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This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: jimmunol.0902274v1 183/9/5977    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Google Scholar Articles by Liu, X.-D. Articles by Eissa, N. T. PubMed PubMed Citation Articles by Liu, X.-D. Articles by Eissa, N. T.

Identification of a Flavin Mononucleotide Module Residue Critical for Activity of Inducible Nitrite Oxide Synthase¹

Xian-De Liu,^* Tuhina Mazumdar,^2* Yi Xu,^* Elizabeth D. Getzoff, and N. Tony Eissa^3*

^*Department of Medicine, Baylor College of Medicine, Houston, TX 77030; and The Scripps Research Institute, La Jolla, CA 92037

Inducible NO synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding domain. For the synthesis of NO, iNOS is active as a homodimer formed by oxygenase domains, while the reductase domain is required to transfer electrons from NADPH. In this study, we identify glutamate 658 in the FMN domain of human iNOS to be a critical residue for iNOS activity and we explore the underlying mechanism for such role. Mutation of glutamate to aspartate almost abolished iNOS activity and reduced dimer formation. Substitution of this residue with noncharged alanine and glutamine, or positively charged lysine did not affect dimer formation and maintained around 60% of iNOS activity. These results suggest that the negative charge specific to glutamate plays an important role in iNOS activity.

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.

¹ This study is supported by National Heat, Lung and Blood Institute, National Institute of Allergy and Infectious Diseases and American Heart Association.

² Current address: Texas A&M Health Science Center, College of Medicine, Houston, Texas 77030.

³ Address correspondence and reprint requests to Dr. N. Tony Eissa, Baylor College of Medicine, One Baylor Plaza, BCM 285 Suite 535E, Houston, TX 77030. E-mail address: teissa{at}bcm.edu

⁴ Abbreviations used in this paper: NOS, NO symthase; iNOS, inducible NOS; H₄B, tetrahydrobuioterin; SILAC, stable isotope labeling by amino acids in cell culture; HEK, human embryonic kidney; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide.

⁵ The online version of this article contains supplemental material.