Electron Transfer and Catalytic Activity of Nitric Oxide Synthases
1998; Elsevier BV; Volume: 273; Issue: 10 Linguagem: Inglês
10.1074/jbc.273.10.5566
ISSN1083-351X
AutoresClinton R. Nishida, Paul R. Ortiz de Montellano,
Tópico(s)Renin-Angiotensin System Studies
ResumoThe nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of ·NO synthesis, cytochromec reduction, and NADPH utilization and in the Ca2+ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of ·NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca2+ independence of inducible NOS requires interactions of calmodulin with both the calmodulin binding motif and the flavoprotein domain, and (d) the effects of tetrahydrobiopterin and l-arginine on electron transfer rates are mediated exclusively by heme domain interactions. The nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of ·NO synthesis, cytochromec reduction, and NADPH utilization and in the Ca2+ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of ·NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca2+ independence of inducible NOS requires interactions of calmodulin with both the calmodulin binding motif and the flavoprotein domain, and (d) the effects of tetrahydrobiopterin and l-arginine on electron transfer rates are mediated exclusively by heme domain interactions. The nitric oxide synthases (NOSs) 1The abbreviations used are: NOS, nitric oxide synthase; nNOS, neuronal NOS; eNOS, endothelial NOS; iNOS, inducible macrophage NOS; CaM, Ca2+-dependent calmodulin; EHC/NR, chimera of eNOS with reductase domain from nNOS; IHC/NR, chimera of iNOS with reductase domain from nNOS; H4B, (6R)-5,6,7,8-tetrahydrobiopterin; NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography. catalyze the oxidation of l-Arg to citrulline and ·NO (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 2Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (815) Google Scholar, 3Masters B.S.S. Annu. Rev. Nutr. 1994; 14: 131-145Crossref PubMed Scopus (82) Google Scholar, 4Knowles R.G. Moncada S. Biochem. J. 1994; 298: 249-258Crossref PubMed Scopus (2507) Google Scholar, 5Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2138) Google Scholar). The three elementary forms of this enzyme are the neuronal (NOS-I or nNOS), endothelial (NOS-III or eNOS), and inducible (NOS-II or iNOS) isoforms. Heme, FMN, FAD, CaM, and H4B are essential as cofactors, and NADPH and O2 are essential as co-substrates for all the NOS isoforms. Each subunit of the homodimeric NOS proteins is composed of three modules: a heme-containing catalytic domain, a consensus CaM binding sequence, and an FMN-, FAD-, and NADPH binding domain with strong sequence similarity to cytochrome P450 reductase (6Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2171) Google Scholar, 7Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar, 8Lowenstein C.J. Glatt C.S. Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6711-6715Crossref PubMed Scopus (622) Google Scholar). The flavin domain uncouples the electrons provided by NADPH and delivers them, one at a time, to the prosthetic heme iron atom. H4B is required for ·NO synthesis by all three proteins, but its role in the catalytic process remains unclear (9Mayer B. Werner E.R. Naunyn-Schmiedeberg's Arch. Pharmacol. 1995; 351: 453-463Crossref PubMed Scopus (125) Google Scholar). Clear differences exist in the Ca2+ and CaM dependence of the three NOS isoforms. The binding of CaM to nNOS and eNOS, the two constitutive isoforms, is Ca2+-dependent and reversible (10Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3125) Google Scholar, 11Busse R. Mulsch A. FEBS Lett. 1990; 265: 133-136Crossref PubMed Scopus (434) Google Scholar), whereas the binding of CaM to iNOS is essentially a Ca2+-independent, irreversible process (12Cho 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 (562) Google Scholar). Thus, the catalytic activities of nNOS and eNOS are regulated by cellular Ca2+-levels, whereas the activity of iNOS is insensitive to the Ca2+ concentration and is primarily regulated by the rate at which the protein is synthesized (12Cho 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 (562) Google Scholar). In addition to the heme, CaM, and reductase domains common to all the isoforms, nNOS has an additional N-terminal domain thought to be involved in subcellular targeting (13Ponting C.P. Phillips C. Trends Biochem. Sci. 1995; 20: 102-103Abstract Full Text PDF PubMed Scopus (163) Google Scholar, 14Brenman J.E. Chao D.S. Xia H. Aldape K. Bredt D.S. Cell. 1995; 82: 743-752Abstract Full Text PDF PubMed Scopus (849) Google Scholar), and eNOS is unique in that it has myristoylation and palmitoylation sites that target it to the membrane (15Liu J. Sessa W.C. J. Biol. Chem. 1994; 269: 11691-11694Abstract Full Text PDF PubMed Google Scholar, 16Robinson L.J. Busconi L. Michel T. J. Biol. Chem. 1995; 270: 995-998Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The catalytic activities of the NOS isoforms differ considerably, regardless of whether activity is measured as ·NO formation or cytochrome c reduction. Regardless of which of these two parameters is measured, the activities of iNOS and nNOS are considerably higher than that of eNOS. Thus, in our hands, the Vmax values for ·NO formation by recombinant iNOS (17Gerber N.C. Nishida C.R. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1997; 343: 249-253Crossref PubMed Scopus (26) Google Scholar), nNOS (18Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1995; 270: 17791-17796Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and eNOS (19Rodrı́guez-Crespo I. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1996; 336: 151-156Crossref PubMed Scopus (45) Google Scholar) were found to be ∼800, ∼400, and ∼130 nmol min−1 mg−1, respectively, and the rates of electron transfer to cytochrome c in the presence of CaM and Ca2+ were ∼45,000, ∼44,000, and ∼1,800 nmol min−1 mg−1, respectively. In each case, the reductase domain was able to transfer electrons to cytochrome c at a rate much greater than the maximum rate of ·NO production, although the nature of the rate-limiting step(s) in ·NO formation and the reason(s) for the differences in the catalytic activities of the isoforms are unclear. The reduction of cytochromec by nNOS and eNOS occurs in the absence of CaM and Ca2+, but the rate is greatly stimulated by the binding of these cofactors (20Abu-Soud H.M. Feldman P.L. Clark P. Stuehr D.J. J. Biol. Chem. 1994; 269: 32318-32326Abstract Full Text PDF PubMed Google Scholar, 21Klatt P. Heinzel B. John M. Kastner M. Bohme E. Mayer B. J. Biol. Chem. 1992; 267: 11374-11378Abstract Full Text PDF PubMed Google Scholar). In the case of iNOS, the cytochromec reductase activity is not Ca2+-dependent. Expression of truncated eNOS and nNOS proteins consisting only of the CaM binding and reductase domains has confirmed that the CaM/Ca2+ activation of electron transfer to cytochrome c does not require the heme domain (22McMillan K. Masters B.S.S. Biochemistry. 1995; 34: 3686-3693Crossref PubMed Scopus (169) Google Scholar, 23Gachhui 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, 24Chen P.F. Tsai A.L. Berka V. Wu K.K. J. Biol. Chem. 1996; 271: 14631-14635Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The specific protein interactions that result in quasi-irreversible binding of CaM to iNOS but not nNOS or eNOS have not been defined. Chimeras in which mouse iNOS residues 503–532 were exchanged with the equivalent residues 725–754 of rat nNOS differed from the parent proteins in that both required an intermediate concentration of Ca2+ to bind CaM and produce ·NO (25Ruan J. Xie Q. Hutchinson N. Cho H. Wolfe G.C. Nathan C. J. Biol. Chem. 1996; 271: 22679-22686Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Furthermore, truncation analysis of iNOS suggested that residues within the sequence 484–726 that lie outside the canonical CaM binding sequence are required for Ca2+-independent CaM binding. In an independent study, Venema et al. (26Venema 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 (152) Google Scholar) replaced the CaM binding domain of murine iNOS with that of bovine eNOS and vice versa. The NOS activity of the iNOS chimera proved to be partially Ca2+-dependent, whereas the eNOS chimera was CaM-independent but completely Ca2+-dependent. These results again suggest that the tight, essentially Ca2+-independent binding of CaM requires interactions in addition to those provided by the consensus iNOS CaM binding sequence. In contrast, surface plasmon resonance studies of the binding to CaM of peptides derived from the CaM binding domains of nNOS and iNOS led to the conclusion that the affinity of iNOS for CaM resides entirely in the CaM binding canonical sequence (27Zoche M. Bienert M. Beyermann M. Koch K.W. Biochemistry. 1996; 35: 8742-8747Crossref PubMed Scopus (35) Google Scholar). To identify contributions of the individual domains of the NOS polypeptide to (a) the differences in the rates of ·NO production by the three isoforms, (b) the basis for the Ca2+-independent binding of CaM in iNOS, and (c) the effects of l-Arg and H4B on electron transfer rates, we constructed, expressed, and purified two chimeric proteins. In these chimeric proteins, the heme and CaM binding domains were contributed by either eNOS or iNOS, and the reductase domain by nNOS. Bovine eNOS (7Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar) and rat brain nNOS (6Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2171) Google Scholar) cDNAs were kind gifts from William C. Sessa (Yale University) and Solomon H. Snyder (Johns Hopkins University), respectively. A mouse iNOS cDNA was provided by Stephen Black (University of California, San Francisco). The mouse iNOS was cloned into the pCWori vector and expressed and purified as described for the human hepatic iNOS isoform (17Gerber N.C. Nishida C.R. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1997; 343: 249-253Crossref PubMed Scopus (26) Google Scholar). The human CaM gene (28Rhyner J.A. Koller M. Durussel-Gerber I. Cox J.A. Strehler E.E. Biochemistry. 1992; 31: 12826-12832Crossref PubMed Scopus (53) Google Scholar) was provided by Emanuel E. Strehler (Mayo Foundation). Enzymes used in DNA manipulation were from New England Biolabs (Beverly, MA). l-Arg was from Aldrich, H4B was from Alexis Biochemicals (San Diego, CA), and HEPES was buffer from Fisher. Bradford protein assay kits were from Bio-Rad. Recombinant human CaM was purified from Escherichia coli by published procedures (28Rhyner J.A. Koller M. Durussel-Gerber I. Cox J.A. Strehler E.E. Biochemistry. 1992; 31: 12826-12832Crossref PubMed Scopus (53) Google Scholar). DNA purification kits and Ni2+-NTA agarose were purchased from QIAGEN (Chatsworth, CA). BL21 competent cells were from Novagen (Madison, WI). Unless otherwise indicated, all other reagents were from Sigma. One of the chimeric proteins was constructed by subcloning a PCR-generated fragment from pBluescriptKS/eNOS (7Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar) into a mutant pBluescriptSK/nNOS cDNA. The eNOS fragment was obtained by PCR amplification using VentRDNA polymerase (New England Biolabs, Beverly, MA) possessing a 3′ to 5′ proofreading exonuclease activity for higher fidelity than was available with Taq polymerase. The eNOS cDNA coding for amino acids Met-1 to Ser-528 was amplified using primer 1 (5′-GC CCCAGCCATATG GCAAAC TTGAAA AGCGTG GGTCAG GA), which introduced a 5′ NdeI site at the starting methionine (in boldface) and a G2A mutation for increased expression, and primer 2 (5′-CGGCC GGTCTC GCTAGC GTACAG GATG), which introduced a 3′NheI splice site. The nNOS cDNA (6Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2171) Google Scholar) was mutated using the Transformer Mutagenesis Kit (CLONTECH); selection primer 3 (5′-GTC GACGGT ATTAAT AAGCTT GATATC), which converted a unique ClaI site in pBluescriptSK to a unique AseI site; and primer 4 (5′-CTG AAGGAC ACAGATCATATG GAAGAG AACACG), which introduced a 5′NdeI site at the starting methionine. Simultaneously, the NheI splice point was created using mutagenesis primer 5 (5′-G ACCATT CTCTAC GCTAGC GAGACA GGCAAA TCAC), which resulted in a T761S mutation and yielded pBluescriptSK/nNOSNheI761. Sequence alignment using the GAP program (29Genetics Computer Group Wisconsin Package, Version 8.0. Genetics Computer Group, Madison, WI1994Google Scholar) revealed that this amino acid is located in a stretch that is identical between nNOS and eNOS (Table I) and therefore was not expected to grossly affect the properties of the chimera. Indeed, the mutation is silent regarding the eNOS sequence and is a conservative mutation from threonine for nNOS. The chimeric gene construct was made by subcloning the NdeI-NheI eNOS fragment into pBluescriptSK/nNOS-NheI761. A 3′ XbaI site in the multiple cloning domain was utilized to subclone the entireNdeI/XbaI chimeric gene into a pCWori expression plasmid that also possessed an insert coding for an N-terminal His6 tag to aid in purification.Table ISequence at the splice sites in the parent and chimeric proteinsProteinSplice point sequencenNOS755ATILYATET763eNOS522ATILYASET530iNOS533ATVLFATET541EHC/NR522ATILYASET530IHC/NR533ATVLFATET541 Open table in a new tab The IHC/NR construction was analogously produced from PCR-generated IHC from pBluescript/iNOS and PCR primers AGTCT CACAT ATGGC TTGCC CGTGC AAGTT TCTGT TCAA and CGGGC GTCGC TAGCA AAGAG GACTG TGGC to generateNdeI and NheI terminal restriction sites, respectively. The NheI splice site introduces a silent mutation at the conserved T539 of iNOS, which is homologous to Thr-761 of nNOS. Subcloning was then performed as for EHC/NR. The His6proteins were expressed in protease-deficient BL21(DE3) cells (Novagen) and purified as described previously (30Hühmer A.F.R. Nishida C.R. Ortiz de Montellano P.R. Schöneich C. Chem. Res. Toxicol. 1997; 10: 618-626Crossref PubMed Scopus (46) Google Scholar). Cytochrome c reduction and NADPH oxidation rates were measured at 37 °C on a Cary 1E spectrometer equipped with a Lauda circulating bath using the extinction coefficients ε550 nm = 21 mm−1cm−1 and ε340 nm = 6200m−1 cm−1, respectively. NOS activity was measured at 37 °C as the ·NO-dependent conversion of ferrous to ferric methemoglobin (31Hevel J.M. Marletta M.A. Methods Enzymol. 1994; 233: 250-258Crossref PubMed Scopus (424) Google Scholar) using an extinction coefficient of Δε401–411 of 60 mm−1 cm−1. Exogenous cofactors, when added, were 5 μm FAD, 5 μm FMN, 1 mm Ca2+, and a 3-fold molar excess of CaM over NOS monomer. For EGTA inhibition experiments, the EGTA stock solution was adjusted to pH 7.5 before addition. The values of Km and Vmax were determined for the EHC/NR chimera purified on ADP-agarose and CaM-Sepharose columns in the absence of H4B and l-Arg. For the IHC/NR chimera, the CaM-Sepharose column could not be used because CaM is already tightly bound to IHC/NR due to coexpression of CaM with the chimera. Imidazole was omitted from all buffers to avoid contamination of the final pure protein with bound imidazole. Buffer D 50 mm HEPES, pH 7.5, 50 mm EDTA, 500 mm NaCl, 10% glycerol, 5 mm 2-mercaptoethanol, and protease inhibitors (100 μm phenylmethylsulfonyl fluoride, 1 μm leupeptin, 1 μm pepstatin, and 1 μg/ml antipain) was used to elute the protein from the Ni2+-NTA column. After re-binding to ADP-Sepharose, the bound protein was washed with at least 5 column volumes of Buffer D. The purification was then continued as described earlier. Size exclusion chromatography was performed by FPLC on an HR200 Superdex (Pharmacia 10/30) column at room temperature using a flow rate of 0.5 ml/min of running buffer composed of 10% glycerol plus 150 mm phosphate-buffered saline and 2 mm dithiothreitol. When applicable, H4B was added to the protein solution to a concentration of 100 μm. The running buffer contained 5 μmH4B. To explore the electron transfer mechanism in NOS, we constructed two chimeras in each of which the heme and CaM binding domains of one NOS isoform were fused to the reductase domain of a second isoform. In one chimera, the eNOS heme and CaM binding domains were fused with the nNOS reductase domain (EHC/NR), and in the other, the iNOS heme and CaM binding domains were fused to the nNOS reductase domain (IHC/NR). The splice points for the chimeras were chosen to minimize whatever structural perturbations might arise from a change in the amino acid at the splice site and were located at regions with high primary sequence identity between isoforms, as determined by the GAP program from the GCG software package (29Genetics Computer Group Wisconsin Package, Version 8.0. Genetics Computer Group, Madison, WI1994Google Scholar). The splice points, indicated by boldface type, are shown in Table I. The sequences of the splice points for the EHC/NR and IHC/NR chimeric proteins share the sequence of at least one of the parent isoforms, with no additions or deletions, and therefore no direct structural perturbation due to the splicing was expected. The chimeric proteins were expressed in E. coli under the same conditions used to express the wild-type proteins (17Gerber N.C. Nishida C.R. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1997; 343: 249-253Crossref PubMed Scopus (26) Google Scholar, 18Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1995; 270: 17791-17796Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 19Rodrı́guez-Crespo I. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1996; 336: 151-156Crossref PubMed Scopus (45) Google Scholar, 32Rodrı́guez-Crespo I. Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 11462-11467Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Because there is no H4B or CaM in E. coli, the proteins are expressed in the absence of these cofactors, although the absence of CaM can be remedied by coexpression of the human CaM gene in the bacterial cells (17Gerber N.C. Nishida C.R. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1997; 343: 249-253Crossref PubMed Scopus (26) Google Scholar, 19Rodrı́guez-Crespo I. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1996; 336: 151-156Crossref PubMed Scopus (45) Google Scholar, 33Wu C. Zhang J. Abu-Soud H. Ghosh D.K. Stuehr D.J. Biochem. Biophys. Res. Commun. 1996; 222: 439-444Crossref PubMed Scopus (89) Google Scholar, 34Fossetta J.D. Niu X.D. Lunn C.A. Zavodny P.J. Narula S.K. Lundell D. FEBS Lett. 1996; 379: 135-138Crossref PubMed Scopus (33) Google Scholar). As the chimeras were tagged with a polyhistidine peptide, their purification was achieved, as was done previously for the wild-type eNOS (19Rodrı́guez-Crespo I. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1996; 336: 151-156Crossref PubMed Scopus (45) Google Scholar, 32Rodrı́guez-Crespo I. Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 11462-11467Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), nNOS (18Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1995; 270: 17791-17796Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and iNOS isoforms (17Gerber N.C. Nishida C.R. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1997; 343: 249-253Crossref PubMed Scopus (26) Google Scholar), by affinity chromatography first on a Ni2+-NTA column and subsequently on a 2′,5′-ADP-agarose column (Fig. 1). The proteins were purified in either the presence or absence of H4B, yielding proteins that were designated, for example, H4B(+)EHC/NR and H4B(−)EHC/NR, respectively. For the EHC/NR chimera, the final protein yield was 5 mg, and the specific activity was 500 nmol min−1 mg−1. SDS-polyacrylamide gel electrophoresis analysis of the protein (Fig. 1,lane 4) indicated that the protein was obtained in a highly purified state. Furthermore, the molecular mass of the protein indicated by SDS-polyacrylamide gel electrophoresis (∼130 kDa) was consistent with the calculated EHC/NR molecular mass of 133 kDa. The active IHC/NR chimera could only be obtained when it was co-expressed with CaM. SDS-polyacrylamide gel electrophoresis analysis of CaM-coexpressed IHC/NR showed that the molecular mass of the purified protein was consistent with the calculated mass of 138 kDa (Fig. 1,lane 8). The presence of H4B during the purification of EHC/NR or IHC/NR had no significant effect on the final protein yield. H4B(+)EHC/NR and H4B(−)EHC/NR are spectroscopically nearly indistinguishable. The only detectable difference is a slightly broader peak in the H4B-free protein (not shown). The Soret band at λmax = 400 nm is similar to that of wild-type eNOS and nNOS (32Rodrı́guez-Crespo I. Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 11462-11467Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and indicates that the protein is primarily high spin. Addition of H4B and l-Arg converts the low spin shoulder to high spin. CaM-coexpressed H4B(+)IHC/NR possesses a spectrum similar to that of EHC/NR. However, CaM-coexpressed H4B(−)IHC/NR exhibited a red-shifted Soret band at λmax = 418 nm similar to that found for H4B(−)iNOS (17Gerber N.C. Nishida C.R. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1997; 343: 249-253Crossref PubMed Scopus (26) Google Scholar, 30Hühmer A.F.R. Nishida C.R. Ortiz de Montellano P.R. Schöneich C. Chem. Res. Toxicol. 1997; 10: 618-626Crossref PubMed Scopus (46) Google Scholar, 35Gachhui R. Ghosh D.K. Wu C. Parkinson J. Crane B.R. Stuehr D.J. Biochemistry. 1997; 36: 5097-5103Crossref PubMed Scopus (72) Google Scholar). These red-shifted Soret maxima, which are not observed with the other H4B(−)NOS isoforms (compare Refs. 17Gerber N.C. Nishida C.R. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1997; 343: 249-253Crossref PubMed Scopus (26) Google Scholar, 32Rodrı́guez-Crespo I. Gerber N.C. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 11462-11467Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 36Gorren A.C. List B.M. Schrammel A. Pitters E. Hemmens B. Werner E.R. Schmidt K. Mayer B. Biochemistry. 1996; 35: 16735-16745Crossref PubMed Scopus (143) Google Scholar), 2C. R. Nishida and P. R. Ortiz de Montellano, unpublished results. indicate that these proteins are primarily low spin. Addition of 100 μm H4B produced nearly equal populations of high and low spin heme, and addition of 1 mml-Arg continued the trend toward a high spin heme. Dithionite reduction of both chimeric proteins in the presence of CO produced the expected 444 nm absorbance maximum of the ferrous-CO complexes. Dimer formation is required for catalytic ·NO production (37Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar). Because dimer formation might be affected by altered protein contacts in the chimeras, the ability of both EHC/NR and IHC/NR to form dimers was evaluated. FPLC size exclusion chromatography showed that dimers were present in both H4B(+) and H4B(−) EHC/NR independent of whether the samples were preincubated with H4B and of whether H4B was added to the FPLC elution buffers (Fig. 2). Nearly all (>90%) of the heme-containing protein was present as the dimer, with approximately 30% of the total protein in the monomeric state. With respect to this H4B-independent dimerization, EHC/NR behaved very similarly to wild-type human eNOS (19Rodrı́guez-Crespo I. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1996; 336: 151-156Crossref PubMed Scopus (45) Google Scholar). In contrast, heme-containing but H4B-free IHC/NR eluted from the gel filtration Sephadex column as both a monomer and a dimer (Fig. 2, A and B). Although quantitation is difficult due to the similar retention times of the monomer and dimer, the proportion of dimer increased for both the heme-containing (compare Fig. 2, A and B) and heme-free protein (compare Fig. 2, C and D) when the analysis was carried out in the presence of H4B. In this regard, IHC/NR behaves like native iNOS, which was shown earlier to exhibit similar H4B-dependent dimerization behavior (37Baek K.J. Thiel B.A. Lucas S. Stuehr D.J. J. Biol. Chem. 1993; 268: 21120-21129Abstract Full Text PDF PubMed Google Scholar, 38Abu-Soud H.M. Loftus M. Stuehr D.J. Biochemistry. 1995; 34: 11167-11175Crossref PubMed Scopus (97) Google Scholar), although we saw considerable dimerization even for H4B- andl-Arg-free iNOS (Fig. 2). The dimerization properties of the chimeras thus resemble those of the parent that contributes the heme and CaM domains. To evaluate the ability of the reductase and heme domains in the chimeras to interact productively and to determine whether CaM binding involves contacts with the protein outside the putative CaM binding domain, we measured the cytochrome c reduction and NOS activities of the chimeric proteins. For both chimeras, the omission of H4B during protein purification did not affect enzyme activity if the proteins were reconstituted with the cofactor. Exogenous flavins were required to achieve maximal activities for both chimeras, which suggests, as for the native isoforms (39Stuehr D.J. Methods Enzymol. 1996; 268: 324-333Crossref PubMed Google Scholar), that some flavin loss occurred during purification despite the presence of flavins in the purification buffers. The cytochrome c reductase activity of EHC/NR was as high as that of wild-type nNOS (Table II). CaM stimulated the EHC/NR cytochrome c reduction rate, as it does for the wild-type isoforms, but the magnitude of the enhancement was greater for the chimera than for the wild-type proteins. In the presence of exogenous flavins, the CaM-dependent enhancement for the chimera was 20–60-fold, whereas the enhancement under similar conditions for eNOS and nNOS was ∼10-fold. The cytochrome c reductase activity of CaM-coexpressed IHC/NR was only slightly lower than that of wild-type nNOS or iNOS (Table II).Table IIActivities of nNOS, iNOS, eNOS, EHC/NR, and IHC/NRProteinCa2+/CaM2-aExogenously added to assay system in addition to NADPH, H4B, and l-Arg.Reductase activity2-bAs measured by the rate of cytochrome c reduction.NOS activity2-cAs measured by the hemoglobin assay for · NO.min−1min−1nNOS−6300+706096eNOS−670+67016iNOS (CaM-coexpressed)+5880105EHC/NR−980+601063IHC/NR (CaM-coexpressed)−473087+459083+Ca2+ only4770872-a Exogenously added to assay system in addition to NADPH, H4B, and l-Arg.2-b As measured by the rate of cytochrome c reduction.2-c As measured by the hemoglobin assay for · NO. Open table in a new tab The kinetic parameters for EHC/NR in the presence of all cofactors indicate that the Km and Vmaxvalues are somewhat higher for EHC/NR than for eNOS: l-ArgKm = 8.8 μm versus 3 μm, and Vmax = 500versus 95–120 nmol min−1 mg−1, respectively. For IHC/NR, l-Arg Km = 9.2 μm and Vmax = 630 nmol min−1 mg−1; these values compare well withKm = 12 μm and Vmax = 800 nmol min−1mg−1 for recombinant hepatic iNOS purified in this laboratory (17Gerber N.C. Nishida C.R. Ortiz de Montellano P.R. Arch. Biochem. Biophys. 1997; 343: 249-253Crossref PubMed Scopus (26) Google Scholar). The ·NO synthase activity of EHC/NR, which was much higher than that of wild-type eNOS, was comparable to that of the wild-type nNOS (Table II). The ability of this chimera to synthesize ·NO was abolished by exogenous EGTA and is therefore full
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