The Ferrous Dioxygen Complex of the Oxygenase
Domain of Neuronal Nitric-oxide
Couture M, Stuehr DJ, Rousseau DL
Department of Physiology and Biophysics, Albert Einstein College of
Medicine, Bronx, New York 10461.
[Record supplied by publisher]
The mechanisms by which nitric-oxide
synthases (NOSs) bind and activate oxygen at their P450-type heme
active site in order to synthesize nitric
oxide from the substrate L-arginine
are mostly unknown. To obtain information concerning the structure and
properties of the first oxygenated intermediate of the enzymatic
cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy
to generate and identify the ferrous dioxygen complex of the oxygenase
domain of nNOS (Fe(2+)O(2) nNOSoxy). We detect a line at 1135 cm(-1)
in the resonance Raman spectrum of the intermediate formed from 0.6 to
3.0 ms after the rapid mixing of the ferrous enzyme
with oxygen that is shifted to 1068 cm(-1) with (18)O(2). This line is
assigned as the O-O stretching mode (nu(O-O)) of the oxygenated
complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy
saturated with L-arginine
, in the presence or absence of (6R)-5,6,7,8-tetrahydro-L-biopterin,
reveal that the nu(O-O) line is insensitive to the presence of the
substrate and the pterin. The optical spectrum of this ferrous
dioxygen species, with a Soret band wavelength maximum at 430 nm,
confirms the identification of the previously reported oxygenated
complexes generated by stopped flow techniques.
- Nitric oxide synthesis by tracheal smooth muscle
cells by a nitric oxide synthase-independent pathway.
Jia Y, Zacour M, Tolloczko B, Martin JG
Meakins-Christie Laboratories, McGill University, and Cystic Fibrosis
Laboratory, Montreal Chest Hospital, Montreal, Quebec, Canada H2X 2P2.
Nitric oxide (NO) is known to be synthesized from L-arginine in a
reaction catalyzed by NO synthase. Liver cytochrome P-450 enzymes also
catalyze the oxidative cleavage of C==N bonds of compounds containing
a -C(NH2)==NOH function, producing NO in vitro. The present study was
designed to investigate whether there was evidence of a similar
pathway for the production of NO in tracheal smooth muscle cells.
Formamidoxime (10(-2) to 10(-4) M), a compound containing
-C(NH2)==NOH, relaxed carbachol-contracted tracheal rings and
increased intracellular cGMP in cultured tracheal smooth muscle cells,
whereas L-arginine had no such effect. NO was detectable in the medium
containing cultured tracheal smooth muscle cells when incubated with
formamidoxime. Ethoxyresorufin (10(-7) to 10(-4) M), an alternate
cytochrome P-450 substrate, inhibited formamidoxime-induced cGMP
accumulation as well as tracheal ring relaxation in cultured tracheal
smooth muscle cells. The NO synthase inhibitors Nomega-nitro-L-arginine
(10(-3) M) and NG-monomethyl-L-arginine (10(-3) M) had no effect on
formamidoxime-induced cGMP accumulation. These results suggest that NO
can be synthesized from formamidoxime in tracheal smooth muscle cells,
presumably by a reaction catalyzed by cytochrome P-450.
P450nor, a Novel Class of Mitochondrial Cytochrome P450 Involved
in Nitrate Respiration in the Fungus Fusarium oxysporum
Sawako Suzuki*, Seigo Kuwazaki*,
Hirofumi Shoun*, , 1,
Masashi Yamaguchi§, Kanji Takeo§
*Institute of Applied Biochemistry Biological
(Tsukuba Advanced Research Alliance) Center, University of
Tsukuba, Tsukuba, Ibaraki, 305-8572, Japan
§Research Center for Pathogenic Fungi
and Microbial Toxicoses, Chiba University, Chiba, 260-8673, Japan
July 1, 1999; received in revised form September 19, 1999)
oxysporum, an imperfect filamentous fungus performs
nitrate respiration under limited oxygen. In the
respiratory system, Cytochrome P450nor (P450nor)
is thought to catalyze the last step; reduction of
nitric oxide to nitrous oxide. We examined its intracellular
localization using enzymatic, spectroscopic,
and immunological analyses to show that P450nor is found
in both the mitochondria and the cytosol. Translational
fusions between the putative mitochondrial targeting
signal on the amino terminus of P450nor and Escherichia
resulted in significant -galactosidase
activity in the mitochondrial fraction of nitrate-respiring
cells, suggesting that one of the isoforms
of P450nor (P450norA) is in anaerobic mitochondrion
of F. oxysporum and acts as nitric oxide reductase.
Furthermore, these findings suggest the involvement
of P450nor in nitrate respiration in mitochondria.
Copyright 1999 Academic Press.Key
Words: cytochrome P450; mitochondria; denitrification; Fusarium
1To whom correspondence and reprint requests should
be addressed. Fax: (81) 298-53-4605. E-mail: firstname.lastname@example.org.
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