The C Termini of Constitutive Nitric-oxide Synthases Control Electron Flow through the Flavin and Heme Domains and Affect Modulation by Calmodulin
2000; Elsevier BV; Volume: 275; Issue: 38 Linguagem: Inglês
10.1074/jbc.m004766200
ISSN1083-351X
AutoresLinda J. Roman, Pavel Martásek, R. Timothy Miller, D. Harris, Melissa A. de la Garza, Thomas M. Shea, Jung-Ja P. Kim, Bettie Sue Siler Masters,
Tópico(s)Electrochemical Analysis and Applications
ResumoThe sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20–40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7–21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs. The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20–40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7–21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs. nitric-oxide synthase rat neuronal nitric-oxide synthase bovine endothelial nitric-oxide synthase murine macrophage inducible nitric-oxide synthase NOS with a truncation at the C terminus nitric oxide NADPH-cytochrome P450 reductase cytochrome c (6R)-5,6,7,8-tetrahydrobiopterin polymerase chain reaction calmodulin wild type 2,6-dichlorophenolindophenol Nitric-oxide synthases (NOSs)1 are bidomain, dimeric enzymes that synthesize NO from l-arginine through a series of monooxygenation reactions (for review see Ref. 1Masters B.S.S. Ignarro L. Nitric Oxide. Academic Press, San Diego, CA2000Google Scholar). The three isoforms, neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), produce NO by the same mechanism but play very different physiological roles due to the type of cell where they are located. nNOS, located in neurons in the brain and at neuromuscular junctions, is involved in neurotransmission (2Garthwaite J. Charles S.L. Chess-Williams R. Nature. 1988; 336: 385-388Crossref PubMed Scopus (2284) Google Scholar, 3Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3128) Google Scholar); iNOS, located in macrophages, is involved in the immune response (4Marletta M.A. Yoon P.S. Iyengar R. Leaf C.D. Wishnok J.D. Biochemistry. 1988; 27: 8706-8711Crossref PubMed Scopus (1422) Google Scholar, 5Stuehr D.J. Gross S.S. Sakuma I. Levi R. Nathan C.F. J. Exp. Med. 1989; 169: 1011-1020Crossref PubMed Scopus (375) Google Scholar); and eNOS, located in endothelial cells, is involved in hemodynamic regulation (6Furchgott R.F. Vanhoutte P.M. Vasodilation: Vascular Smooth Muscle, Peptides, Autonomic Nerves, and Endothelium. Raven Press, Ltd., New York1988: 401-414Google Scholar, 7Ignarro L.J. Byrns R.E. Woods K.S. Vanhoutte P.M. Vasodilation: Vascular Smooth Muscle, Peptides, Autonomic Nerves, and Endothelium. Raven Press, Ltd., New York1988: 427-435Google Scholar). The NO produced by nNOS and eNOS exerts its effects through the stimulation of guanylate cyclase, whereas the NO produced by iNOS exerts its effects directly or by combining with superoxide anion radical to form peroxynitrite anion, both potent oxidants deleterious to proteins and DNA. The NOSs consist of two domains, a heme and H4B-containing oxygenase (or heme) domain, which binds the substratel-arginine, and a flavin-containing reductase (or flavoprotein) domain, which binds the prosthetic group flavins FAD and FMN and the cofactor NADPH. Electrons are transferred into NOS at the FAD moiety and are subsequently passed through the FMN to the heme domain. A calmodulin-binding region bisects the two domains. Calmodulin (CaM) is required for NO production and mediates the transfer of electrons from the FMN of NOS to the heme domain (8Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). CaM binds NOS in a 1:1 stoichiometry with very high affinity. CaM binds to sequences within the nNOS, eNOS, and iNOS CaM-binding sites withKD values of 1–2, 4, and 0.1 nm, respectively (9Sheta E. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar, 10Zhang M. Vogel H.J. J. Biol. Chem. 1994; 269: 981-985Abstract Full Text PDF PubMed Google Scholar, 11Vorherr T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (142) Google Scholar, 12Venema R.C. Sayegh H.S. Kent J.D. Harrison D.G. J. Biol. Chem. 1996; 271: 6435-6440Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 13Anagli J. Hofmann F. Quadroni M. Vorherr T. Carafoli E. Eur. J. Biochem. 1995; 233: 701-708Crossref PubMed Scopus (41) Google Scholar, 14Zoche M. Bienert M. Beyermann M. Koch K.W. Biochemistry. 1996; 35: 8742-8747Crossref PubMed Scopus (35) Google Scholar). Two of the isoforms (nNOS and eNOS) are constitutive; induction of NO synthesis activity requires an influx of calcium to promote calmodulin binding (3Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3128) Google Scholar, 15Förstermann U. Pollock J.S. Schmidt H.H.H.W. Heller M. Murad F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1788-1792Crossref PubMed Scopus (551) Google Scholar). The iNOS enzyme is induced at the transcriptional level, and calmodulin is bound even at basal calcium concentrations (16Cho 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). The basis for this dramatic difference of the calcium requirements between the inducible and constitutive NOSs has not been totally clarified, but differences in the CaM-binding sequences themselves do not explain it because swapping the binding sites between the inducible and constitutive NOSs does not completely confer the CaM binding properties of one to the other (12Venema R.C. Sayegh H.S. Kent J.D. Harrison D.G. J. Biol. Chem. 1996; 271: 6435-6440Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Although the three isoforms are about 50–60% identical at the amino acid level (17Dinerman J.L. Lowenstein C.J. Snyder S.H. Circ. Res. 1993; 73: 217-222Crossref PubMed Scopus (330) Google Scholar), recently published crystal structures of the heme domains of iNOS and eNOS (18Crane 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, 19Raman C.S. Li H. Martásek P. Král V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 20Fischmann 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, 21Li H. Raman C.S. Glaser C.B. Blasko E. Young T.A. Parkinson J.F. Whitlow M. Poulos T.L. J. Biol. Chem. 1999; 274: 21276-21284Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) and that of nNOS 2H. Li, P., Martásek, B. S. S. Masters, T. L. Poulos, and C. S. Raman, manuscript in preparation. demonstrate very minor structural differences; yet, these isoforms catalyze NO synthesis at vastly differing rates. The reductase domains of the NOSs have not yet been crystallized, but they appear to be structurally very similar to the NADPH-cytochrome P450 reductase (CPR), with the nNOS flavoprotein domain sharing 36% identity and 58% close homology at the amino acid level (22Bredt 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). Like those of CPR, the flavins of NOSs transfer electrons to artificial electron acceptors, such as cytochrome c, 2,6-dichlorophenolindophenol (DCIP), and ferricyanide (9Sheta E. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar, 23Miller R.T. Martásek P. Omura T. Masters B.S.S. Biochem. Biophys. Res. Commun. 1999; 265: 184-188Crossref PubMed Scopus (74) Google Scholar); these rates of reduction also differ very greatly from CPR and between the NOS isoforms. In addition to mediating electron transfer between the flavin and heme domains, CaM also has an effect on the flavin domain itself, as its binding increases the rates of reduction of cytc, DCIP, and ferricyanide by eNOS and nNOS and by flavoprotein constructs containing the CaM-binding sequences (24McMillan K. Masters B.S.S. Biochemistry. 1995; 34: 3686-3693Crossref PubMed Scopus (169) Google Scholar). To account for such differences in calcium dependence, NO production, and cyt c reduction in what are otherwise very homologous structures, attention has now focused on the search for control mechanisms in several regions that exist in the flavin domains of NOSs that have no counterpart in CPR. A putative autoinhibitory domain described by Salerno et al. (25Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M.E. Martásek 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), consisting of about 45 amino acids located in the middle of the FMN-binding region, is present in the constitutive NOSs (nNOS and eNOS) but is absent in iNOS and CPR. This autoinhibitory domain was proposed to control the calmodulin binding/activation of the constitutive isoforms. Deletion of this region in nNOS resulted in an enzyme that attained maximal NO synthesis at lower levels of free calcium and even retained 30% of its activity in the absence of calcium/CaM (26Daff S. Sagami I. Shimizu T. J. Biol. Chem. 1999; 274: 30589-30595Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), suggesting that the insert inhibits electron transfer from FMN to the heme in the absence of CaM and also destabilizes CaM binding at low calcium concentrations. The autoinhibitory peptide, however, does not limit the rates of NO synthesis and cyt c reduction; its effect is only on the calcium/CaM dependence. Another notable difference between the NOS isoforms and CPR is that each of the NOS isoforms contains about 21–42 additional amino acids at the C terminus (42, 33, and 21 amino acids in bovine eNOS, rat nNOS, and murine iNOS, respectively), forming a tail that is not present in CPR. Xie et al. (27Xie Q.W. Cho H. Kashiwabara Y. Baum M. Weidner J.R. Elliston K. Mumford R. Nathan C. J. Biol. Chem. 1994; 269: 28500-28505Abstract Full Text PDF PubMed Google Scholar) showed that deletion of 22 or 23 residues from the C terminus of iNOS, which removed the tail plus 1 or 2 additional residues, reduced the rate of NO synthesis 26 or 66%, respectively. We have shown that removing the C-terminal 21 amino acids from murine iNOS results in an enzyme with significantly higher cytochrome c reduction and NO synthesis activities (10- and 1.2-fold, respectively; see Ref. 28Roman L.J. Miller R.T. de la Garza M.A. Kim J.-J.P. Masters B.S.S. J. Biol. Chem. 2000; 275: 21914-21919Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The kinetic consequences of this truncation were an increased rate of electron transfer to and between the flavin moieties and from the flavins to the heme. In addition, the air-stable semiquinone form of the flavin domain, observed with the wild-type enzyme, was greatly destabilized, and the heme-nitrosyl complex, formed by the wild-type enzyme during NO synthesis, was decreased in the truncated form. However, iNOS lacks the autoinhibitory domain, and experiments cannot be done in the absence of CaM due to the expression system used, which coexpresses iNOS and CaM. Thus, any effect of the C-terminal tail on the CaM dependence of the enzyme could not be examined. To investigate the functional role of the additional 33 and 42 residues at the C terminus of nNOS and eNOS, respectively, we have removed them from nNOS and eNOS and compared the resulting enzymes to the intact proteins. The mechanistic implications of this deletion are discussed. (6R)-5,6,7,8-Tetrahydrobiopterin (H4B) was from Research Biochemicals International (Natick, MA), and all other chemicals were obtained from Sigma and were of the highest grade available. Pfu turbo polymerase was from Stratagene (La Jolla, CA); ligase and restriction enzymes were purchased from either Promega (Cambridge, MA) or New England Biolabs (Beverly, MA). Shrimp alkaline phosphatase was from United States Biochemical Corp. The rat nNOS cDNA was provided by Drs. Solomon Snyder and David Bredt at The Johns Hopkins Medical School, Baltimore. The bovine eNOS cDNA was provided by Dr. William Sessa at Yale University, New Haven. PCWori+ was given by Dr. Michael Waterman at Vanderbilt University in Nashville, TN. nNOSpCW-tr1 and eNOSpCW-tr1, the plasmids for the expression of the truncated nNOS (residues 1–1397) and eNOS (residues 1–1163) in Escherichia coli,were constructed as follows. For nNOS-tr1, the initial 1397 codons of nNOSpCW (29Roman L.J. Sheta E.A. Martásek 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) (from the ATG start codon to the final residue minus 33) were amplified by PCR. Primer tr1-1 (upstream primer, withNdeI site) was 5′-TGCTTAGGAGGTCATATG-3′, and primer tr1-2 (return primer, with XbaI site) was 5′- CTAGTCTAGATTATCCAAAGATGTCCTCGTG-3′. Primers were synthesized by the Center for Advanced DNA Technologies at the University of Texas Health Science Center, San Antonio. Reaction mixtures included 50 pmol of each primer, 20 ng of nNOSpCW template, 200 μm dNTPs, 1× Pfu turbo polymerase buffer, and 2.5 units ofPfu turbo polymerase in 50 μl total volume. The mixture was preincubated for 1 min at 94 °C prior to the addition ofPfu turbo polymerase, followed by amplification for 18 cycles: 95 °C for 45 s, 55 °C for 60 s, and 68 °C for 10 min. The PCR product was gel-purified using the GeneClean II kit (Bio 101, Vista, CA) and digested with NdeI andXbaI. PCWori+ DNA was digested withNdeI and XbaI, and the ends were dephosphorylated. The resulting nNOS cDNA and pCW plasmid were ligated, and the ensuing products were used to transform E. coli XL10-gold competent cells (Stratagene) using the manufacturer's instructions. Positive colonies were identified by restriction digest analysis of plasmid DNA isolated from small (2 ml) cultures of select colonies. For eNOS-tr1, glycine in position 1163 of bovine eNOS represents the residue aligned with the terminal serine of CPR. Preparation of parental plasmid bov-eNOSpCWori+ was described previously (30Martásek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar). The sense primer 5′-GACATCCTGAGAAACCGAGCTG-3′ was located between nucleotides 3276 and 3297 and the antisense primer 5-TATATGAATTCTCATCAGCCGAAAATGTCCTCGTGATAGC-3′ encompassed the codon for Gly-1163 followed by two stop codons and contains anEcoRI restriction site. A DNA product amplified by PCR using the parental plasmid as the template and above-mentioned oligonucleotides was digested by XhoI and EcoRI. The longer XhoI-EcoRI fragment was gel-purified. The parental plasmid was digested by XhoI-EcoRI; the longer fragment was gel-purified and ligated with theXhoI-EcoRI fragment from PCR. DNA from the new construct was sequenced in the PCR-amplified region using automated sequencing at the Center for Advanced DNA Technologies at the University of Texas Health Science Center, San Antonio. groELS was co-transformed with nNOSpCW-tr1 or eNOSpCW-tr1 into E. coli BL21 cells via electroporation using an Invitrogen Electroporator II (San Diego, CA) according to the manufacturer's instructions and plated on LB agar containing 50 μg/ml ampicillin and 35 μg/ml chloramphenicol. Protein expression and purification are as described in Roman et al. (29Roman L.J. Sheta E.A. Martásek 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) for nNOS and nNOS-tr1. Protein expression and purification for eNOS and eNOS-tr1 are as described in Martásek et al. (30Martásek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar). The peak fraction of dimeric protein from the gel filtration column was used for all analyses described. Both nNOS-tr1 and eNOS-tr1 were purified in tandem with their wild-type forms to reduce differences resulting from preparation to preparation. Absolute spectra and CO difference spectra were performed as described (29Roman L.J. Sheta E.A. Martásek 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). The molar protein concentrations for nNOS and nNOS-tr1 were determined based on heme content via reduced CO difference spectra, where ε = 100 mm−1 cm−1for ΔA445–470. The molar protein concentrations for eNOS and eNOS-tr1 were determined based on spectral determination at Soret maximum after subtraction of extrapolated base line, using ε = 100 mm−1. This protein concentration determination was in good agreement with that of CO difference spectum. All spectral analyses were performed using a Shimadzu model 2101 UV-visible dual-beam spectrophotometer. Stopped-flow reactions were performed aerobically under turnover conditions at 23 °C, as described in Miller et al. (23Miller R.T. Martásek P. Omura T. Masters B.S.S. Biochem. Biophys. Res. Commun. 1999; 265: 184-188Crossref PubMed Scopus (74) Google Scholar) and Roman et al.(28Roman L.J. Miller R.T. de la Garza M.A. Kim J.-J.P. Masters B.S.S. J. Biol. Chem. 2000; 275: 21914-21919Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), using an Applied Photophysics SX.18MV diode array stopped-flow spectrophotometer, which had a dead time of 2 ms. Reactions contained 2 μm enzyme, 100 μm NADPH, 10 μm H4B, and 100 μml-arginine in 50 mm HEPES, pH 7.4, and 100 mm NaCl. Where indicated, 3 μm CaM was also added. Heme reduction was monitored at 397 nm, and flavin reduction was monitored at the 485 nm shoulder, rather than the absorption peak value at 455 nm, to avoid spectral interference from the heme. Nitric oxide formation (hemoglobin capture assay and/or [3H]arginine to citrulline assay) and cytochrome c reduction were measured at 23 °C as described by Sheta et al. (9Sheta E. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar) and Martásek et al. (31Martásek P. Miller R.T. Roman L.J. Shea T. Masters B.S.S. Methods Enzymol. 1999; 301: 70-78Crossref PubMed Scopus (22) Google Scholar), with the exception that the cytochrome creduction assays were performed in a buffer containing 50 mm HEPES, pH 7.4, using an extinction coefficient of 21 mm−1 for reduced minus oxidized cyt c at 550 nm. NaCl was 0, 100, or 250 mm, as indicated in the figure or table legends. Ferricyanide reduction was performed the same as cyt c reduction except that 100 mm ferricyanide was used, and the extinction coefficient was 1.02 mm−1. Oxidation of NADPH was monitored at 340 nm in the presence of 50 mm HEPES, pH 7.4, 100 μm NADPH, and 26 nm truncated or 134 nm wild-type enzyme at 23 °C. In the case of NADPH oxidation under cyt creduction conditions, 250 mm NaCl was included. The rate was determined using an extinction coefficient of 6.2 mm−1 at 340 nm for NADPH. The reoxidation of reduced flavins was monitored at 485 nm for all enzymes in the presence of 50 mm HEPES, pH 7.4, 250 mm NaCl, 20 or 100 μm NADPH, and 2 μm enzyme at 23 °C. Fig. 1 shows the C-terminal final 8 amino acids of CPR and the co-alignment of several NOS sequences. Depicted are the murine iNOS, rat nNOS, and the bovine eNOS along with two non-mammalian sequences for comparison. All known NOSs appear to have additional C-terminal residues as compared with CPR. Also shown in the boxed region is a highly conserved 8-amino acid sequence just prior to the NOS tail. The penultimate amino acid in CPR, tryptophan, has been shown to be important for reductase activity. Specifically, it has been proposed to be involved in modulating the hydride transfer from NADPH to FAD and, in the crystal structure of CPR (32Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (671) Google Scholar), it appears to be a shielding or stacking residue for the FAD. The aromatic property of this residue is completely conserved in all known NOSs, and deletion of the C terminus of iNOS including this residue to the C-terminal end yields a protein with only 30% of wild-type NO synthesis activity (27Xie Q.W. Cho H. Kashiwabara Y. Baum M. Weidner J.R. Elliston K. Mumford R. Nathan C. J. Biol. Chem. 1994; 269: 28500-28505Abstract Full Text PDF PubMed Google Scholar), 3L. J. Roman and B. S. S. Masters, unpublished observations.whereas deletion of only the additional tail residues, i.e.the final 21 amino acids, yields a protein with 15% higher activity (28Roman L.J. Miller R.T. de la Garza M.A. Kim J.-J.P. Masters B.S.S. J. Biol. Chem. 2000; 275: 21914-21919Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The path of electron transfer through the NOSs is from NADPH to the FAD moiety, to FMN, and finally to the NOS heme (Scheme FS1). Like CPR, the NOSs also have the ability to reduce artificial electron acceptors, such as cyt c, DCIP, and ferricyanide. Cyt c and DCIP both accept electrons from the FMN, whereas ferricyanide can accept electrons directly from the FAD, as shown by its reduction by a CPR in which FMN has been removed (33Vermilion J.L. Coon M.J. J. Biol. Chem. 1978; 253: 8812-8819Abstract Full Text PDF PubMed Google Scholar, 34Alexander L.M. Hersh L.B. Masters B.S.S. Coon M.J. Conney A.H. Estabrook R.W. Gelboin H.V. Gillette J.R. O'Brien P.J. Microsomes, Drug Oxidations, and Chemical Carcinogenesis. Academic Press, Inc., New York1980: 285-288Google Scholar). When the iNOS isoform was expressed without the C-terminal tail (minus the 21 residues of the C terminus), the rate of cyt c reduction was increased 10-fold (28Roman L.J. Miller R.T. de la Garza M.A. Kim J.-J.P. Masters B.S.S. J. Biol. Chem. 2000; 275: 21914-21919Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). It was not possible to examine the effect of CaM with iNOS-tr1 since the enzyme was expressed in the presence of CaM, which remains tightly bound. The constitutive enzymes, nNOS and eNOS, are easily expressed in the absence of CaM, whose binding is modulated by the calcium concentration. The rate of cyt c reduction by wild-type nNOS is stimulated about 10-fold by CaM (345versus 3860 min−1, in the absence and presence of CaM, respectively; Fig.2 A) and that of eNOS is stimulated about 3-fold (173 versus 478 min−1, in the absence and presence of CaM, respectively; Fig. 2 A). Surprisingly, both nNOS-tr1- and eNOS-tr1-catalyzed cyt c reduction are inhibited by CaM. Although the rates in the presence of CaM are similar for both the wild-type and truncated enzymes (3860 versus 4604, respectively, for nNOS and nNOS-tr1, and 478 versus 665, respectively, for eNOS and eNOS-tr1; Fig. 2 A), nNOS-tr1 reduces cyt c at a 56% higher rate than nNOS-tr1 in the presence of CaM and 21-fold faster than wild-type in its absence. Likewise, eNOS-tr1 reduces cyt c at a 85% higher rate than eNOS-tr1 in the presence of CaM and 7-fold faster than wild-type in its absence.Figure 2Reduction of artificial electron acceptors by intact and truncated NOSs. A, cytochrome creduction; B, ferricyanide reduction. The open bars are in the absence of CaM, and the solid bars are in the presence of CaM. The data for nNOS-wt and nNOS-tr1 are plotted against the left axis, and the data for eNOS-wt and eNOS-tr1 are plotted against the right axis. These data represent the mean ± S.E. of at least three separate assays. Assay conditions were as described under "Experimental Procedures," and concentration of NaCl was 250 mm. The concentration of enzyme used for the nNOS experiments was 1 nm, and where present, the concentration of CaM was 50 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since the most significant differences in rate between the wild-type and tr1 forms are seen in the absence of CaM, it was possible that the loss of the tail affected the binding of cyt c under these conditions. The Km and kcatvalues for cyt c in cyt c reduction by either wild-type or truncated enzyme were determined in the absence of CaM. There is no difference in the Km value in the absence of CaM between the wild-type and truncated forms for either nNOS or eNOS (approximately 2–3 μm; TableI), and thus the C-terminal tail probably does not directly affect binding of cyt c.Table IKinetic constants for Cyt c reductionEnzyme−CaM+CaMkcatKmEfficiencyaEfficiency iskcat/Km.kcatKmEfficiencymin−1μmmin−1μm−1min−1μmmin−1μm−1nNOS-wt2133.364.527245.4504nNOS-tr135482.1169022771.12070eNOS-wt282.411.71181.865.5eNOS-tr12872.2130.41520.9168.9These experiments were performed in the absence of NaCl. The addition of 250 mm NaCl increases the rate of NOS-mediated cytc reduction by about 3-fold (see rates in Fig. 2). The concentrations of enzymes used were as follows: nNOS-wt, 10 nm; nNOS-tr1, 1 nm; eNOS-wt, 30 nm; eNOS-tr1, 30 nm. These concentrations were chosen so that the absolute rate of cyt c reduction was in a reasonable range. The concentration of CaM was 60 nm for nNOS-wt, 30 nm for nNOS-tr1, and 100 nm for both eNOS enzymes. Assay conditions were as described under "Experimental Procedures."a Efficiency iskcat/Km. Open table in a new tab These experiments were performed in the absence of NaCl. The addition of 250 mm NaCl increases the rate of NOS-mediated cytc reduction by about 3-fold (see rates in Fig. 2). The concentrations of enzymes used were as follows: nNOS-wt, 10 nm; nNOS-tr1, 1 nm; eNOS-wt, 30 nm; eNOS-tr1, 30 nm. These concentrations were chosen so that the absolute rate of cyt c reduction was in a reasonable range. The concentration of CaM was 60 nm for nNOS-wt, 30 nm for nNOS-tr1, and 100 nm for both eNOS enzymes. Assay conditions were as described under "Experimental Procedures." To investigate further the effect of CaM on cyt c reduction, assays with both the wild-type and truncated enzymes were titrated with CaM, as shown in Fig. 3. With the wild-type enzyme, CaM is a nonessential activator of cyt creduction; it is not required for activity, but it potentiates the formation of reduced product. The kinetic constants for the wild-type enzymes were determined by varying the cyt c concentration in the absence of CaM and at saturating CaM concentrations, and the resulting data are shown in Table I. In the case of the wild-type nNOS, CaM at a saturating concentration increases the Km for cyt cby 1.6-fold and kcat by 13-fold. In the case of eNOS-wt, saturating CaM decreases the Km for cytc by 25% and increases kcat about 4-fold. As shown in Fig. 3, CaM clearly has an inhibitory effect on cytc reduction by nNOS-tr1, even at low concentrations of CaM. CaM and cytochrome c bind independently and reversibly to different sites on NOS to produce NOS·CaM, NOS·cytc, and NOS·CaM·cyt c complexes. Since both the NOS·cyt c and NOS·cyt c·CaM complexes can form product (reduced cyt c), with the NOS·cytc·CaM complex being less effective than NOS·cytc, and the activity reaches a minimum that is not zero, CaM must be a partial noncompetitive inhibitor of cyt creduction by the NO
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