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J. Biol. Chem., Vol. 281, Issue 14, 9623-9632, April 7, 2006

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Structure and Reactivity of a Thermostable Prokaryotic Nitric-oxide Synthase That Forms a Long-lived Oxy-Heme Complex^*

From the Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853

In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed, and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from L-arginine via the stable intermediate N-hydroxy L-arginine (NOHA). The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates L-arginine and NOHA. The substrate-induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO forms only in the presence of H₄B. The crystal structure of gsNOS at 3.2 AÅ of resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme. Thus, a more constrained heme pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 °C. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation.

Received for publication, September 13, 2005 , and in revised form, January 9, 2006.

The atomic coordinates and structure factors (code 2FLQ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the National Science Foundation (MCB-0133 564). 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 Fig. 1.

¹ To whom correspondence should be addressed: G 60 S. T. Olin, Dept. of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853. Tel.: 607-254-8634; Fax: 607-255-1248; E-mail: bc69{at}cornell.edu.

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