Interactions between the Isolated Oxygenase and Reductase Domains of Neuronal Nitric-oxide Synthase: ASSESSING THE ROLE OF CALMODULIN

Elena A Rozhkova,Norikazu Fujimoto,Ikuko Sagami,Simon N Daff,Toru Shimizu

doi:10.1074/jbc.m200642200

Elena A Rozhkova, Norikazu Fujimoto + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m200642200

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Abstract

Nitric-oxide synthase (NOS) is a fusion protein composed of an oxygenase domain with a heme-active site and a reductase domain with an NADPH binding site and requires Ca(2+)/calmodulin (CaM) for NO formation activity. We studied NO formation activity in reconstituted systems consisting of the isolated oxygenase and reductase domains of neuronal NOS with and without the CaM binding site. Reductase domains with 33-amino acid C-terminal truncations were also examined. These were shown to have faster cytochrome c reduction rates in the absence of CaM. N(G)-hydroxy-l-Arg, an intermediate in the physiological NO synthesis reaction, was found to be a viable substrate. Turnover rates for N(G)-hydroxy-l-Arg in the absence of Ca(2+)/CaM in most of the reconstituted systems were 2.3-3.1 min(-1). Surprisingly, the NO formation activities with CaM binding sites on either reductase or oxygenase domains were decreased dramatically on addition of Ca(2+)/CaM. However, NADPH oxidation and cytochrome c reduction rates were increased by the same procedure. Activation of the reductase domains by CaM addition or by C-terminal deletion failed to increase the rate of NO synthesis. Therefore, both mechanisms appear to be less important than the domain-domain interaction, which is controlled by CaM binding in wild-type neuronal NOS, but not in the reconstituted systems.

Highlights

Nitric-oxide synthase (NOS) is a fusion protein composed of an oxygenase domain with a heme-active site and a reductase domain with an NADPH binding site and requires Ca2؉/calmodulin (CaM) for Nitric oxide (NO) formation activity
The OxCaM-Fe(III) complex had a Soret peak around 405 nm and the addition of L-Arg moved the peak to 395 nm, indicating that the substrate binding site is not altered in this domain on elimination of the reductase domain
It is suggested that the NHA and H4B binding sites of the oxygenase domain in the reconstituted system were not altered by isolating the domain from the full-length wild-type protein

Summary

EXPERIMENTAL PROCEDURES

The complete cDNA for rat nNOS (GenBank௢ accession no. X59949) in pBluescript SK(Ϫ) was kindly provided by Dr S. Expression plasmids for the nNOS reductase domain with (designated RedCaM) or without (designated Red) CaM-binding site (residues 721–1429 and 746 –1429, respectively) and their C-terminal truncated domains (residues 721–1395 for RedCaM⌬33 and residues 746 –1395 for Red⌬33, respectively) were generated as previously described (16 – 20). Full-length nNOS of wild type and the nNOS oxygenase domains, OxCaM and Ox, were expressed in E. coli cell line, BL21, which contains another plasmid, pGroESL, for expression of chaperone proteins as described previously [12, 23, 24, 28]. Catalytic assays were carried out at 25 °C in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 0.1 ␮M heme domain, 0.5 ␮M reductase domain or CPR, 0.1 mM NADPH, 0.5 mM NHA, 5 ␮M H4B, 20 ␮M DTT, 10 units/ml SOD, and 100 units/ml catalase in the presence or absence of 1 mM CaCl2 and 10 ␮g/ml CaM.

RESULTS

26 Ϯ 1 86 Ϯ 9

DISCUSSION