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• W2002775305 abstract "Neuronal nitric-oxide synthase (nNOS) differs from inducible NOS (iNOS) in both its dependence on the intracellular Ca2+ concentration and the production rate of NO. To investigate what difference(s) exist between the two NOS flavin domains at the electron transfer level, we isolated the recombinant human NOS flavin domains, which were co-expressed with human calmodulin (CaM). The flavin semiquinones, FADH• and FMNH•, in both NOSs participate in the regulation of one-electron transfer within the flavin domain. Each semiquinone can be identified by a characteristic absorption peak at 520 nm (Guan, Z.-W., and Iyanagi, T. (2003) Arch. Biochem. Biophys. 412, 65–76). NADPH reduction of the FAD and FMN redox centers by the CaM-bound flavin domains was studied by stopped-flow and rapid scan spectrometry. Reduction of the air-stable semiquinone (FAD-FMNH•) of both domains with NADPH showed that the extent of conversion of FADH2/FMNH• to FADH•/FMNH2 in the iNOS flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN) with NADPH resulted in the initial formation of a small amount of disemiquinone, which then decayed. The rate of intramolecular electron transfer between the two flavins in the iNOS flavin domain was faster than that of the nNOS flavin domain. In addition, the formation of a mixture of the two- and four-electron-reduced states in the presence of excess NADPH was different for the two NOS flavin domains. The data indicate a more favorable formation of the active intermediate FMNH2 in the iNOS flavin domain. Neuronal nitric-oxide synthase (nNOS) differs from inducible NOS (iNOS) in both its dependence on the intracellular Ca2+ concentration and the production rate of NO. To investigate what difference(s) exist between the two NOS flavin domains at the electron transfer level, we isolated the recombinant human NOS flavin domains, which were co-expressed with human calmodulin (CaM). The flavin semiquinones, FADH• and FMNH•, in both NOSs participate in the regulation of one-electron transfer within the flavin domain. Each semiquinone can be identified by a characteristic absorption peak at 520 nm (Guan, Z.-W., and Iyanagi, T. (2003) Arch. Biochem. Biophys. 412, 65–76). NADPH reduction of the FAD and FMN redox centers by the CaM-bound flavin domains was studied by stopped-flow and rapid scan spectrometry. Reduction of the air-stable semiquinone (FAD-FMNH•) of both domains with NADPH showed that the extent of conversion of FADH2/FMNH• to FADH•/FMNH2 in the iNOS flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN) with NADPH resulted in the initial formation of a small amount of disemiquinone, which then decayed. The rate of intramolecular electron transfer between the two flavins in the iNOS flavin domain was faster than that of the nNOS flavin domain. In addition, the formation of a mixture of the two- and four-electron-reduced states in the presence of excess NADPH was different for the two NOS flavin domains. The data indicate a more favorable formation of the active intermediate FMNH2 in the iNOS flavin domain. Nitric-oxide synthase (NOS 1The abbreviations used are: NOS, nitric-oxide synthase; human nNOS, human neuronal nitric oxide synthase; human iNOS, human inducible nitric oxide synthase; CaM, Ca2+-dependent calmodulin; CPR, NADPH-cytochrome P450 reductase.; EC 1.14.13.39) catalyzes the oxidation of l-arginine to nitric oxide and citruline. Three genetically distinct NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) have been identified from neurons, endothelial cells, and macrophages, respectively (1Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2173) Google Scholar, 2Lamas S. Marsden P.A. Li G.K. Tempst P. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6348-6352Crossref PubMed Scopus (921) Google Scholar, 3Lyons C.R. Orloff G.J. Cunningham J.M. J. Biol. Chem. 1992; 267: 6370-6374Abstract Full Text PDF PubMed Google Scholar, 4Roman L.J. Martàsek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1189Crossref PubMed Scopus (177) Google Scholar). Each NOS monomer comprises an N-terminal oxygenase domain with the binding sites for a P450-like cysteine thiolate-ligated heme, tetrahydrobiopterin cofactor (H4B), and l-arginine. The C-terminal reductase domain contains the binding sites for FAD, FMN, and the NADPH cofactor (5Klatt P. Schmidt K. Uray G. Mayer B. J. Biol. Chem. 1993; 268: 14781-14787Abstract Full Text PDF PubMed Google Scholar, 6Bredt D.S. Ferris C.D. Snyder S.H. J. Biol. Chem. 1992; 267: 10976-10981Abstract Full Text PDF PubMed Google Scholar, 7Schmidt H.H.H.W. Smith R.M. Nakane M. Murad F. Biochemistry. 1992; 31: 3243-3249Crossref PubMed Scopus (145) Google Scholar, 8Stuehr D.J. Cho H.J. Kwon N.S. Weise M.F. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7773-7777Crossref PubMed Scopus (733) Google Scholar, 9Hevel J.M. Marletta M.A. Biochemistry. 1992; 31: 7160-7165Crossref PubMed Scopus (169) Google Scholar, 10McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Crossref PubMed Scopus (357) Google Scholar, 11Stuehr D.J. Ikeda-Saito M. J. Biol. Chem. 1992; 267: 20547-20550Abstract Full Text PDF PubMed Google Scholar). The two domains are linked by a functional peptide of 20–25 amino acids, which binds calmodulin (CaM) (1Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2173) Google Scholar, 6Bredt D.S. Ferris C.D. Snyder S.H. J. Biol. Chem. 1992; 267: 10976-10981Abstract Full Text PDF PubMed Google Scholar, 7Schmidt H.H.H.W. Smith R.M. Nakane M. Murad F. Biochemistry. 1992; 31: 3243-3249Crossref PubMed Scopus (145) Google Scholar). The presence of functional domains within the enzyme has allowed a detailed biochemical, biophysical, and crystallographic study of the isolated domains. Recently, the x-ray crystallographic structure of the eNOS and iNOS oxygenase domains (12Crane B.R. Arvai A.S. Ghosh D.K. Wu C.Q. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1998; 279: 2121-2126Crossref PubMed Scopus (626) Google Scholar, 13Raman C.S. Li H. Martàsek P. Kràl V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 14Fischmann T.O. Hruza A. Niu X.D. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Reichert P. Lundell D.J. Narula S.K. Weber P.C. Nat. Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (410) Google Scholar) and the C-terminal FAD/NADPH binding domain of nNOS were reported (15Zhang J. Martàsek P. Paschke R. Shea T. Masters B.S.S. Kim J-J.P. J. Biol. Chem. 2001; 276: 37506-37513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The latter structure is superimposable on those of NADPH-cytochrome P450 reductase (CPR). The x-ray crystal structure of CPR, which contains one molecule each of FAD and FMN (16Iyanagi T. Mason H.S. Biochemistry. 1973; 12: 2297-2308Crossref PubMed Scopus (280) Google Scholar), indicates that direct electron transfer between the two flavins is most likely, given their close proximity (17Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (671) Google Scholar). The genes encoding nNOS and eNOS are constitutively expressed, and their enzymatic activities are regulated by the intracellular free Ca2+ concentration through binding of Ca2+/CaM (4Roman L.J. Martàsek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1189Crossref PubMed Scopus (177) Google Scholar, 18Cho H.J. Xie Q.W. Calaycay J. Mumford R.A. Swiderek K.M. Lee T.D. Nathan C. J. Exp. Med. 1992; 176: 599-604Crossref PubMed Scopus (564) Google Scholar, 19Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2147) Google Scholar). In contrast, iNOS activity is regulated predominantly at the transcriptional level and binds CaM with such high affinity that it is already fully activated at the free Ca2+ level of a resting cell (20Nathan C. Xie Q-W. Cell. 1994; 78: 915-918Abstract Full Text PDF PubMed Scopus (2758) Google Scholar). Thus, nNOS and eNOS have typically been used to investigate the mechanism of CaM activation of NOS. The CaM-dependent activation mechanism is modulated by several unusual structural features within the NOS flavin domain, such as an autoinhibitory loop in the FMN binding subdomain that is absent in iNOS, kinase-dependent phosphorylation sites, and a C-terminal extension (tail) (4Roman L.J. Martàsek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1189Crossref PubMed Scopus (177) Google Scholar, 21Salerno J.C. Harris D E. Irizarry K. Patel B. Morales A.J. Smith S.M.E. Martàsek P. Roman L.J. Masters B.S.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 22Roman L.J. Martàsek P. Miller R.T. Harris D.E. de la Garza M.A. Shea T.M. Kim J.-J. Masters B.S.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). All three NOS isoforms share 50–60% overall sequence similarity, and they have an analogous electron transfer pathway (NADPH → FAD → FMN → heme) but have different NO production rates. Turnover numbers for NO synthesis of ∼200 min–1, 100 min–1, and 20 min–1 for iNOS, nNOS, and eNOS, respectively, have been reported (4Roman L.J. Martàsek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1189Crossref PubMed Scopus (177) Google Scholar). A chimeric enzyme in which the heme domain of one isoform was connected to the flavin domain of another (23Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: 5566-5571Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) indicated that an increased flavin domain activity could lead to a corresponding increase in NO synthase activity. This raises the possibility that there are differences in the flavin-mediated electron transfer between the NOS isoforms. The electron transfer from the flavin domain to the heme domain is thought to be stimulated by the binding of CaM. CaM can influence two possible electron transfer steps: from NADPH to FAD or/and from FAD to FMN (24Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar, 25Matsuda H. Iyanagi T. Biochim. Biophys. Acta. 1999; 1473: 345-355Crossref PubMed Scopus (84) Google Scholar, 26Guan Z-W. Iyanagi T. Arch. Biochem. Biophys. 2003; 412: 65-76Crossref PubMed Scopus (39) Google Scholar). The NOS flavin domain is related structurally and functionally to CPR (16Iyanagi T. Mason H.S. Biochemistry. 1973; 12: 2297-2308Crossref PubMed Scopus (280) Google Scholar, 17Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (671) Google Scholar). Both enzymes have cytochrome c and ferricyanide reductase activities, and each flavin has different redox properties (27Iyanagi T. Makino N. Mason H.S. Biochemistry. 1974; 13: 1701-1710Crossref PubMed Scopus (158) Google Scholar, 28Noble M.A. Munro A.W. Rivers S.L. Robledo L. Daff S.N. Yellowlees L.J. Shimizu T. Sagami I. Guillemette J.G. Chapman S.K. Biochemistry. 1999; 38: 16413-16418Crossref PubMed Scopus (122) Google Scholar). However, the nNOS flavin domain is controlled by Ca2+-dependent CaM binding, which can stimulate the reduction of cytochrome c (4Roman L.J. Martàsek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1189Crossref PubMed Scopus (177) Google Scholar, 25Matsuda H. Iyanagi T. Biochim. Biophys. Acta. 1999; 1473: 345-355Crossref PubMed Scopus (84) Google Scholar, 26Guan Z-W. Iyanagi T. Arch. Biochem. Biophys. 2003; 412: 65-76Crossref PubMed Scopus (39) Google Scholar). This suggests that CaM may enhance NOS cytochrome c reduction by directly activating the reductase domain itself. Craig et al. (30Craig D.H. Chapman S.K. Daff S. J. Biol. Chem. 2002; 277: 33987-33994Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) have shown that the reductase domain will be conformationally locked and unable to transfer electrons efficiently to cytochrome c. CaM causes a shift in the orientation of the FMN subdomain, thereby unlocking the reductase activity, enabling efficient transfer of electrons to cytochrome c. Furthermore, the crystal structure of the FAD/NADPH subdomain suggests that a C-terminal tail bisects the FMN and FAD redox centers (15Zhang J. Martàsek P. Paschke R. Shea T. Masters B.S.S. Kim J-J.P. J. Biol. Chem. 2001; 276: 37506-37513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 22Roman L.J. Martàsek P. Miller R.T. Harris D.E. de la Garza M.A. Shea T.M. Kim J.-J. Masters B.S.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In the presence of CaM, this lock is removed, facilitating electron transfer between FAD and FMN. Intramolecular electron transfer between the two flavins in CPR has been studied using laser flash photolysis (31Bhattacharyya A.K. Lipka J.J. Waskell L. Tollin G. Biochemistry. 1991; 30: 759-765Crossref PubMed Scopus (42) Google Scholar) and temperature-jump relaxation spectroscopy (32Gutierrez A. Paine M. Wolf C.R. Scrutton N.S. Roberts G.C.K. Biochemistry. 2002; 41: 4626-4637Crossref PubMed Scopus (80) Google Scholar). Pulse radiolysis was used to study the electron transfer in nNOS (33Kobayashi K. Tagawa S. Daff S. Sagami I. Shimizu T. J. Biol. Chem. 2001; 276: 39864-39871Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These data suggest that domain movement within the protein may play a major role in the mechanism of intramolecular electron transfer. Adak et al. (34Adak S. Sharma M. Meade A.L. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13516-13521Crossref PubMed Scopus (48) Google Scholar) speculated that a conserved phenyl side chain, Phe1399 of rat nNOS (corresponding to Trp677 of human CPR), located close to the isoalloxazine ring of FAD, forms part of a conformational trigger mechanism that controls CaM-dependent electron transfer. Although this work has established the importance of the flavin domain in regulating CaM-dependent electron transfer, it is still unclear as to how the FAD and FMN centers communicate in the transfer of electrons within the nNOS and iNOS flavin domains. In this study, we have measured the rapid kinetic behavior of each FAD and FMN semiquinone bound to the flavin domains of human nNOS and iNOS and provide direct evidence that CaM binding stimulates the one-electron transfer between the two flavins. Materials—2′,5′-ADP-Sepharose 4B was purchased from Amersham Biosciences, and DE52-cellulose was from Whatman Inc. NADPH and NADP+ were obtained from Yeast Co. Ltd. (Tokyo, Japan). 2′,3′-AMP was purchased from Sigma. Cytochrome c was obtained from Roche Applied Science. All restriction and modification enzymes used for recombinant DNA technology were purchased from Takara Shuzo (Osaka, Japan), Toyobo (Osaka, Japan), and Nippon Gene (Toyama, Japan), and all other chemicals were of the highest grade available from commercial suppliers. Plasmids—The plasmid MDN75–3′-5′ (XbaI/MunI) human nNOS, containing the human neuronal NOS cDNA in pBluescript I SK (–) was kindly provided by Dr. Philip A. Marsden (University of Toronto) (35Wang 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). The plasmid, containing human iNOS cDNA in pBluescript II SK (–) was kindly supplied by Dr. Richard A. Shapiro (University of Pittsburgh). Human calmodulin cDNA, pKK CaM III was kindly supplied by Dr. E. Strehler (Mayo Clinic Foundation, Rochester, MN). The expression vector (tac promoter) pCWori+ plasmid DNA was kindly supplied by Dr. F. W. Dahlquist (University of Oregon, Eugene, OR). Molecular Biology—The expression plasmid for human CaM III was constructed using standard procedures. The human CaM III cDNA fragment, cut from plasmid pKK CaM III with restriction enzymes EcoRI and ScaI, was ligated into expression vector pACY. The expression plasmids for the recombinant human nNOS and iNOS flavin domains (containing NADPH, FAD, FMN, and CaM binding sites) were constructed as follows. The DNA fragments of 2145 nucleotides (2598–4742) for the human nNOS flavin domain and 1953 nucleotides (1510–3462) for the human iNOS flavin domain were amplified by PCR with restriction sites NdeI/XbaI and NdeI/HindIII, respectively. The oligonucleotides used as primers were as follows: nNOS, sense (5′-AAGAATTCTCATATGTCCACCAACGGGACC-3′) and antisense (5′-ACAGAATTCGGTCTAGAGGGTCCAG-3′); iNOS, sense (5′-AGAATTCATATGGAGAAGCGGAGACC-3′) and antisense (5′-GACTCCCGGATGTAGATCTCTTAAGA-3′). The PCR products were resolved by agarose gel electrophoresis and gel-extracted, and the nNOS and iNOS fragments were digested with NdeI/XbaI and NdeI/HindIII, respectively. The digestion products were ligated into expression vector pCWori+ using the appropriate restriction sites within the polylinker region. For expression studies, the plasmid constructs were transformed into Escherichia coli BL21 harboring the compatible plasmid pACY-CaM III. Transformed clones were selected and maintained on LB medium containing 50 μg/ml ampicillin and 20 μg/ml tetracycline. Recombinant clones were verified by restriction mapping and DNA sequencing. Protein Expression and Purification—The human CaM III, human nNOS FAD/NADPH binding domain, and CaM-free human nNOS flavin (FAD/FMN) domain were expressed and purified as described previously (26Guan Z-W. Iyanagi T. Arch. Biochem. Biophys. 2003; 412: 65-76Crossref PubMed Scopus (39) Google Scholar). The human nNOS and iNOS flavin domains, which were co-expressed with the recombinant plasmid pACY-CaM III in E. coli cells were purified by DE52 and 2′,5′-ADP Sepharose 4B chromatography. Recombinant E. coli BL21 harboring the compatible plasmids was cultured in LB medium, containing 50 μg/ml ampicillin and 20 μg/ml tetracycline, at 30 °C for 24 h. Expression was induced at A 600 = 0.1–0.2 with 0.5 mm isopropyl-β–d–thiogalactopyranoside. Cells were harvested by centrifugation, washed in buffer A (10 mm Hepes, pH 7.0, 5% glycerol, 1 mm CaCl2, 0.2 mm phenylmethylsulfonyl fluoride), and frozen at –80 °C until required. The E. coli cells were disrupted by sonication (10 pulses of 30 s) in buffer A on ice. Cell debris was removed by centrifugation 100,000 × g for 30 min at 4 °C, and the supernatant was applied to a DE52 column (35 × 20 cm) equilibrated with buffer A. The enzyme adsorbed on the column as a blue band and was eluted with 0.1 m NaCl in buffer A. The active fractions were pooled and loaded onto a 2′,5′-ADP-Sepharose 4B (2 × 4 cm) affinity column and then oxidized using ferricyanide. After washing with 200 ml of 0.1 m NaCl, 1 mm 2′,3′-AMP in buffer A, the enzyme was eluted with 100 ml of 0.3 m NaCl, 10 mm 2′,3′-AMP in buffer A. The active fractions were identified by SDS-PAGE, and 2′,3′-AMP was removed by centrifugation at 4 °C using an Ultrafree-15 centrifugal filter device. Based on SDS-PAGE analysis, the purified enzymes bound CaM (17–18 kDa) in a ratio of 1:1 and had an estimated molecular mass of about 80 and 74 kDa for the human nNOS and iNOS flavin domains, respectively. In addition, both the purified flavin domains contained FAD and FMN with a ratio of 1:1 determined by capillary electrophoresis (36Britz-McKibbin P. Markuszewski M.J. Iyanagi T. Matsuda K. Nishioka T. Terabe S. Anal. Chem. 2003; 57: 87-95Google Scholar). The enzymes were further purified by a DE-52 column prior to experiments. Quantitative Analysis of CaM Bound to NOS Flavin Domain—The quantities of CaM bound to NOS flavin domain were analyzed by SDS-PAGE using purified human CaM III as a standard. The intensity of the protein bands was quantified using Gel Plotting Macros software. The co-expressed nNOS and iNOS flavin domain bound CaM with a ratio ([E]/[CaM]) of 1.0:1.2 and 1.0:1.1, respectively. The values represent the means ± S.D. of three independent measurements each. Spectrophotometric Methods—Enzyme concentrations were calculated from the absorbance at 457 nm of the NOS flavin domains, using an extinction coefficient of 22.9 mm–1 cm–1 for the human nNOS flavin domain (26Guan Z-W. Iyanagi T. Arch. Biochem. Biophys. 2003; 412: 65-76Crossref PubMed Scopus (39) Google Scholar) and 23.1 mm–1 cm–1 for the human iNOS flavin domain. The extinction coefficient of the CaM-bound oxidized human iNOS flavin domain was determined by comparing the absorbance at the flavin peak at 457 nm in the holoenzyme with the absorbance of the free flavins (ϵ450 = 11.3 mm–1 cm–1 for FAD and ϵ450 = 12.5 mm–1 cm–1 for FMN) released by the addition of 5% sodium lauroylsarcosine. The concentration of NADPH was determined using an extinction coefficient of 6.2 mm–1 cm–1 at 340 nm. The concentration of cytochrome c was measured by monitoring the absorbance change at 550 nm, using a difference extinction coefficient of 21 mm–1 cm–1 between the reduced and oxidized forms. The percentage conversion of FADH2/FMNH• to FADH•/FMNH2 (see Fig. 7) was calculated using a difference extinction coefficient of 2.3 mm–1 cm–1 at 520 nm between the FAD and FMN semiquinone forms (26Guan Z-W. Iyanagi T. Arch. Biochem. Biophys. 2003; 412: 65-76Crossref PubMed Scopus (39) Google Scholar). The percentage conversion of the oxidized enzyme to the fully reduced form (see Fig. 8) was calculated from the extinction coefficient of 8.6 mm–1 cm–1 at 596 nm for nNOS disemiquinone (FADH•/FMNH•). The extinction coefficient of disemiquinone at 596 nm was determined by comparing the absorbance of disemiquinone at 596 nm with the absorbance at 457 nm of the oxidized enzymes (see Fig. 2B).Fig. 8Reduction of the CaM-bound oxidized human nNOS (A1 and A2) and iNOS (B1 and B2) flavin domains. The spectra were recorded by using rapid scan (A1 and B1) and stopped-flow methods (A2 and B2) after 5 μm enzyme with 50 μm NADPH in 10 mm Hepes buffer containing 1 mm CaCl2, pH 7.0, 25 °C. A1, spectra were recorded at 16, 40, 88, 136, and 184 ms (spectra 1–5). B1, spectra were recorded at 16, 40, 64, 88, 136, and 184 ms (spectra 1–6) after the initial mixing event and at a long wavelength period (520–700 nm). A2 and B2, the spectra were recorded at two wavelengths (457 and 596 nm) and over a time period of 500 ms. The arrows indicate the predicated start point for the absorbance changes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2Absorption spectra of the semiquinone species derived from the human nNOS flavin domain. A, the spectrum for FAD semiquinone (FADH•) was recorded at 5 min after mixing the human nNOS FAD/NADPH domain with a 10-fold molar excess of NADPH in 10 mm Hepes buffer, pH 7.0, 25 °C. B, the spectrum for air-stable semiquinone (FAD-FMNH•) was recorded at 10 min after mixing CaM-bound human nNOS flavin domain with a 5-fold molar excess of NADPH in 10 mm Hepes buffer, pH 7.0, 25 °C, and the spectrum for disemiquinone (FADH•-FMNH•) was plotted based on the absorbance of FAD and FMN semiquinone species at several wavelengths. The extinction coefficient of disemiquinone at 596 nm was calculated as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) K m and k cat values were determined from a Lineweaver-Burk plot of the data (Table I). The K m and k cat values for NADPH were measured between 0.5 and 4.0 μm NADPH at a constant concentration of 100 μm ferricyanide as an electron acceptor. The K m and k cat values for ferricyanide and cytochrome c were determined in the concentration ranges of 0.2–2.0 mm ferricyanide and 0.25–200 μm cytochrome c and at a constant concentration of 300 μm NADPH as electron donor, respectively. Spectra analysis was performed using a Shimazu model MSP UV-2000 spectrophotometer in 10 mm Hepes buffer, pH 7.0, 25 °C.Table IApparent kinetic parameters for the CaM-bound human nNOS and iNOS flavin domainsElectron acceptorsKmnNOS flavin domainiNOS flavin domainμ mK3Fe(CN)6293.6 ± 84.1364.4 ± 80.4Cytochrome c0.2 ± 0.0414.9 ± 2.4NADPHaNADPH was used as an electron donor.0.2 ± 0.050.84 ± 0.18k catmin-1K3Fe(CN)68861.8 ± 654.413,294.9 ± 838.7Cytochrome c836.5 ± 34.14170.5 ± 576a NADPH was used as an electron donor. Open table in a new tab Stopped-flow Spectroscopy—Stopped-flow reactions were performed using a Union Giken model RA401 stopped-flow spectrophotometer equipped with a photodiode array detector, which has a dead time of 5 ms. For rapid scan experiments, the spectra were collected between 440 and 700 nm (see Fig. 5) and between 500 and 700 nm (see Figs. 7 and 8). The one-electron-reduced air-stable semiquinone form of the two flavin domains were generated by incubating the oxidized flavin domains with a 5-fold molar excess of NADPH for 10 min at 25 °C. All stopped-flow experiments were performed in 10 mm Hepes buffer, pH 7.0, and at a constant temperature of 25 °C. The apparent rate constants for the reduction of the human nNOS and iNOS flavin domains and their air-stable semiquinone forms with NADPH were determined by plotting the absorbance changes at 457 and 520 nm against time (t) as previously described (26Guan Z-W. Iyanagi T. Arch. Biochem. Biophys. 2003; 412: 65-76Crossref PubMed Scopus (39) Google Scholar). The results are summarized in Tables II and III.Table IIApparent rate constants for reduction of air-stable semiquinone (SQ) of the CaM-bound NOS flavin domains457 nm520 nmturnover/snNOS-SQFast phase25.8 (65.5%)50.2Slow phase12.0 (34.5%)iNOS-SQFast phase23.5 (75.8%)44.1Slow phase11.2 (24.2%) Open table in a new tab Table IIIApparent rate constants for reduction of CaM bound oxidized NOS flavin domainsEnzymeRate constantsFast component (percentage of total)Fast phaseSlow phases-1%nNOS flavin domain25.711.870.0iNOS flavin domain72.016.860.0 Open table in a new tab Photoreduction—The flavin cofactor in the enzyme was photoreduced in an anaerobic cuvette containing 20 μm protein, 1 μm 5-deazariboflavin, 5 mm EDTA, 1 μm indigodisulfonate, in 10 mm Hepes buffer, pH 7.0 (see Fig. 4). The solutions were made anaerobic by successive flushing with oxygen-free argon gas with gentle agitation for more than 60 min. The absorption spectra were measured before and after illumination at 25 °C with a 300-watt halogen lamp at room temperature. Preparation of CaM-bound Enzymes—Both the neuronal and inducible NOS have a functional peptide of 20–25 amino acids located between the N-terminal oxygenase domain and C-terminal reductase domain, which binds calmodulin (CaM). CaM binds NOS in a 1:1 stoichiometry with very high affinity (K D values typically 1–2, 0.1, and 4 nm for nNOS, iNOS, and eNOS, respectively) (4Roman L.J. Martàsek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1189Crossref PubMed Scopus (177) Google Scholar). In this work, the expression construct for either the human nNOS and iNOS flavin domain was co-expressed with human calmodulin III (CaM III) in an E. coli host and purified as described under “Experimental Procedures.” Both of the flavin domains absorbed to a 2′,5′-ADP-Sepharose 4B affinity column to form a greenish band, indicating the formation of an air-stable semiquinone form (25Matsuda H. Iyanagi T. Biochim. Biophys. Acta. 1999; 1473: 345-355Crossref PubMed Scopus (84) Google Scholar, 26Guan Z-W. Iyanagi T. Arch. Biochem. Biophys. 2003; 412: 65-76Crossref PubMed Scopus (39) Google Scholar, 37Miller R.T. Martàsek P. Omura T. Masters B.S.S. Biochem. Biophys. Res. Commun. 1999; 265: 184-188Crossref PubMed Scopus (74) Google Scholar), which was later confirmed spectrophotometrically. The enzymes were oxidized with ferricyanide solution before elution from the affinity column. The final products were at least 95% homogeneous as judged by SDS-PAGE. The apparent molecular masses of the human nNOS and iNOS flavin domains were ∼80 and 74 kDa, respectively, which are in good agreement with the predicted size of the polypeptides based on the gene sequence (78.7 and 71.7 kDa, respectively). In addition, both the co-expressed human nNOS and iNOS flavin domains bound CaM with a ratio of 1:1 based on SDS-PAGE analysis (see “Experimental Procedures” and Fig. 1). Reductase Activities of the CaM-bound Enzymes—Both NOS flavin domains can transfer electrons to several artificial electron acceptors. The apparent steady-state kinetic parameters for ferricyanide and cytochrome c were determined under turnover conditions and are listed in Table I. These values were not significantly changed by the further addition of Ca2+/CaM (data not shown). The iNOS flavin domain showed about 5-fold greater activity for cytochrome c and 1.5-fold greater ferricyanide reductase activity than the nNOS flavin domain. We have confirmed that ferricyanide mainly accepted electrons from FAD and cytochrome c mainly accepted electrons from FMN (26Guan Z-W. Iyanagi T. Arch. Biochem. Biophys. 2003; 412: 65-76Crossref PubMed Scopus (39) Google Scholar). The data strongly suggest that intramolecular (i.e. FAD to FMN) electron transfer is faster in the iNOS flavin domain than it" @default.
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• W2002775305 title "Mechanistic Studies on the Intramolecular One-electron Transfer between the Two Flavins in the Human Neuronal Nitric-oxide Synthase and Inducible Nitric-oxide Synthase Flavin Domains" @default.
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