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Characterization of Mouse nNOS2, a Natural Variant of Neuronal Nitric-oxide Synthase Produced in the Central Nervous System by Selective Alternative Splicing

1999; Elsevier BV; Volume: 274; Issue: 25 Linguagem: Inglês

10.1074/jbc.274.25.17559

ISSN

1083-351X

Autores

Toshio Iwasaki, Hiroyuki Hori, Yoko Hayashi, Takeshi Nishino, Koji Tamura, Soichi Oue, Tetsutarō Iizuka, Tsutomu Ogura, Hiroyasu Esumi,

Tópico(s)

ATP Synthase and ATPases Research

Resumo

Mouse neuronal nitric-oxide synthase 2 (nNOS2) is a unique natural variant of constitutive neuronal nitric-oxide synthase (nNOS) specifically expressed in the central nervous system having a 105-amino acid deletion in the heme-binding domain as a result of in-frame mutation by specific alternative splicing. The mouse nNOS2 cDNA gene was heterologously expressed in Escherichia coli, and the resultant product was characterized spectroscopically in detail. Purified recombinant nNOS2 contained heme but showed no l-arginine- and NADPH-dependent citrulline-forming activity in the presence of Ca2+-promoted calmodulin, elicited a sharp electron paramagnetic resonance (EPR) signal at g = 6.0 indicating the presence of a high spin ferriheme as isolated and showed a peak at around 420 nm in the CO difference spectrum, instead of a 443-nm peak detected with the recombinant wild-type nNOS1 enzyme. Thus, although the heme domain of nNOS2 is capable of binding heme, the heme coordination geometry is highly abnormal in that it probably has a proximal non-cysteine thiolate ligand both in the ferric and ferrous states. Moreover, negligible spectral perturbation of the nNOS2 ferriheme was detected upon addition of either l-arginine or imidazole. These provide a possible rational explanation for the inability of nNOS2 to catalyze the cytochrome P450-type monooxygenase reaction. Mouse neuronal nitric-oxide synthase 2 (nNOS2) is a unique natural variant of constitutive neuronal nitric-oxide synthase (nNOS) specifically expressed in the central nervous system having a 105-amino acid deletion in the heme-binding domain as a result of in-frame mutation by specific alternative splicing. The mouse nNOS2 cDNA gene was heterologously expressed in Escherichia coli, and the resultant product was characterized spectroscopically in detail. Purified recombinant nNOS2 contained heme but showed no l-arginine- and NADPH-dependent citrulline-forming activity in the presence of Ca2+-promoted calmodulin, elicited a sharp electron paramagnetic resonance (EPR) signal at g = 6.0 indicating the presence of a high spin ferriheme as isolated and showed a peak at around 420 nm in the CO difference spectrum, instead of a 443-nm peak detected with the recombinant wild-type nNOS1 enzyme. Thus, although the heme domain of nNOS2 is capable of binding heme, the heme coordination geometry is highly abnormal in that it probably has a proximal non-cysteine thiolate ligand both in the ferric and ferrous states. Moreover, negligible spectral perturbation of the nNOS2 ferriheme was detected upon addition of either l-arginine or imidazole. These provide a possible rational explanation for the inability of nNOS2 to catalyze the cytochrome P450-type monooxygenase reaction. Nitric-oxide synthase (NOS) 1The abbreviations used are: NOS, nitric-oxide synthase; H4BP, 6(R)-5,6,7,8-tetrahydro-l-biopterin; NO, nitric oxide; iNOS, inducible NOS; nNOS1, wild-type neuronal NOS; nNOS2, natural variant of constitutive neuronal NOS, having a 105-amino acid deletion in the heme-binding domain as a result of the in-frame mutation by specific alternative splicing of exons 9 and 10; Sf9 , Spodoptera frugiperda. is a complex flavo-hemoprotein that catalyzes the conversion ofl-arginine to citrulline in the presence of molecular oxygen, NADPH, tetrahydrobiopterin (H4BP), and Ca2+-promoted calmodulin with concomitant production of nitric oxide (NO) (1Marletta M.A. J. Biol. Chem. 1993; 268: 12231-12234Abstract Full Text PDF PubMed Google Scholar, 2Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2147) Google Scholar, 3Masters B.S.S. Annu. Rev. Nutr. 1994; 14: 131-145Crossref PubMed Scopus (82) Google Scholar, 4Knowles R.G. Moncada S. 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Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar), where NO is produced at the heme center in the presence of oxygen, H4BP, and l-arginine (6Griffith O. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 8Stuehr D.J. Annu. Rev. Pharmacol. 1997; 37: 339-359Crossref Scopus (453) Google Scholar). The heme-binding domain of NOS shows negligible structural homology to regular cytochrome P450 (6Griffith O. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 8Stuehr D.J. Annu. Rev. Pharmacol. 1997; 37: 339-359Crossref Scopus (453) Google Scholar, 10Crane B.R. Arvai A.S. Gachhui R. Wu C. Ghosh D.K. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1997; 278: 425-431Crossref PubMed Scopus (337) Google Scholar, 11Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar), although a wide range of spectroscopic evidence supports coordination of an endogenous thiolate sulfur donor ligand to the central heme iron (12Stuehr D.J. Ikeda-Saito M. J. Biol. Chem. 1992; 267: 20547-20550Abstract Full Text PDF PubMed Google Scholar, 13Wang J. Stuehr D.J. Ikeda-Saito M. Rousseau D.L. J. Biol. Chem. 1993; 268: 22255-22258Abstract Full Text PDF PubMed Google Scholar, 14Sono M. Stuehr D.J. Ikeda-Saito M. Dawson J.H. J. Biol. Chem. 1995; 270: 19943-19948Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Migita C.T. Salerno J.C. Masters B.S.S. Martasek P. McMillan K. Ikeda-Saito M. Biochemistry. 1997; 36: 10987-10992Crossref PubMed Scopus (56) Google Scholar). Recent high resolution x-ray crystal structure determinations of the monomeric and dimeric forms of the heme-binding domain fragment of inducible NOS (iNOS) have proven that the overall protein topology is very different from that of either regular cytochrome P450 or chloroperoxidase, although the proximal ligand to the central ferriheme iron is a conserved cysteine residue (10Crane B.R. Arvai A.S. Gachhui R. Wu C. Ghosh D.K. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1997; 278: 425-431Crossref PubMed Scopus (337) Google Scholar, 11Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar). The endogenous thiolate sulfur donor ligand to the central heme iron provides a rational basis for the proposed two-step sequential catalytic mechanism of NOS, where the first step involving the formation of the intermediateN ω-hydroxyarginine may follow a cytochrome P450-type monooxygenase mechanism (1Marletta M.A. J. Biol. Chem. 1993; 268: 12231-12234Abstract Full Text PDF PubMed Google Scholar, 16Andersson L.A. Dawson L.A. Struct. Bonding. 1991; 64: 1-40Google Scholar, 17Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2887Crossref PubMed Scopus (2125) Google Scholar, 18Dawson J.H. Sono M. Chem. Rev. 1987; 87: 1255-1276Crossref Scopus (494) Google Scholar, 19Poulos T.L. J. Biol. Inorg. Chem. 1996; 1: 356-359Crossref Scopus (243) Google Scholar, 20Rietjens I.M.C.M. Osman A.M. Veeger C. Zakharieva O. Antony J. Grodzicki M. Trautwein A.X. J. Biol. Inorg. Chem. 1996; 1: 372-376Crossref Scopus (46) Google Scholar). The substrate (l-arginine) and pterin cofactor (H4BP) binding sites have been defined by the crystal structure of the dimeric iNOS heme domain (11Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar), and the former site (conserved glutamate residue) at the C-terminal part of the NOS heme domain has also been subjected to site-directed mutagenesis studies (21Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1997; 272: 6114-6118Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Gachhui R. Ghosh D.K. Wu C. Parkinson J. Crane B.R. Stuehr D.J. Biochemistry. 1997; 36: 5097-5103Crossref PubMed Scopus (72) Google Scholar). Aside from the protein chemistry, the in vivo regulation of NOS, from the viewpoint of control of NO synthesis, has been the subject of extensive investigation because of diverse physiological functions of NO in cells. Thus, NO produced by NOS isoforms induces vascular smooth muscle relaxation, serves as a messenger in the central and peripheral nervous systems, and also acts as a cytotoxic agent in the immune system to kill tumor cells and intracellular parasites (2Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2147) Google Scholar,23Dawson T.M. Zhang J. Dawson V.L. Snyder S.H. Prog. Brain Res. 1994; 103: 365-369Crossref PubMed Scopus (72) Google Scholar, 24Moncada S. Higgs E.A. FASEB J. 1995; 9: 1319-1330Crossref PubMed Scopus (722) Google Scholar, 25Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1542) Google Scholar, 26Ignarro L.J. Kidney Int. 1996; 55: S2-S5Google Scholar, 27Dawson T.M. Dawson V.L. Annu. Rev. Med. 1996; 47: 219-227Crossref PubMed Scopus (133) Google Scholar). In the case of nNOS, the genes are either constitutively or stage- and tissue-specifically expressed in different neuronal cell types and in skeletal muscle (5Nathan C. Xie Q.-W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar, 28Brenman J.E. Xia H. Chao D.S. Black S.M. Bredt D.S. Dev. Neurosci. 1997; 19: 224-231Crossref PubMed Scopus (144) Google Scholar). Although the coding region of the nNOS gene encodes a 160-kDa protein, the mRNA is considerably larger (∼9.5 kilobase pairs), and significant molecular diversity is found, mostly in the 5′-noncoding region. This diversity is produced by specific alternative splicing at different splice sites and may affect the stability of individual mRNA species (5Nathan C. Xie Q.-W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar). It has been reported that several different nNOS are produced in different cell types by selective alternative splicing in the coding region (2Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2147) Google Scholar, 7Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Z. Huang F. Xia H. Peters M.F. Froehner S.C. Bredt D.S. Cell. 1996; 84: 8410-8413Abstract Full Text Full Text PDF Scopus (1446) Google Scholar, 28Brenman J.E. Xia H. Chao D.S. Black S.M. Bredt D.S. Dev. Neurosci. 1997; 19: 224-231Crossref PubMed Scopus (144) Google Scholar, 29Ogura T. Yokoyama T. Fujisawa H. Kurashima Y. Esumi H. Biochem. Biophys. Res. Commun. 1993; 193: 1014-1022Crossref PubMed Scopus (108) Google Scholar, 30Fujisawa H. Ogura T. Kurashima Y. Yokoyama T. Yamashita J. Esumi H. J. Neurochem. 1994; 63: 140-145Crossref PubMed Scopus (60) Google Scholar, 31Ogilvie P. Schilling K. Billingsley M.L. Schmidt H.H. FASEB J. 1995; 9: 799-806Crossref PubMed Scopus (95) Google Scholar, 32Silvagno F. Xia H. Bredt D.S. J. Biol. Chem. 1996; 271: 11204-11208Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 33Wang Y. Goligorsky M.S. Lin M. Wilcox J.N. Marsden P.A. J. Biol. Chem. 1997; 272: 11392-11401Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 34Lee M.A. Cai L. Hubner N. Lee Y.A. Lindpaintner K. J. Clin. Invest. 1997; 100: 1507-1512Crossref PubMed Scopus (83) Google Scholar, 35Eliasson M.J. Blackshaw S. Schell M.J. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3396-3401Crossref PubMed Scopus (256) Google Scholar). The presence of at least six different variants of alternatively spliced nNOS mRNA species, which are expressed in a tissue-specific and developmentally regulated manner, has been reported (28Brenman J.E. Xia H. Chao D.S. Black S.M. Bredt D.S. Dev. Neurosci. 1997; 19: 224-231Crossref PubMed Scopus (144) Google Scholar). Among them, a natural mutant of mouse nNOS created by specific alternative splicing and designated nNOS2 by Ogura et al. (29Ogura T. Yokoyama T. Fujisawa H. Kurashima Y. Esumi H. Biochem. Biophys. Res. Commun. 1993; 193: 1014-1022Crossref PubMed Scopus (108) Google Scholar) is one of the most unusual and interesting. First, it has been shown that the primary structure of mouse nNOS2 lacks the C-terminal half of the highly conserved “dihydrofolate reductase module” region in the heme-binding domain (corresponding to residues 504–608 in mouse or rat nNOS1) due to in-frame mutation with skipping of exons 9 and 10 by alternative splicing (29Ogura T. Yokoyama T. Fujisawa H. Kurashima Y. Esumi H. Biochem. Biophys. Res. Commun. 1993; 193: 1014-1022Crossref PubMed Scopus (108) Google Scholar) (see Fig. 1). Second, the missing 105-amino acid residue region contains Glu-597, which is involved in l-arginine binding (11Crane B.R. Arvai A.S. Ghosh D.K. Wu C. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar, 21Chen P.-F. Tsai A.-L. Berka V. Wu K.K. J. Biol. Chem. 1997; 272: 6114-6118Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Gachhui R. Ghosh D.K. Wu C. Parkinson J. Crane B.R. Stuehr D.J. Biochemistry. 1997; 36: 5097-5103Crossref PubMed Scopus (72) Google Scholar). Third, despite the difference of the heme domain sequence of nNOS2 from that of nNOS1, their calmodulin-binding and cytochrome P450 reductase domains remain intact at the primary structure level (29Ogura T. Yokoyama T. Fujisawa H. Kurashima Y. Esumi H. Biochem. Biophys. Res. Commun. 1993; 193: 1014-1022Crossref PubMed Scopus (108) Google Scholar, 30Fujisawa H. Ogura T. Kurashima Y. Yokoyama T. Yamashita J. Esumi H. J. Neurochem. 1994; 63: 140-145Crossref PubMed Scopus (60) Google Scholar, 31Ogilvie P. Schilling K. Billingsley M.L. Schmidt H.H. FASEB J. 1995; 9: 799-806Crossref PubMed Scopus (95) Google Scholar). Fourth, the alternatively spliced product is specifically expressed in mouse central nervous system cells (29Ogura T. Yokoyama T. Fujisawa H. Kurashima Y. Esumi H. Biochem. Biophys. Res. Commun. 1993; 193: 1014-1022Crossref PubMed Scopus (108) Google Scholar, 30Fujisawa H. Ogura T. Kurashima Y. Yokoyama T. Yamashita J. Esumi H. J. Neurochem. 1994; 63: 140-145Crossref PubMed Scopus (60) Google Scholar), and its homolog has also been identified in rat and human central nervous system andDrosophila head cells (36Regulski M. Tully T. Proc. Natl. Acad, Sci. U. S. A. 1995; 92: 9072-9076Crossref PubMed Scopus (216) Google Scholar), implying an unknown conserved function in the central nervous system. Nevertheless, none of these natural variants of NOS isoforms has been characterized spectroscopically in detail. It is therefore of particular interest to investigate whether or not the alternatively spliced product nNOS2 has a native heme coordination geometry and retains the ability to function as a true “nitric-oxide synthase.” In this paper, we report the heterologous overexpression of mouse nNOS2 cDNA gene inEscherichia coli for the first time and the molecular and spectroscopic characterization of the recombinant enzyme. Synthetic DNA oligomers were purchased from either SCI-MEDIA (Tokyo, Japan) or Nissinbo (Tokyo, Japan), and DNA modification enzymes and restriction enzymes were from either New England Biolabs or Takara Biomedicals (Otsu, Japan). 2′,5′-ADP-Sepharose 4B, Sephacryl S-200HR, DEAE-Sepharose Fast Flow, and Ampure SA were from Amersham Pharmacia Biotech. Calmodulin, FAD, FMN, l-arginine, and l-citrulline were from Sigma, and 6(R)-5,6,7,8-tetrahydro-l-biopterin (H4BP) was from Schircks Laboratories (Jona, Switzerland).l-[U-14C]Arginine was obtained from NEN life Science Products. Water was purified by a Milli-Q purification system (Millipore). Other chemicals used in this study were of analytical grade. The baculovirus-Spodoptera frugiperda (Sf9) insect cell expression system (37King L.A. Possee R.D. The Baculovirus Expression System: A Laboratory Guide. Chapman & Hall, New York1992Crossref Google Scholar) and E. coli pCWori+ expression system (38Muchmore D.C. McIntosh L.P. Russell C.B. Anderson D.E. Dahlquist F.W. Methods Enzymol. 1989; 177: 44-73Crossref PubMed Scopus (474) Google Scholar, 39Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1995; 270: 17791-17796Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 40Roman L.J. Sheta E.A. Martasek P. Gross S.S. Liu Q. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar) were employed for the expression of mouse full-length wild-type nNOS (nNOS1) and the alternatively spliced form (nNOS2). Unless otherwise stated, vectors were constructed by using E. coli HB101 as the host strain. The site-directed mutagenesis was done by using a Muta-Gene Phagemid in vitro mutagenesis kit (Bio-Rad). All of the altered DNA sequences were analyzed by using a Sequenase version 2.0 DNA sequencing kit (U. S. Biochemical Co). The plasmid vectors, pTZ19RNOS1 carrying the full-length cDNA for mouse nNOS and pTZ19RNOS2 carrying the alternatively spliced cDNA reported previously (29Ogura T. Yokoyama T. Fujisawa H. Kurashima Y. Esumi H. Biochem. Biophys. Res. Commun. 1993; 193: 1014-1022Crossref PubMed Scopus (108) Google Scholar), and the baculovirus transfer vector pJVP10Z (41Summers M.D. Smith G.E. A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experiment Station Bulletin No. 1555. Texas A & M University, College Station, Texas1987Google Scholar) (kindly provided by Dr. S. Kawamoto, Yokohama City University) were used. The transfer vectors carrying the cDNA for nNOS1 and nNOS2 were individually constructed for recombination with AcNPV as follows: an NheI site was newly generated upstream of the first Met codon of the cDNA for nNOS1 with a phosphorylated DNA oligomer (NOSNhe I: 5′- GTA AAA CGA CGG CCA GTG AGC TAG CAT GGA AGA GCA CAC G-3′). The DNA fragment coding the altered 5′-leader sequence and N-terminal region of nNOS was replaced by the corresponding region of nNOS2 using two unique restriction enzyme sites, ScaI and AflII sites. The obtained plasmids (pTZ19RNOS1/NheI, and pTZ19RNOS2/NheI) were excised by NheI and XbaI digestion, and the DNA fragments encoding nNOS genes were individually ligated into theNheI site of pJVP10Z using the compatibility betweenXbaI and NheI sites. The directions of the cDNA were identified by DNA sequencing. The coinfection with AcNPV DNA and the constructed transfer vectors was conducted by using a linear transfection module (Invitrogen), and the screening of the recombinant virus was carried out according to the manufacturer's manual, Max Bac baculovirus expression system manual (Invitrogen) and the literature (37King L.A. Possee R.D. The Baculovirus Expression System: A Laboratory Guide. Chapman & Hall, New York1992Crossref Google Scholar). The recombinant enzymes were expressed as described in the literature (42Richards M.K. Marletta M.A. Biochemistry. 1994; 33: 14723-14732Crossref PubMed Scopus (100) Google Scholar, 43Nakane M. Pollock J.S. Klinghofer V. Basha F. Marsden P.A. Hokari A. Ogura T. Esumi H. Carter G.W. Biochem. Biophys. Res. Commun. 1995; 206: 511-517Crossref PubMed Scopus (47) Google Scholar, 44Riveros-Moreno V. Heffernan B. Torres B. Chubb A. Charles I. Moncada S. Eur. J. Biochem. 1995; 230: 52-57Crossref PubMed Scopus (52) Google Scholar). A heterologous expression system for nNOS1 and nNOS2 inE. coli was constructed with pCWori+ vector (38Muchmore D.C. McIntosh L.P. Russell C.B. Anderson D.E. Dahlquist F.W. Methods Enzymol. 1989; 177: 44-73Crossref PubMed Scopus (474) Google Scholar, 39Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1995; 270: 17791-17796Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 40Roman L.J. Sheta E.A. Martasek P. Gross S.S. Liu Q. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar) and the chaperonin expression vector pKY206 (pACYC184) (Nippon Gene, Toyama, Japan) carrying the E. coli chaperonin groELSgenes (kindly provided by Dr. K. Ito, Kyoto University) as reported (39Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1995; 270: 17791-17796Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 40Roman L.J. Sheta E.A. Martasek P. Gross S.S. Liu Q. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar), with the following minor modifications. First, anNdeI site was newly generated to include the first Met codon of the cDNA for the wild-type nNOS in pTZ19RNOS1 by using the mutation primer (NOSNde I: 5′-GAC GGC CAG TGA GAA CAT ATG GAA GAG CAC ACG-3′). Because the obtained plasmid (pTZ19RNOS1/NdeI) has two NdeI sites, the plasmid was partially digested withNdeI, followed by complete digestion with XbaI. The NdeI-XbaI fragment containing the full-length nNOS1 cDNA was then inserted between the NdeI andXbaI sites of the multicloning linker of pCWori+. The pCWori vectors for nNOS2 were prepared by exchanges of theAflII-XbaI fragment encoding the alternatively spliced region. The resultant pCWori vectors and pKY206 were introduced by repeated transformation into the host strain, E. colistrain BL21 (Takara Biomedicals), which lacks the two proteaseslon and ompT. The recombinant enzymes were expressed as described in the literature (39Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1995; 270: 17791-17796Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 40Roman L.J. Sheta E.A. Martasek P. Gross S.S. Liu Q. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar). NOS activity was measured by monitoring the conversion of l-[14C]arginine to l-[14C]citrulline as described previously (45Hori H. Iwasaki T. Kurahashi Y. Nishino T. Biochem. Biophys. Res. Commun. 1997; 234: 476-480Crossref PubMed Scopus (10) Google Scholar). The standard assay was performed at 25 °C in assay mixture containing 16.7 mm HEPES-NaOH buffer, pH 7.4, 4.2 mm Tris-HCl buffer, pH 7.4, 667 μm EDTA, 167 μm EGTA, 667 μm dithiothreitol, 16.7 μml-[U-14C]arginine, 667 μm NADPH, 1.2 mm CaCl2, 6.7 μg of calmodulin, 1.25 μm FAD, 1.25 μm FMN, 2.5 μm H4BP and the enzyme, in a total volume of 30 μl. The specific activity ofl-[U-14C]Arg used in the assays was 11.84 GBq/mmol. The NADPH-dependent diaphorase activity was measured using dichlorophenolindophenol as an artificial electron acceptor by monitoring the NADPH-dependent reduction of dichlorophenolindophenol at A 600 nm, essentially as described previously (9Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar, 46Gachhui R. Presta A. Bentley D.F. Abu-Soud H.M. McArthur R. Brudvig G. Ghosh D.K. Stuehr D.J. J. Biol. Chem. 1996; 271: 20594-20602Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The standard assay was performed at 25 °C in an assay mixture containing 50 mm Tris-HCl buffer, pH 7.4, 100 μm dithiothreitol, 85 μm NADPH, 50 μm dichlorophenolindophenol , 100 μg of bovine serum albumin, 5 μm FAD, 5 μm FMN, and the enzyme in a total volume of 1 ml. The effect of Ca2+-calmodulin complex on NADPH diaphorase activity was measured in the same assay mixture, except for the presence of 1 mm CaCl2 and 10 μg of calmodulin. Recombinant nNOS1 and nNOS2 produced using the baculovirus-insect cell expression system were partially purified on a 2′,5′-ADP-Sepharose 4B column (Amersham Pharmacia Biotech), followed by a fast desalting column, Ampure SA (Amersham Pharmacia Biotech). Purification of recombinant nNOS1 and nNOS2 produced in E. coli strain BL21 was performed on ice or at 4 °C essentially as described in the literature (40Roman L.J. Sheta E.A. Martasek P. Gross S.S. Liu Q. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar), except that purification was conducted using 2′,5′-ADP-Sepharose 4B column chromatography (Amersham Pharmacia Biotech), followed by Sephacryl S200HR and DEAE-Sepharose Fast Flow column chromatography (Amersham Pharmacia Biotech) (47Iwasaki T. Hori H. Hayashi Y. Nishino T. J. Biol. Chem. 1999; 274: 7705-7713Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). H4BP (10 μm) was supplied in the ultrasonification step. The catalytic activity and the purity of the purified wild-type enzyme, nNOS1, were comparable with those previously reported for recombinant nNOS1 by others (40Roman L.J. Sheta E.A. Martasek P. Gross S.S. Liu Q. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar). Absorption spectra were recorded using a Hitachi U3210 spectrophotometer or a Beckman DU-7400 spectrophotometer. EPR measurements were carried out using a JEOL JEX-RE1X spectrometer equipped with an Air Products model LTR-3 Heli-Tran cryostat system, in which the temperature was monitored with a Scientific Instruments series 5500 temperature indicator/controller as reported previously (47Iwasaki T. Hori H. Hayashi Y. Nishino T. J. Biol. Chem. 1999; 274: 7705-7713Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 48Iwasaki T. Wakagi T. Isogai Y. Tanaka K. Iizuka T. Oshima T. J. Biol. Chem. 1994; 269: 29444-29450Abstract Full Text PDF PubMed Google Scholar). EPR spectra of several different batches of recombinant nNOS samples were also measured at JEOL Ltd. (Tokyo, Japan), using a JEOL JES-TE200 spectrometer equipped with an ES-CT470 Heli-Tran cryostat system, in which the temperature was monitored with a Scientific Instruments digital temperature indicator/controller model 9650 and the magnetic field was monitored with a JEOL NMR field meter ES-FC5. All spectral data were processed using KaleidaGraph software version 3.05 (Abelbeck Software). Purified nNOS was estimated by using the Coomassie protein assay reagent (Pierce) with bovine serum albumin as a standard. The homology search against data bases was performed with the BEAUTY and BLAST network service (49Worley K.C. Wiese B.A. Smith R.F. Genome Res. 1995; 5: 173-184Crossref PubMed Scopus (227) Google Scholar). The multiple sequence alignments were performed using the CLUSTAL X graphical interface (50Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35617) Google Scholar) with small manual adjustments. The alternatively spliced product of mouse nNOS variant, nNOS2, is specifically expressed in the central nervous system and has a 105-amino acid deletion in the C-terminal highly conserved region of the heme-binding domain (29Ogura T. Yokoyama T. Fujisawa H. Kurashima Y. Esumi H. Biochem. Biophys. Res. Commun. 1993; 193: 1014-1022Crossref PubMed Scopus (108) Google Scholar) (Fig. 1). We first produced the nNOS2 cDNA gene product in a baculovirus-Sf9 insect cell expression system and partially purified nNOS2 as described under “Experimental Procedures.” In contrast to the case of the recombinant wild-type enzyme nNOS1, partially purified nNOS2 lacked citrulline-forming activity at 25–37 °C (data not shown). Because essentially the same result has been obtained with recombinant mouse nNOS2 expressed in cell culture (29Ogura T. Yokoyama T. Fujisawa H. Kurashima Y. Esumi H. Biochem. Biophys. Res. Commun. 1993; 193: 1014-1022Crossref PubMed Scopus (108) Google Scholar, 30Fujisawa H. Ogura T. Kurashima Y. Yokoyama T. Yamashita J. Esumi H. J. Neurochem. 1994; 63: 140-145Crossref PubMed Scopus (60) Google Scholar, 51Ogura T. Fujisawa H. Yokoyama T. Kurashima Y. Esumi H. Moncada S. Proceedings of the Third International Meeting on Biology of Nitric Oxide. Portland Press, London1994Google Scholar), we constructed the pCWori+ vector harboring the cDNA of the full-length nNOS2 gene, and produced the recombinant mouse nNOS2 in an E. coli expression system co-producingE. coli GroELS for detailed spectroscopic characterization. The recombinant enzyme was purified as described under “Experimental Procedures” and was used for further characterization (see below). Fig.2 shows typical optical absorption spectra of purified mouse nNOS2 produced in E. coli strain BL21. The ferric form of nNOS2 purified either in the presence or absence of l-arginine contained bound protoheme IX, whose content varied considerably from preparation to preparation. Thus, a broad Soret peak centered at 416 nm was clearly visible in most preparations (represented by preparation 1) of purified nNOS2, 2The typical optical spectra of purified nNOS2 (preparation 1) were essentially identical to those of partially purified nNOS2 heme domain produced in E. coli as a glutathione S-transferase fusion protein in the absence of H4BP, except for the absence of the spectral contribution of the diflavin reductase domain (T. Iwasaki, H. Hori, and T. Nishino, unpublished results). whereas a small shoulder around 420 nm could be detected in a few instances in which the spectral contribution was primarily from the bound diflavin centers in the reductase domain (represented by preparation 2) (Fig.2 A). In either case, the occupancy of bound heme in purified nNOS2 was consistently much lower than that of r

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