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Evolution of NO Synthesis & Utilization 
J Biol Chem 2000 Feb 4;275(5):3201-3205 Related Articles, LinkOut

The Ferrous Dioxygen Complex of the Oxygenase Domain of Neuronal Nitric-oxide Synthase.

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 or N(omega)-hydroxy-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.
Am J Physiol 1998 Nov;275(5 Pt 1):L895-901 Related Articles, Books, LinkOut
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.
Cytochrome P450nor, a Novel Class of Mitochondrial Cytochrome P450 Involved in Nitrate Respiration in the Fungus Fusarium oxysporum
pp. 340-346 (doi:10.1006/abbi.1999.1499) 
Naoki Takaya* Sawako Suzuki* Seigo Kuwazaki* Hirofumi Shoun*, 1 Fumiaki Maruo Masashi Yamaguchi Kanji Takeo

*Institute of Applied Biochemistry Biological Sciences TARA (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
IDEAL Related Articles
(Received July 1, 1999; received in revised form September 19, 1999)
Abstract

Fusarium 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 coli-galactosidase 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 oxysporum

1To whom correspondence and reprint requests should be addressed. Fax: (81) 298-53-4605. E-mail: p450nor@sakura.cc.tsukuba.ac.jp.

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