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Papers In Press, published online ahead of print May 1, 2003
Biochemistry-7760, University of Texas Health Science Center, San Antonio, TX 78229
Corresponding Author: roman{at}uthscsa.edu
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 oxido-reductase (CYPOR), which transfers electrons to microsomal cytochromes P450. The crystal structure of the 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 is 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, while 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.
J. Biol. Chem, 10.1074/jbc.M212309200
Submitted on December 3, 2002
Revised on April 7, 2003
Accepted on May 1, 2003
Chimeric enzymes of cytochrome P450 Oxido-reductase and neuronal nitric oxide synthase reductase domain reveal structural and functional differences between them
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