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J. Biol. Chem., Vol. 281, Issue 15, 9953-9962, April 14, 2006

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A Weak Fe–O Bond in the Oxygenated Complex of the Nitric-oxide Synthase of Staphylococcus aureus^*

From the Department of Biochemistry and Microbiology and CREFSIP Research Center, Université Laval, Quebec City, Quebec G1K 7P4, Canada

Little is known about the intermediates formed during catalysis by nitric-oxide synthase (NOS). We report here the characterization by resonance Raman spectroscopy of the oxygenated complex of the NOS from Staphylococcus aureus (saNOS) as well as the kinetics of formation and decay of the complex. An oxygenated complex transiently formed after mixing reduced saNOS with oxygen and decayed to the ferric enzyme with kinetics that were dependent on the substrate L-arginine and the cofactor H₄B. The oxygenated complex displayed a Soret absorption band centered at 430 nm. Resonance Raman spectroscopy revealed that it can be described as a ferric superoxide form () with a single _O–O mode at 1135 cm^–1. In the presence of L-arginine, an additional _O–O mode at 1123 cm^–1 was observed, indicating an increased back-bonding electron donation to the bound oxygen induced by the substrate. With saNOS, this is the first time that the _Fe–O mode of a NOS has been observed. The low frequency of this mode, at 517 cm^–1, points to an oxygenated complex that differs from that of P450_cam. The electronic structure of the oxygenated complex and the effect of L-arginine are discussed in relation to the kinetic properties of saNOS and other NOS.

Received for publication, December 30, 2005 , and in revised form, February 9, 2006.

^* This work was supported by the National Sciences and Engineering Research Council of Canada (Grant 250073), the Canadian Foundation for Innovation (equipment Grant 7128), and the Fonds Québécois de la Recherche sur la Nature et les Technologies (Grant 78927 to M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Microbiology and Centre de Recherche sur la Fonction, la Structure et l'Ingénierie des Protéines (CREFSIP), Pavillon Marchand, Room 4165, Université Laval, Quebec City, Quebec G1K 7P4, Canada. Tel.: 418-656-2131 (ext. 13515); Fax: 418-656-7176; E-mail: manon.couture{at}bcm.ulaval.ca.

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