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• W1974614963 abstract "In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed, and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from l-arginine via the stable intermediate N-hydroxy l-arginine (NOHA). The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates l-arginine and NOHA. The substrate-induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO forms only in the presence of H4B. The crystal structure of gsNOS at 3.2 AÅ of resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme. Thus, a more constrained heme pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 °C. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation. In an effort to generate more stable reaction intermediates involved in substrate oxidation by nitric-oxide synthases (NOSs), we have cloned, expressed, and characterized a thermostable NOS homolog from the thermophilic bacterium Geobacillus stearothermophilus (gsNOS). As expected, gsNOS forms nitric oxide (NO) from l-arginine via the stable intermediate N-hydroxy l-arginine (NOHA). The addition of oxygen to ferrous gsNOS results in long-lived heme-oxy complexes in the presence (Soret peak 427 nm) and absence (Soret peak 413 nm) of substrates l-arginine and NOHA. The substrate-induced red shift correlates with hydrogen bonding between substrate and heme-bound oxygen resulting in conversion to a ferric heme-superoxy species. In single turnover experiments with NOHA, NO forms only in the presence of H4B. The crystal structure of gsNOS at 3.2 AÅ of resolution reveals great similarity to other known bacterial NOS structures, with the exception of differences in the distal heme pocket, close to the oxygen binding site. In particular, a Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme. Thus, a more constrained heme pocket may slow ligand dissociation and increase the lifetime of heme-bound oxygen to seconds at 4 °C. Similarly, the ferric-heme NO complex is also stabilized in gsNOS. The slow kinetics of gsNOS offer promise for studying downstream intermediates involved in substrate oxidation. Nitric-oxide synthases (NOSs) 2The abbreviations used are: NOS, nitric-oxide (NO) synthase; bsNOS, B. subtilis NOS; gsNOS, G. stearothermophilus NOS; SVD, single-value decomposition; H4B, (6R)-tetrahydro-l-biopterin; H2B, dihydro-l-biopterin; Compound I, [[Fe(IV)=O]+. heme radical; NOHA, Nω-hydroxy-l-arginine; eNOS, endothelial NOS; iNOS, inducible NOS; nNOS, neuronal NOS; NOSoxy, NOS oxygenase. are highly regulated proteins that catalyze the two-step oxidation of l-arginine to nitric oxide (NO) and citrulline via the stable intermediate Nω-hydroxy l-arginine (1Rousseau D.L. Li D. Couture M. Yeh S.R. J. Inorg. Biochem. 2005; 99: 306-323Crossref PubMed Scopus (95) Google Scholar, 2Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2001; 357: 593-615Crossref PubMed Scopus (3276) Google Scholar, 3Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). NO functions in mammals as a potent signaling molecule and a cytotoxic agent to protect against pathogens. Mammalian NOSs consist of a reductase domain that has binding sites for FAD, FMN, and NADPH and an oxygenase domain that binds iron protoporphyrin IX (heme), substrate l-arginine, and the cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H4B) (1Rousseau D.L. Li D. Couture M. Yeh S.R. J. Inorg. Biochem. 2005; 99: 306-323Crossref PubMed Scopus (95) Google Scholar, 2Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2001; 357: 593-615Crossref PubMed Scopus (3276) Google Scholar, 3Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). Proteins similar to mammalian NOSs have been found in a variety of lower eukaryotes including insects, fungi (4Muller U. Prog. Neurobiol. (N.Y.). 1997; 51: 363-381Crossref PubMed Scopus (181) Google Scholar, 5Klessig D.F. Durner J. Noad R. Navarre D.A. Wendehenne D. Kumar D. Zhou J.M. Shah J. Zhang S. Kachroo P. Trifa Y. Pontier D. Lam E. Silva H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8849-8855Crossref PubMed Scopus (570) Google Scholar, 6Golderer G. Werner E.R. Leitner S. Grobner P. Werner-Felmayer G. Genes Dev. 2001; 15: 1299-1309Crossref PubMed Scopus (70) Google Scholar, 7Ninnemann H. Maier J. Photochem. Photobiol. 1996; 64: 393-398Crossref PubMed Scopus (183) Google Scholar), and bacteria (8Takami H. Nakasone K. Takaki Y. Maeno G. Sasaki R. Masui N. Fuji F. Hirama C. Nakamura Y. Ogasawara N. Kuhara S. Horikoshi K. Nucleic Acids Res. 2000; 28: 4317-4331Crossref PubMed Scopus (447) Google Scholar, 9Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Danchin A. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3126) Google Scholar). Genes responsible for cofactor (H4B) biosynthesis are present in bacteria such as Bacillus subtilis and Geobacillus kaustophilus but not in other NOS-containing bacteria such as Deinococcus radiodurans (9Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. Borriss R. Boursier L. Brans A. Braun M. Brignell S.C. Bron S. Brouillet S. Bruschi C.V. Caldwell B. Capuano V. Carter N.M. Choi S.K. Codani J.J. Connerton I.F. Danchin A. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3126) Google Scholar, 10Takami H. Takaki Y. Chee G.J. Nishi S. Shimamura S. Suzuki H. Matsui S. Uchiyama I. Nucleic Acids Res. 2004; 32: 6292-6303Crossref PubMed Scopus (165) Google Scholar, 11Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar). Bacterial NOS-like proteins are similar to the oxygenase domain of mammalian NOSs but contain no associated reductase module. Reductase partners for bacterial NOSs are yet to be identified. NOSs from D. radiodurans, B. subtilis, Staphylococcus aureus, and Bacillus anthracis are well characterized and have been shown to produce nitrogen oxides (NOx) in vitro (11Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, 12Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2002; 277: 16167-16171Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 13Bird L.E. Ren J. Zhang J. Foxwell N. Hawkins A.R. Charles I.G. Stammers D.K. Structure (Camb). 2002; 10: 1687-1696Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 14Midha S. Mishra R. Aziz M.A. Sharma M. Mishra A. Khandelwal P. Bhatnagar R. Biochem. Biophys. Res. Commun. 2005; 336: 346-356Crossref PubMed Scopus (19) Google Scholar). Conservation of nearly all the key residues involved in substrate and cofactor binding among mammalian and bacterial NOSs (11Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, 13Bird L.E. Ren J. Zhang J. Foxwell N. Hawkins A.R. Charles I.G. Stammers D.K. Structure (Camb). 2002; 10: 1687-1696Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 15Pant K. Bilwes A.M. Adak S. Stuehr D.J. Crane B.R. Biochemistry. 2002; 41: 11071-11079Crossref PubMed Scopus (123) Google Scholar) suggests a similar mechanism of NO formation in the two classes of proteins. Interestingly, D. radiodurans NOS can support l-Arg-based NOx formation with cofactors other than H4B, such as the ubiquitous cofactor tetrahydrofolate and even tryptophan (11Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, 16Buddha M.R. Tao T. Parry R.J. Crane B.R. J. Biol. Chem. 2004; 279: 49567-49570Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The ability to react with l-tryptophan may be significant as NOSs from certain Streptomyces strains participate in biosynthetic tryptophan nitration (17Kers J.A. Wach M.J. Krasnoff S.B. Widom J. Cameron K.D. Bukhalid R.A. Gibson D.M. Crane B.R. Loria R. Nature. 2004; 429: 79-82Crossref PubMed Scopus (205) Google Scholar). The NOS reaction sequence is well understood (Fig. 1), although the nature of the heme-oxygen complexes directly involved in substrate oxidation remains largely unknown (18Stuehr D.J. Santolini J. Wang Z.Q. Wei C.C. Adak S. J. Biol. Chem. 2004; 279: 36167-36170Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). l-arginine is first hydroxylated at the guanidino nitrogen, and then the resultant Nω-hydroxy-l-arginine (NOHA), an enzyme-bound intermediate (19Stuehr D.J. Kwon N.S. Nathan C.F. Griffith O.W. Feldman P.L. Wiseman J. J. Biol. Chem. 1991; 266: 6259-6263Abstract Full Text PDF PubMed Google Scholar), is further oxidized to NO and citrulline. In both the l-arginine and NOHA reactions, reduction of the Fe(III) heme enables oxygen binding and formation of a heme-dioxygen complex, which is best described as a ferric superoxy species (Fe(III)-O2·¯) (1Rousseau D.L. Li D. Couture M. Yeh S.R. J. Inorg. Biochem. 2005; 99: 306-323Crossref PubMed Scopus (95) Google Scholar, 20Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 21Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 22Ledbetter A.P. McMillan K. Roman L.J. Masters B.S. Dawson J.H. Sono M. Biochemistry. 1999; 38: 8014-8021Crossref PubMed Scopus (56) Google Scholar, 23Boggs S. Huang L. Stuehr D.J. Biochemistry. 2000; 39: 2332-2339Crossref PubMed Scopus (66) Google Scholar). This intermediate does not react with l-arginine but may (24Huang H. Hah J.M. Silverman R.B. J. Am. Chem. Soc. 2001; 123: 2674-2676Crossref PubMed Scopus (62) Google Scholar) or may not (25Wei C.C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) react with NOHA. The addition of oxygen to reduced eNOS forms two distinct heme-oxy species, which have been interpreted as the ferrous-dioxygen complex and the ferric-superoxy complex (26Marchal S. Gorren A.C. Sorlie M. Andersson K.K. Mayer B. Lange R. J. Biol. Chem. 2004; 279: 19824-19831Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). H4B acts as an electron donor to the ferric-superoxy species in both steps of NO synthesis.(Fig. 1). In the first step the reductase domain reduces the H4B+. radical (18Stuehr D.J. Santolini J. Wang Z.Q. Wei C.C. Adak S. J. Biol. Chem. 2004; 279: 36167-36170Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 27Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 28Hurshman A.R. Krebs C. Edmondson D.E. Huynh B.H. Marletta M.A. Biochemistry. 1999; 38: 15689-15696Crossref PubMed Scopus (216) Google Scholar, 29Schmidt P.P. Lange R. Gorren A.C. Werner E.R. Mayer B. Andersson K.K. J. Biol. Inorg. Chem. 2001; 6: 151-158Crossref PubMed Scopus (96) Google Scholar, 30Wei C.C. Wang Z.Q. Wang Q. Meade A.L. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2001; 276: 315-319Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 31Hurshman A.R. Krebs C. Edmondson D.E. Marletta M.A. Biochemistry. 2003; 42: 13287-13303Crossref PubMed Scopus (52) Google Scholar, 32Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (167) Google Scholar, 33Berka V. Yeh H.C. Gao D. Kiran F. Tsai A.L. Biochemistry. 2004; 43: 13137-13148Crossref PubMed Scopus (47) Google Scholar). In the second step a downstream reaction intermediate (25Wei C.C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), possibly a ferrous-heme NO complex (34Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (169) Google Scholar), reduces the H4B+. radical. Reduction of the Fe(III)-O2·¯. species at cryogenic temperatures results in a ferric heme-peroxo species that rapidly reacts at higher temperatures with either l-arginine or NOHA to form products (35Davydov R. Ledbetter-Rogers A. Martasek P. Larukhin M. Sono M. Dawson J.H. Masters B.S. Hoffman B.M. Biochemistry. 2002; 41: 10375-10381Crossref PubMed Scopus (108) Google Scholar). Unlike heme-oxygenases such as cytochrome P-450 or heme oxygenase (36Davydov R. Makris T.M. Kofman V. Werst D.E. Sligar S.G. Hoffman B.M. J. Am. Chem. Soc. 2001; 123: 1403-1415Crossref PubMed Scopus (405) Google Scholar, 37Davydov R. Kofman V. Fujii H. Yoshida T. Ikeda-Saito M. Hoffman B.M. J. Am. Chem. Soc. 2002; 124: 1798-1808Crossref PubMed Scopus (147) Google Scholar), a ferric heme-hydroperoxo species has not been observed in cryo annealing experiments. Bacterial NOSs retain NO in their heme pockets for longer times compared with their mammalian counterparts (38Wang Z.Q. Wei C.C. Sharma M. Pant K. Crane B.R. Stuehr D.J. J. Biol. Chem. 2004; 279: 19018-19025Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In B. subtilis NOS (bsNOS), the release of NO is 20-fold slower than that in mammalian NOSs due to a bacterially conserved Val to Ile switch, which offers more steric hindrance for the heme-bound NO to diffuse away from the heme. An Ile to Val mutation in bsNOS increases the rate of NO release 3.6 times, and a Val to Ile mutation in mouse iNOSoxy decreases the rate of NO release by 3 times (38Wang Z.Q. Wei C.C. Sharma M. Pant K. Crane B.R. Stuehr D.J. J. Biol. Chem. 2004; 279: 19018-19025Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Thus, the NOS heme pocket can tune the reactivity of heme ligands. Otherwise unstable reaction intermediates of cytochromes P-450, another class of well studied heme-containing monooxygenases, have been observed at cryogenic temperatures after radiolytic reduction of the heme (39Denisov I.G. Makris T.M. Sligar S.G. J. Biol. Chem. 2001; 276: 11648-11652Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 40Denisov I.G. Makris T.M. Sligar S.G. Methods Enzymol. 2002; 357: 103-115Crossref PubMed Scopus (29) Google Scholar, 41Denisov I.G. Makris T.M. Sligar S.G. J. Biol. Chem. 2002; 277: 42706-42710Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Additionally, in cysteine-ligated heme proteins, such as P450cam and chloroperoxidase, rapid formation of Compound I ([Fe(IV)=O]+. heme/thiolate radical) and related species can be achieved on reaction with peracids (42Spolitak T. Dawson J.H. Ballou D.P. J. Biol. Chem. 2005; 280: 20300-20309Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 43Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1469Crossref PubMed Scopus (180) Google Scholar, 44Kellner D.G. Hung S.C. Weiss K.E. Sligar S.G. J. Biol. Chem. 2002; 277: 9641-9644Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 45Palcic M.M. Rutter R. Araiso T. Hager L.P. Dunford H.B. Biochem. Biophys. Res. Commun. 1980; 94: 1123-1127Crossref PubMed Scopus (114) Google Scholar). In these cases Compound I (absorption peak ∼367 nm) forms in ∼10 ms after rapidly mixing 3-chloroperbenzoic acid with the ferric enzyme. Within 40 ms, this species converts to an inactive product with a peak ∼406 nm (42Spolitak T. Dawson J.H. Ballou D.P. J. Biol. Chem. 2005; 280: 20300-20309Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). In NOS, the heme-oxy species that react with l-arginine or NOHA have not been observed, although it is widely thought that the Compound I species is involved, in analogy to cytochrome P-450-type reactions (43Egawa T. Shimada H. Ishimura Y. Biochem. Biophys. Res. Commun. 1994; 201: 1464-1469Crossref PubMed Scopus (180) Google Scholar, 44Kellner D.G. Hung S.C. Weiss K.E. Sligar S.G. J. Biol. Chem. 2002; 277: 9641-9644Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 46Sono M. Roach M.P. Coulter E.D. Dawson J.H. Chem. Rev. 1996; 96: 2841-2888Crossref PubMed Scopus (2108) Google Scholar). Thermophilic prokaryotes can be exploited as a source of thermostable enzymes that have slow reaction profiles at temperatures below 25 °C (44Kellner D.G. Hung S.C. Weiss K.E. Sligar S.G. J. Biol. Chem. 2002; 277: 9641-9644Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). With this motivation, we have cloned, expressed, and characterized a thermophilic nitric-oxide synthase like protein from Geobacillus stearothermophilus (gsNOS). Herein, we characterize biophysical and biochemical properties of gsNOS and demonstrate its enhanced thermostability and slower reaction kinetics compared with other bacterial NOSs. We also show that the N-terminal extension contributes to this thermal stability. The most striking property of gsNOS is that it forms a stable heme-oxygen complex that persists on the timescale of seconds at 4 °C. The crystal structure of gsNOS at 3.2 Å of resolution reveals very high similarity to the structure of bsNOS and provides insight into the slower reactivity of gsNOS. Materials and Methods—Dioxane and sodium chloride were obtained from Mallinckrodt, (±)2-methyl-2,4-pentane diol from Hampton Research and Tris (hydroxymethyl) aminomethane from Fisher. All other chemicals were obtained from Sigma-Aldrich unless otherwise noted. All UV-visible spectra and kinetic data were recorded using an Agilent 8453 UV-visible spectroscopy system. Single-value decomposition (SVD) analysis was done using the program SPECFIT (47Gampp H. Maeder M. Meyer C.J. Zuberbuhler A.D. Talanta. 1985; 32: 95-101Crossref PubMed Scopus (542) Google Scholar, 48Gampp H. Maeder M. Meyer C.J. Zuberbuhler A.D. Talanta. 1985; 32: 257-264Crossref PubMed Scopus (424) Google Scholar, 49Gampp H. Maeder M. Meyer C.J. Zuberbuhler A.D. Talanta. 1985; 32: 1133-1139Crossref PubMed Scopus (453) Google Scholar, 50Gampp H. Maeder M. Meyer C.J. Zuberbuhler A.D. Talanta. 1986; 33: 943-951Crossref PubMed Scopus (506) Google Scholar). Molecular Biology—The NOS gene of G. stearothermophilus (ATCC strain number 12980) was amplified by PCR from genomic DNA. The 5′ primer generated an NdeI site before the start codon, and the 3′ primer generated an XhoI site after the stop codon. The amplified fragment was cloned into the pET28 expression vector (Novagen) and transformed into Escherichia coli BL21(DE3) cells. Protein Expression and Purification—The full-length NOS (gsNOS) and a shorter construct with the first 13 residues removed from the N terminus (gsNOS+13) were overexpressed in E. coli BL21(DE3) cells with a His6 tag. The proteins were purified using nickel-chelate chromatography and then size-exclusion chromatography after removal of the His6 tag with thrombin. Both the constructs could be concentrated to ∼100 mg/ml, as estimated by the Bradford assay. Crystallization— gsNOS produced orthorhombic crystals of dimensions 200-400 μm in 24-48 h at 22 °C when grown by vapor diffusion from 45-50 mg/ml of protein in 50 mm Tris (pH 7.5), 150 mm NaCl, mixed with freshly dissolved 1-2 mm l-arginine and 1-2 mm l-tryptophan (Trp). Trp was added to help stabilize the pterin-binding site due to evidence that it will bind there in the D. radiodurans NOS (16Buddha M.R. Tao T. Parry R.J. Crane B.R. J. Biol. Chem. 2004; 279: 49567-49570Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The reservoir was mixed a 1:1 with protein solution and contained 40-46% dioxane and 10% (±)2-methyl-2,4-pentane diol. gsNOS crystals were of space group P21212 with cell dimensions 154.0 Å × 118.7 Å × 49.8 Å. The crystals contained two NOS subunits per asymmetric unit. Structure Determination—Diffraction data were collected at 100 K with synchrotron radiation (λ = 1.002 Å) on beamline X-25 of the National Synchrotron Light Source at Brookhaven National Laboratory. The data sets were reduced and scaled using HKL2000 (51Otwinowski A. Minor W. Methods Enzymol. 1997; 276: 307-325Crossref PubMed Scopus (38570) Google Scholar). Initial phases were determined by molecular replacement (AmoRe) (52Navaza J. Acta Crystallogr. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar) with the structure of NOS from B. subtilis as the probe (PDB entry 1M7V). The model was then refined in CNS (53Brunger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. 1998; 54: 905-921Crossref PubMed Scopus (16966) Google Scholar) using standard positional and thermal factor refinement, and the structure was adjusted with XFIT to Fobs-Fcalc and 2Fobs-Fcalc maps. The addition of l-arginine, heme, and water molecules amidst cycles of refinement produced the final model. Nitrite Formation with Peroxide—30-50 μm enzyme was incubated at different temperatures with 1 mm l-arginine and 20 mm H2O2, and the reaction was stopped at different times by adding Griess reagents R1 (sulfanilamide) and R2 (N-(1-naphthyl)ethylenediamine). The product (pink dye) formation was monitored by measuring the absorbance at 540 nm. The amount of nitrite generated in the solution was calculated using the Griess assay kit from Cayman Chemicals. Each activity reported represents an average from at least three experiments. Single Turnover Experiments—Concentrated full-length gsNOS was cycled through a degassing chamber and left in an anaerobic glove box for ∼30 min. All buffer solutions were extensively degassed and placed under argon. Degassed buffer solution was used to dilute the protein solution suitably for UV-visible spectroscopy. To observe the spectral changes the enzyme undergoes during reaction with oxygen, gsNOS (13 μm) was reduced by titration with sodium dithionite (30-50 μm). The cuvette containing reduced gsNOS (13 μm) was sealed using a rubber septum inside the glove box and then transferred to a UV-visible spectrophotometer. The temperature of the cell with the sample in the UV-visible spectrophotometer was lowered to 4 °C. Nitrogen gas was blown around the cuvette to prevent water condensation due to lowered temperature. Ice-cold air saturated buffer was injected into the cuvette through the rubber septum to start the reaction, at which point the sample contained ∼8 μm gsNOS, ∼20-30 μm dithionite, and ∼160 μm oxygen (54Weiss R. Deep Sea Res. A. 1970; 17: 721-735Google Scholar). The solutions were mixed rapidly using a magnetic stir bar in the cuvette. gsNOS was PCR cloned and expressed with a His6 affinity tag in E. coli. The protein has 65% sequence identity when compared with the NOS protein from the related mesophile B. subtilis (Supplemental Fig. 1). After nickel nitrilotriacetic acid affinity purification and proteolytic cleavage of the His tag, gsNOS (∼43 kDa/subunit) elutes on a gel filtration column as a dimer (apparent molecular mass ∼86 kDa). The UV-visible spectra of free gsNOS (absorption maxima at 403 and 519 nm), imidazole-bound enzyme (427 and 553 nm), l-arginine-bound enzyme (399 and 517 nm), and reduced enzyme with l-arginine bound (415 and 552 nm) (Fig. 2) are very similar to those of mammalian and other bacterial NOS oxygenase domains (1Rousseau D.L. Li D. Couture M. Yeh S.R. J. Inorg. Biochem. 2005; 99: 306-323Crossref PubMed Scopus (95) Google Scholar, 11Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, 12Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2002; 277: 16167-16171Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 20Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 21Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 22Ledbetter A.P. McMillan K. Roman L.J. Masters B.S. Dawson J.H. Sono M. Biochemistry. 1999; 38: 8014-8021Crossref PubMed Scopus (56) Google Scholar). Shifts in the Soret peak indicate that the heme coordinates imidazole (427 nm) and can be displaced by l-arginine (Fig. 2). gsNOS shows high thermal stability. Molar ellipticity measurements (θ222) of NOS dimers indicate irreversible loss of secondary structure with increasing temperature (Fig. 3). We define the melting temperature as that at which half of the ellipticity (θ222) is lost as the temperature is raised. Whereas bsNOS melts at 60 °C, full-length gsNOS melts at 80 °C. gsNOS+13, in which an N-terminal amino acid extension has been removed, melts at an intermediate temperature of 66 °C (Fig. 3). In all cases, loss of secondary structure, as evidenced by CD, is irreversible. Activity of gsNOS and bsNOS were compared at different temperatures by evaluating the rate of nitrite produced from substrate l-arginine in the presence of hydrogen peroxide (Table 1). The amount of nitrite formed was quantitated by the Griess reaction (11Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar) at 25 °C after incubation of the l-arginine-saturated enzyme with peroxide at various temperatures and times. Product formation was linear with time. We followed the conversion of l-arginine rather than NOHA to NOx because NOHA breakdown at elevated temperatures resulted in significant background product formation. At increased temperatures, gsNOS gives a lower rate of product formation than bsNOS. At 50 °C, the bsNOS activity deviates greatly from that of gsNOS, which increases gradually with temperature. At temperatures above 50 °C, bsNOS denatures and precipitates. gsNOS reactions with peroxide and NOHA instead of l-arginine give ∼2 times more nitrite formation at room temperature.TABLE 1Rate constants of nitrite production by NOSs from various sources as a function of temperaturekcatTemperatureNO2− productionNO2− + NO3− production, nNOSbRef. 27.gsNOSbsNOSdeiNOSaRef. 16.°Cheme−1min−1 × 100heme−1min−1 × 100heme−1min−1 × 100heme−1min−1 × 100252.6 ± 0.14.6 ± 0.17.5 ± 0.528 ± 3353.6 ± 0.213.3 ± 0.35010.3 ± 0.150.3 ± 1.1a Ref. 16Buddha M.R. Tao T. Parry R.J. Crane B.R. J. Biol. Chem. 2004; 279: 49567-49570Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar.b Ref. 27Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar. Open table in a new tab Substrate-free gsNOS—In the absence of any substrate, the spectrum recorded ∼5 s after the introduction of oxygen showed a red-shifted Soret peak at 413 nm (Fig. 4), and the line shape was considerably different than substrate-free reduced gsNOS, which has a Soret peak at 411 nm and a broad shoulder from 440 to 490 nm (Fig. 4). Furthermore, the extinction coefficient for the Soret band of the intermediate is less than that of both the ferric- and ferrous-free enzyme. The 413 species was stable for ∼1 min at 4 °C and slowly decayed back to the ferric form (403 nm) with a change in line shape. Conversion to the ferric form of the enzyme was not complete even after 3 min at 4 °C and required increasing the temperature of the cell to 25 °C. Because the new 413 species results from mixing air-saturated buffer with the reduced protein, it likely represents the Fe(II)-O2 complex; although given the limited time resolution, the initial spectrum observed on the addition of oxygen could include some contribution from free ferric enzyme. Nevertheless, combination of spectra from the substrate-free ferric and ferrous forms cannot explain the optical features of the intermediate observed in the presence of oxygen. When the same reaction was carried out in the presence of 40 μm H4B, which is known to accelerate the decay of the ferrous-oxy species in mammalian and bacterial NOSs (21Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 32Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (167) Google Scholar), the conversion of the 413 species to" @default.
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• W1974614963 creator A5067178930 @default.
• W1974614963 creator A5071526287 @default.
• W1974614963 date "2006-04-01" @default.
• W1974614963 modified "2023-10-18" @default.
• W1974614963 title "Structure and Reactivity of a Thermostable Prokaryotic Nitric-oxide Synthase That Forms a Long-lived Oxy-Heme Complex" @default.
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• W1974614963 doi "https://doi.org/10.1074/jbc.m510062200" @default.
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