Dou are gratefully acknowledged for their contribution of hemoglobin structure graphics. This work was supported by grants from the Children’s Hospital Research Foundation Trustees, the American Heart Association (9730193N), and the National Institutes of Health (R01 GM65090).

NODSFRBM EducationProgram Gardner 20


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