The 42-Amino Acid Insert in the FMN Domain of Neuronal Nitric-oxide Synthase Exerts Control over Ca2+/Calmodulin-dependent Electron Transfer

Simon Daff,Ikuko Sagami,Toru Shimizu

doi:10.1074/jbc.274.43.30589

Abstract

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca(2+) concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca(2+), whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40-50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca(2+) dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Delta40 and Delta42) retained maximal NO synthesis activity at lower concentrations of free Ca(2+) than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca(2+)/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca(2+) concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca(2+)/CaM. However, reduction of heme in the Delta40 mutant occurred spontaneously on addition of NADPH in the absence of Ca(2+)/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca(2+)/CaM and by destabilizing CaM binding at low Ca(2+) concentrations, consistent with its role as an autoinhibitory domain.

Highlights

The nitric-oxide synthases (NOSs)1 are a family of dimeric enzymes found in a variety of organisms and cell types
Construction of the Mutants—The alignment shown in Fig. 1 compares the amino acid sequences of the FMN-binding subdomains of the three NOS isoforms and that of cytochrome
All three NOS isoforms consist of a diflavin-binding reductase domain coupled directly to a heme-binding monooxygenase domain via a CaM-binding linker region

Summary

EXPERIMENTAL PROCEDURES

Materials—(6R)-5,6,7,8-Tetrahydro-L-biopterin was purchased from Schircks Laboratories (Jona, Switzerland). The ⌬40 nNOS mutant plasmid (pSD1⌬40) was generated by ligating the ScaI site at Thr871 in the protein sequence to the BanI site at Arg829 after blunt-ending with the Klenow fragment of DNA polymerase I. Assays of Enzyme Activity—The rate of NO formation was determined from the NO-mediated conversion of oxyhemoglobin to methemoglobin, monitored at 401 nm using a methemoglobin minus oxyhemoglobin extinction coefficient of 49 mMϪ1 cmϪ1 [29], with 10 ␮M oxyhemoglobin, 0.1 mM NADPH, and 1 mM L-arginine. The ferricyanide reduction rate was determined by monitoring the decrease at 420 nm using an extinction coefficient of 1.01 mMϪ1 cmϪ1, with concentrations of ferricyanide at 1 mM and NADPH at 0.5 mM unless otherwise indicated. The temperature of the glove box was maintained at 15 °C throughout

RESULTS

86 Ϯ 20 56 Ϯ 12

DISCUSSION