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J. Biol. Chem., Vol. 278, Issue 28, 25700-25707, July 11, 2003

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Chimeric Enzymes of Cytochrome P450 Oxidoreductase and Neuronal Nitric-oxide Synthase Reductase Domain Reveal Structural and Functional Differences^*

Linda J. Roman , Jennifer McLain and Bettie Sue Siler Masters

From the Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229-3900

The nitric-oxide synthases (NOSs) are comprised of an oxygenase domain and a reductase domain bisected by a calmodulin (CaM) binding region. The NOS reductase domains share 60% sequence similarity with the cytochrome P450 oxidoreductase (CYPOR), which transfers electrons to microsomal cytochromes P450. The crystal structure of the neuronal NOS (nNOS) connecting/FAD binding subdomains reveals that the structure of the nNOS-connecting subdomain diverges from that of CYPOR, implying different alignments of the flavins in the two enzymes. We created a series of chimeric enzymes between nNOS and CYPOR in which the FMN binding and the connecting/FAD binding subdomains are swapped. A chimera consisting of the nNOS heme domain and FMN binding subdomain and the CYPOR FAD binding subdomain catalyzed significantly increased rates of cytochrome c reduction in the absence of CaM and of NO synthesis in its presence. Cytochrome c reduction by this chimera was inhibited by CaM. Other chimeras consisting of the nNOS heme domain, the CYPOR FMN binding subdomain, and the nNOS FAD binding subdomain with or without the tail region also catalyzed cytochrome c reduction, were not modulated by CaM, and could not transfer electrons into the heme domain. A chimera consisting of the heme domain of nNOS and the reductase domain of CYPOR reduced cytochrome c and ferricyanide at rates 2-fold higher than that of native CYPOR, suggesting that the presence of the heme domain affected electron transfer through the reductase domain. These data demonstrate that the FMN subdomain of CYPOR cannot effectively substitute for that of nNOS, whereas the FAD subdomains are interchangeable. The differences among these chimeras most likely result from alterations in the alignment of the flavins within each enzyme construct.

Received for publication, December 3, 2002 , and in revised form, April 7, 2003.

^* This work was supported by National Institutes of Grants HL30050 and GM52419 and Robert A. Welch Foundation Research Grant AQ-1192 (to B. S. S. M.). 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.

To whom correspondence may be addressed: Dept. of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 79229-3900. Tel.: 210-567-6979; Fax: 210-567-6984; E-mail: Roman{at}UTHSCSA.edu.

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