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

Linda J Roman,Jennifer Mclain,Bettie Sue Siler Masters

doi:10.1074/jbc.m212309200

Abstract

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 approximately 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.

Highlights

Despite the fact that all three nitric-oxide synthases (NOSs) isoforms share 50 – 60% overall sequence similarity, each of the NOS isoforms is intrinsically different from the other with regard to rates of NO synthesis, cytochrome c reduction, electron transfer, and hemenitrosyl complex formation
Expression and Spectral Characterization of Chimeric Enzymes—All of the chimeric enzymes expressed very well in E. coli, with yields comparable with those of wild-type neuronal NOS (nNOS) and cytochrome P450 oxidoreductase (CYPOR) (i.e. 100 –150 nmol/liter cells), and all were of the correct mass as visualized by SDS-PAGE
We have produced a series of chimeric enzymes by swapping the FMN binding and FAD/NADPH binding subdomains between nNOS and CYPOR

Summary

Introduction

Despite the fact that all three NOS isoforms share 50 – 60% overall sequence similarity, each of the NOS isoforms is intrinsically different from the other with regard to rates of NO synthesis, cytochrome c reduction, electron transfer, and hemenitrosyl complex formation. 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. In addition to the CaM binding sequence, an autoregulatory insert was identified in the FMN binding region of the constitutively expressed isoforms, nNOS and eNOS, but not in. Tel.: 210-567-6979; Fax: 210-567-6984; If the autoregulatory insert is completely removed, the rate of electron flow increases, indicating that this element intrinsically regulates electron flow to the heme, even in the presence of CaM (9 –11)

Methods

Results

Conclusion