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A Conserved Tryptophan 457 Modulates the Kinetics and Extent of N-Hydroxy-l-Arginine Oxidation by Inducible Nitric-oxide Synthase

2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês

10.1074/jbc.m111967200

ISSN

1083-351X

Autores

Zhiqiang Wang, Chin‐Chuan Wei, Dennis J. Stuehr,

Tópico(s)

Metal-Catalyzed Oxygenation Mechanisms

Resumo

In the oxygenase domain of mouse inducible nitric-oxide synthase (iNOSoxy), a conserved tryptophan residue, Trp-457, regulates the kinetics and extent of l-Arg oxidation to Nω-hydroxy-l-arginine (NOHA) by controlling electron transfer between bound (6R)-tetrahydrobiopterin (H4B) cofactor and the enzyme heme FeIIO2 intermediate (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819–12825). To investigate whether NOHA oxidation to citrulline and nitric oxide (NO) is regulated by a similar mechanism, we performed single turnover reactions with wild type iNOSoxy and mutants W457F and W457A. Ferrous proteins containing NOHA plus H4B or NOHA plus 7,8-dihydrobiopterin (H2B), were mixed with O2-containing buffer, and then heme spectral transitions and product formation were followed versus time. All three proteins formed a FeIIO2 intermediate with identical spectral characteristics. In wild type, H4B increased the disappearance rate of the FeIIO2intermediate relative to H2B, and its disappearance was coupled to the formation of a FeIIINO immediate product prior to formation of ferric enzyme. In W457F and W457A, the disappearance rate of the FeIIO2 intermediate was slower than in wild type and took place without detectable build-up of the heme FeIIINO immediate product. Rates of FeIIO2 disappearance correlated with rates of citrulline formation in all three proteins, and reactions containing H4B formed 1.0, 0.54, and 0.38 citrulline/heme in wild type, W457F, and W457A iNOSoxy, respectively. Thus, Trp-457 modulates the kinetics of NOHA oxidation by iNOSoxy, and this is important for determining the extent of citrulline and NO formation. Our findings support a redox role for H4B during NOHA oxidation to NO by iNOSoxy. In the oxygenase domain of mouse inducible nitric-oxide synthase (iNOSoxy), a conserved tryptophan residue, Trp-457, regulates the kinetics and extent of l-Arg oxidation to Nω-hydroxy-l-arginine (NOHA) by controlling electron transfer between bound (6R)-tetrahydrobiopterin (H4B) cofactor and the enzyme heme FeIIO2 intermediate (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819–12825). To investigate whether NOHA oxidation to citrulline and nitric oxide (NO) is regulated by a similar mechanism, we performed single turnover reactions with wild type iNOSoxy and mutants W457F and W457A. Ferrous proteins containing NOHA plus H4B or NOHA plus 7,8-dihydrobiopterin (H2B), were mixed with O2-containing buffer, and then heme spectral transitions and product formation were followed versus time. All three proteins formed a FeIIO2 intermediate with identical spectral characteristics. In wild type, H4B increased the disappearance rate of the FeIIO2intermediate relative to H2B, and its disappearance was coupled to the formation of a FeIIINO immediate product prior to formation of ferric enzyme. In W457F and W457A, the disappearance rate of the FeIIO2 intermediate was slower than in wild type and took place without detectable build-up of the heme FeIIINO immediate product. Rates of FeIIO2 disappearance correlated with rates of citrulline formation in all three proteins, and reactions containing H4B formed 1.0, 0.54, and 0.38 citrulline/heme in wild type, W457F, and W457A iNOSoxy, respectively. Thus, Trp-457 modulates the kinetics of NOHA oxidation by iNOSoxy, and this is important for determining the extent of citrulline and NO formation. Our findings support a redox role for H4B during NOHA oxidation to NO by iNOSoxy. nitric oxide NO synthase neuronal NOS cytokine-inducible NOS the oxygenase domain of inducible nitric-oxide synthase l-arginine Nω-hydroxy-l-arginine (6R)-tetrahydro-l-biopterin ferrous-dioxy specie ferric-NO dithiothreitol 7,8-dihydro-l-biopterin Nitric oxide (NO)1 is important in physiology and pathology (1.Nathan C. Shiloh M.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8841-8848Crossref PubMed Scopus (1132) Google Scholar, 2.Stopper H. Moller M. Bommel H.M. Schmidt H.H. Toxicol. Lett. 1999; 106: 59-67Crossref PubMed Scopus (17) Google Scholar, 3.Sasaki M. Gonzalez-Zulueta M. Huang H. Herring W.J. Ahn S. Ginty D.D. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8617-8622Crossref PubMed Scopus (160) Google Scholar). Three main NO synthases (NOSs) (EC1.14.13.39) have been characterized in animals: 1) neuronal NOS (nNOS, type I), 2) cytokine-inducible NOS (iNOS, type II), and 3) endothelial NOS (eNOS, type III). Each NOS is distinguished by its tissue location, regulation, and function (4.Butt E. Bernhardt M. Smolenski A. Kotsonis P. Frohlich L.G. Sickmann A. Meyer H.E. Lohmann S.M. Schmidt H.H.H.W. J. Biol. Chem. 2000; 275: 5179-5187Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 5.Harris M.B. Ju H. Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B.E. Venema R.C. J. Biol. Chem. 2001; 276: 16587-16591Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 6.Ganster R.W. Taylor B.S. Shao L. Geller D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8638-8643Crossref PubMed Scopus (277) Google Scholar, 7.Wang Y. Newton D.C. Robb G.B. Kau C.L. Miller T.L. Cheung A.H. Hall A.V. VanDamme S. Wilcox J.N. Marsden P.A. Proc. Natl. Acad. Sci. U. S. 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Biol. 1998; 2: 656-663Crossref PubMed Scopus (199) Google Scholar). In the first reaction, Arg undergoes hydroxylation to form water and Nω-hydroxy-l-arginine (NOHA) as an enzyme-bound intermediate. In the second reaction, NOHA undergoes oxidation to generate water, NO, and citrulline (Scheme FS1). Both reactions take place within a NOS oxygenase domain homodimer (NOSoxy), which contains two equivalent active sites comprised of iron protoporphyrin IX (heme), the cofactor (6R)-tetrahydrobiopterin (H4B), and the substrate binding site (14.Fischmann T.O. Hruza A. DaNiu X. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Paul R. Lundell D.J. Narula S.K. Weber P.C. Nat. Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (396) Google Scholar, 15.Li H. Raman C.S. Martasek P. Masters B.S. Poulos T.L. Biochemistry. 2001; 40: 5399-5406Crossref PubMed Scopus (74) Google Scholar, 16.Crane B.R. Arvai A.S. Ghosh S. Getzoff E.D. Stuehr D.J. Tainer J.A. Biochemistry. 2000; 39: 4608-46021Crossref PubMed Scopus (141) Google Scholar). Electrons derived from NADPH are provided to NOSoxy by attached reductase domains, which bind FMN, FAD, and NADPH (17.Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2001; 276: 23246-23252Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar,18.Zhang J. Martasek P. Paschke R. Shea T. Masters B.S. Kim J.J. J. Biol. Chem. 2001; 276: 37506-37513Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Crystal structures of NOS oxygenase domains (14.Fischmann T.O. Hruza A. DaNiu X. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Paul R. Lundell D.J. Narula S.K. Weber P.C. Nat. Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (396) Google Scholar, 15.Li H. Raman C.S. Martasek P. Masters B.S. Poulos T.L. Biochemistry. 2001; 40: 5399-5406Crossref PubMed Scopus (74) Google Scholar, 16.Crane B.R. Arvai A.S. Ghosh S. Getzoff E.D. Stuehr D.J. Tainer J.A. Biochemistry. 2000; 39: 4608-46021Crossref PubMed Scopus (141) Google Scholar) show that Arg and NOHA are held directly above the heme, consistent with the heme activating O2 for their oxidation. Accordingly, O2 activation in both reactions of NO synthesis is modeled after cytochrome P450 monooxygenase chemistry (Fig. 1). The transfer of an electron to the ferric NOS heme enables O2 binding and formation of a detectable ferrous-dioxy specie (FeIIO2) (heme specie I) (19.Abu-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 (157) Google Scholar, 20.Couture M. Stuehr D.J. Rousseau D.L. J. Biol. Chem. 2000; 275: 3201-3205Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 21.Ledbetter A.P. McMillan K. Roman L.J. Masters B.S.S. Dawson J.H. Sono M. Biochemistry. 1999; 38: 8014-8021Crossref PubMed Scopus (56) Google Scholar, 22.Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 23.Abu-Soud H.M. Ichimori K. Presta A. Stuehr D.J. J. Biol. Chem. 2000; 275: 17349-17357Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The reduction of FeIIO2 by a second electron is thought to form an iron-peroxo specie (heme specie II), which upon protonation and O–O bond scission can generate water and a high valence iron-oxo specie (heme specie III). The first electron provided to heme comes from the NOS reductase domain, whereas the second electron can come from the reductase domain or from H4B (22.Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 24.Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 25.Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (167) Google Scholar). Because heme species II or III (or related model species) can oxidize Arg and NOHA (24.Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 25.Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (167) Google Scholar, 26.Groves J.T. Wang C.C.-Y. Curr. Opin. Chem. Biol. 2000; 4: 687-695Crossref PubMed Scopus (178) Google Scholar, 27.Fukuto J.M. Methods Enzymol. 1996; 268: 365-375Crossref PubMed Google Scholar, 28.Huang H. Hah J.M. Silverman R.B. J. Am. Chem. Soc. 2001; 123: 2674-2676Crossref PubMed Scopus (60) Google Scholar), it is important to understand the second electron transfer in NOS and how it impacts downstream events like substrate oxidation. Experiments that view a single catalytic turnover are particularly helpful in this regard. In such experiments, ferrous NOSoxy proteins, which contain substrate and H4B, are rapidly mixed with oxygenated buffer to start the reaction. The progress of heme intermediates, electron transfer, and product formation are followed by rapid spectral acquisition and chemical quenching techniques. Our single turnover study of the Arg reaction showed that H4B reduces an FeIIO2intermediate, and this electron transfer is kinetically coupled to Arg hydroxylation (29.Wei 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 (131) Google Scholar). Moreover, a conserved Trp residue (Trp-457 in mouse iNOSoxy), which interacts with H4B by π stacking, was found to control the tempo of FeIIO2reduction by H4B (30.Wang Z.Q. Wei C.C. Ghosh S. Meade A.L. Hemann C. Hille R. Stuehr D.J. Biochemistry. 2001; 40: 12819-12825Crossref PubMed Scopus (41) Google Scholar). This finding appears to be important, because a slower reduction of FeIIO2by H4B in iNOSoxy mutants W457A and W457F was associated with slower and less complete Arg hydroxylation. Crystal structures of the Trp-457 iNOSoxy mutants suggest that they alter H4B redox function by causing structural changes that destabilize the H4B radical (31.Aoyagi M. Arvai A.S. Ghosh S. Stuehr D.J. Tainer J.A. Getzoff E.D. Biochemistry. 2001; 40: 12826-12832Crossref PubMed Scopus (33) Google Scholar). Thus, it appears that protein residues like Trp-457 can control the tempo and extent of Arg hydroxylation by influencing H4B reduction of the FeIIO2 intermediate. Although H4B has a clear redox function in the Arg reaction, whether it behaves similarly during NOHA oxidation is still unknown. Bound H4B is clearly required for NOS to convert NOHA to NO plus citrulline in both single and multiple turnover reactions (22.Bec N. Gorren A.C. Voelker C. Mayer B. Lange R. J. Biol. Chem. 1998; 273: 13502-13508Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 24.Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 25.Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (167) Google Scholar, 32.Abu-Soud H.M. Presta A. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10811-10816Crossref PubMed Scopus (72) Google Scholar, 33.Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar, 34.Hurshman A.R. Krebs C. Edmondson D.E. Huynh B.H. Marletta M.A. Biochemistry. 1999; 38: 15689-15696Crossref PubMed Scopus (212) Google Scholar). However, H4B reduction of FeIIO2 cannot be assigned, because there is only minimal H4B radical build-up during NOHA oxidation by NOS (34.Hurshman A.R. Krebs C. Edmondson D.E. Huynh B.H. Marletta M.A. Biochemistry. 1999; 38: 15689-15696Crossref PubMed Scopus (212) Google Scholar, 35.Bec N. Gorren A.F.C. Mayer B. Schmidt P.P. Andersson K.K. Lange R. J. Inorg. Biochem. 2000; 81: 207-211Crossref PubMed Scopus (59) Google Scholar, 36.Schmidt 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 (94) Google Scholar). Given this limitation, our Trp-457 mutants may provide an alternative approach to investigate H4B function in the second reaction of NO synthesis. Therefore, we studied NOHA oxidation under single turnover conditions in wild type, W457A, and W457F iNOSoxy. Our goal was 2-fold. (a) Define consecutive heme transitions that occur during the NOHA reaction and their temporal and quantitative relationship to product formation. (b) Determine whether Trp-457 mutations impact these parameters. Our results provide a kinetic framework for NOHA oxidation by iNOSoxy and are consistent with a redox role for H4B in the second reaction of NO synthesis. DTT was obtained from Sigma. H4B and 7,8-dihydrobiopterin (H2B) were purchased from the laboratory of Dr. B. Schirck (Jona, Switzerland), NOHA was a gift from Dr. Bruce King (Wake Forest University, Winston Salem, NC). All other chemicals were from Sigma or from sources reported previously (29.Wei 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 (131) Google Scholar, 30.Wang Z.Q. Wei C.C. Ghosh S. Meade A.L. Hemann C. Hille R. Stuehr D.J. Biochemistry. 2001; 40: 12819-12825Crossref PubMed Scopus (41) Google Scholar). Wild type and mutant iNOSoxy proteins (amino acids 1–498) containing a six-histidine tag at their C termini were overexpressed in Escherichia coli BL21 using the pCWori vector and purified as reported previously (37.Ghosh S. Wolan D. Adak S. Crane B.R. Kwon N.S. Tainer J.A. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 1999; 274: 24100-24112Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Concentrations of iNOSoxy proteins were determined from the absorbance at 444 nm of their ferrous-CO complexes, using an extinction coefficient of 76 mm−1 cm−1. W457A and W457F iNOSoxy proteins were preincubated with H4B or H2B (as required) on ice overnight before performing experiments because of their lower affinity toward H4B (30.Wang Z.Q. Wei C.C. Ghosh S. Meade A.L. Hemann C. Hille R. Stuehr D.J. Biochemistry. 2001; 40: 12819-12825Crossref PubMed Scopus (41) Google Scholar, 37.Ghosh S. Wolan D. Adak S. Crane B.R. Kwon N.S. Tainer J.A. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 1999; 274: 24100-24112Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). NOHA was added to the protein samples just prior to dithionite reduction to minimize its hydrolysis to citrulline. Mutant and wild type iNOSoxy proteins were reduced to ferrous forms as described previously (29.Wei 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 (131) Google Scholar, 30.Wang Z.Q. Wei C.C. Ghosh S. Meade A.L. Hemann C. Hille R. Stuehr D.J. Biochemistry. 2001; 40: 12819-12825Crossref PubMed Scopus (41) Google Scholar). The ferric proteins were placed in a cuvette, made anaerobic by repeated cycles of vacuum and N2 purging, diluted in N2-purged Hepes buffer (50 mm), pH 7.5, containing DTT, H4B or H2B, and NOHA. Graded amounts of a dithionite solution were then added under anaerobic conditions, and heme reduction was monitored spectroscopically. Full heme reduction typically required a slight molar excess of dithionite. All single turnover experiments were performed three or four times using different batches of iNOSoxy proteins prepared in this manner. Rapid-scanning measurements were carried out in a Hi-Tech (SF-61) stopped-flow apparatus equipped for anaerobic work and coupled to a Hi-Tech MG-6000 diode array detector. Ferrous iNOSoxy proteins prepared as described above were transferred into the stopped-flow instrument using a gas-tight syringe. The reactions mixed equal volumes of anaerobic enzyme solutions with air-saturated Hepes buffer at 10 °C. Final (post-mixing) concentrations for wild type iNOSoxy were 14 μm protein, 100 μm H4B or H2B, 0.5 mm DTT, and 100 μm NOHA. Final concentrations for W457A and W457F mutants were 7–9 μm protein, 1.5 mm H4B or H2B, 3 mmDTT, and 0.5 mm NOHA. Ninety-six spectral scans were obtained after each mixing. Sequential spectral data were fit to different reaction models using the Specfit global analysis program provided by Hi-Tech Ltd. that could calculate the number of different enzyme species, their spectra, and their concentrations versus time during the single turnover reactions. In some cases, reacted samples were collected from the exit loop of the instrument and frozen immediately on dry ice for product analysis by high performance liquid chromatography. The kinetics of NOHA oxidation during the single turnover reaction was studied using a Hi-Tech RFQ-63 rapid-quench instrument as described previously (29.Wei 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 (131) Google Scholar, 30.Wang Z.Q. Wei C.C. Ghosh S. Meade A.L. Hemann C. Hille R. Stuehr D.J. Biochemistry. 2001; 40: 12819-12825Crossref PubMed Scopus (41) Google Scholar). In general, solutions of ferrous iNOSoxy proteins containing NOHA plus H4B or H2B were mixed with an equal volume of oxygen-saturated buffer at 10 °C followed by rapid quenching with the same volume of 1 n HCl. Reactions contained 0.1–0.13 mm wild type or Trp-457 mutants plus 2 mmH4B or H2B, 3 mm DTT, 3 mm NOHA, and 0.1 mml-glutamate as an internal standard. Ferrous protein solutions were prepared as described above for the rapid-scanning experiments. An analysis of amino acids present in the quench-flow reactions was done as described elsewhere (29.Wei 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 (131) Google Scholar, 30.Wang Z.Q. Wei C.C. Ghosh S. Meade A.L. Hemann C. Hille R. Stuehr D.J. Biochemistry. 2001; 40: 12819-12825Crossref PubMed Scopus (41) Google Scholar) with some modifications. Isopropyl alcohol (5 μl) was added to each quenched reaction sample (total volume ∼90 μl). The samples were vortexed for 30 min and centrifuged at 10,000 × g for 15 min. The supernatant was removed and had 1 μl of 30 mm methyl red added. Acid was partially neutralized immediately prior to analysis by slowly adding 5n NaOH to each sample until the color changed from pink to yellow. Amino acids in aliquots taken from single turnover reactions run in anaerobic vessels from stopped-flow rapid-scanning experiments or from rapid-quench experiments (acid neutralized) were derivatized with o-phthalaldehyde and then analyzed by reverse-phase high performance liquid chromatography with fluorescence detection (29.Wei 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 (131) Google Scholar). Samples generated in anaerobic vessels or in the stopped-flow instrument were filtered through an Amicon Centricon device (10,000 molecular weight cut-off) prior to derivatization. For high performance liquid chromatography, we used a Hewlett-Packard ODS-Hypersil column that was eluted with a gradient of buffer A (5 mm ammonium acetate, pH 6.0, 20% methanol) and buffer B (100% methanol). Retention times and concentrations of amino acids were calculated based on NOHA and citrulline standard solutions. Citrulline in samples generated by rapid quenching was quantitated relative to the l-glutamate standard. Nitrite was measured using the Griess colorimetric assay (32.Abu-Soud H.M. Presta A. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10811-10816Crossref PubMed Scopus (72) Google Scholar). We first determined the stoichiometry of NOHA conversion to citrulline for wild type iNOSoxy and for the W457A and W457F mutant iNOSoxy proteins. Mechanistic considerations predict that ferrous iNOSoxy should convert one NOHA to citrulline/heme in a single turnover reaction (32.Abu-Soud H.M. Presta A. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10811-10816Crossref PubMed Scopus (72) Google Scholar, 33.Gorren A.C. Bec N. Schrammel A. Werner E.R. Lange R. Mayer B. Biochemistry. 2000; 39: 11763-11770Crossref PubMed Scopus (74) Google Scholar, 38.Boggs S. Huang L. Stuehr D.J. Biochemistry. 2000; 39: 2332-2339Crossref PubMed Scopus (65) Google Scholar). Anaerobic samples of dithionite-reduced (ferrous) iNOSoxy proteins containing NOHA plus H4B were mixed with O2-containing buffer, and the reactions were allowed to go to completion. Wild type iNOSoxy generated 1.0 ± 0.03 (n = 3) citrulline/heme from NOHA in the single turnover reaction. This result matches the stoichiometry we observed for nNOSoxy NOHA reactions run in a similar manner (38.Boggs S. Huang L. Stuehr D.J. Biochemistry. 2000; 39: 2332-2339Crossref PubMed Scopus (65) Google Scholar). W457F and W457A iNOSoxy proteins generated 0.54 ± 0.10 (n = 3) and 0.38 ± 0.06 (n = 4) citrulline/heme, respectively. The rank order of nitrite formed in the reactions mimicked citrulline production (wild type > W457F > W457A, data not shown). Control experiments run without dithionite or without enzyme produced no citrulline from NOHA, and the addition of superoxide dismutase did not lower product formation (data not shown). Wild type and mutant iNOSoxy reactions that contained H2B all produced some citrulline from NOHA (up to 0.4 citrulline/heme), but in these cases SOD addition decreased citrulline production by 60%, suggesting NOHA had reacted in a non-enzymatic manner with superoxide. Thus, all three iNOSoxy proteins oxidized NOHA to citrulline and nitrite in the single turnover reaction. The enzymatic reaction required H4B, and catalysis by Trp-457 mutants was less efficient than wild type. We next examined heme transitions associated with catalysis during the NOHA single turnover reaction. The solutions of ferrous iNOSoxy containing NOHA and H4B were rapidly mixed with O2-containing buffer in a stopped-flow spectrophotometer equipped with a rapid-scanning diode array detector, and spectral data were subject to global analysis using software provided by the instrument manufacturer. For wild type iNOSoxy, the spectral data were best fit to an A → B → C → D model with three monophasic transitions, and thus discerned four distinct spectral species. These were in order of appearance the ferrous enzyme, two transient intermediates, and ferric enzyme (Fig. 2A). The Soret and visible absorbance features of the first and second transient species match those of FeIIO2 and ferric-NO (FeIIINO) complexes of iNOS, nNOS, or endothelial NOS that have been observed under similar circumstances (19.Abu-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 (157) Google Scholar, 20.Couture M. Stuehr D.J. Rousseau D.L. J. Biol. Chem. 2000; 275: 3201-3205Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 23.Abu-Soud H.M. Ichimori K. Presta A. Stuehr D.J. J. Biol. Chem. 2000; 275: 17349-17357Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 29.Wei 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 (131) Google Scholar, 38.Boggs S. Huang L. Stuehr D.J. Biochemistry. 2000; 39: 2332-2339Crossref PubMed Scopus (65) Google Scholar). Fig. 2B depicts the calculated concentrations of the four species versus time during the single turnover reaction. Calculated rates for the three heme transitions are listed in Table I. Oxygen binding to ferrous iNOSoxy represents the first transition. Maximal accumulation of FeIIO2 specie was only 55% total enzyme because of its similar rates of formation and disappearance (Table I). The next transition represents conversion of the FeIIO2 species into the FeIIINO specie. This product-forming step represents kcat for the reaction (29.Wei 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 (131) Google Scholar, 30.Wang Z.Q. Wei C.C. Ghosh S. Meade A.L. Hemann C. Hille R. Stuehr D.J. Biochemistry. 2001; 40: 12819-12825Crossref PubMed Scopus (41) Google Scholar). Relative rates of FeIIINO formation and decay are such that 85% FeIIINO product accumulated prior to NO dissociation from the heme.Table IObserved rates (s−1) for heme transitions during NOHA single turnover reactions catalyzed by wild type iNOSoxy and Trp-457 mutantsFeII → FeIIO2FeIIO → FeIIINOFeIIINO → FeIIIWT + NOHA + H4B (n = 2)47.3 ± 3.736.7 ± 1.12.3 ± 0.1FeII → FeIIO2FeIIO2 → FeIIIWT + NOHA + H2B (n = 4)64.0 ± 1.611.0 ± 0.1WT + Arg + H4B1-aData were taken from Refs.29 and 30.52.7 ± 2.212.5 ± 0.2WT + Arg + H2B1-aData were taken from Refs.29 and 30.0.30 ± 0.08FeII → FeIIO2FeIIO2 → FeIIIW457A + NOHA + H4B (n = 2)73.0 ± 4.96.7 ± 0.2W457A + NOHA + H2B (n = 2)191 ± 1313.3 ± 0.2W457A + Arg + H4B1-aData were taken from Refs.29 and 30.95.4 ± 1.53.0 ± 0.1W457A + Arg + H2B1-aData were taken from Refs.29 and 30.0.052 ± 0.003FeII → FeIIO2FeIIIO2 → FeIIIW457F + NOHA + H4B (n = 3)68.7 ± 1.18.6 ± 0.1W457F + NOHA + H2B (n = 1)ND2.62 ± 0.01W457F + Arg + H4B1-aData were taken from Refs.29 and 30.60.3 ± 4.76.5 ± 0.1W457F + Arg + H2B1-aData were taken from Refs.29 and 30.0.37 ± 0.01Ferrous iNOSoxy proteins containing NOHA and the indicated pteridine were rapid-mixed at 10 °C with air-saturated buffer to start the reactions. Subsequent heme transformations were followed by stopped-flow rapid-scanning spectroscopy. Rates were calculated by Specfit global analysis of diode array spectral data. Values are the means ± S.D. with number of independent experiments in parentheses involving 8–15 individual reactions/experiment. WT, wild type; ND, not determined.1-a Data were taken from Refs.29.Wei 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 (131) Google Scholar and 30.Wang Z.Q. Wei C.C. Ghosh S. Meade A.L. Hemann C. Hille R. Stuehr D.J. Biochemistry. 2001; 40: 12819-12825Crossref PubMed Scopus (41) Google Scholar. Open table in a new tab Ferrous iNOSoxy proteins containing NOHA and the indicated pteridine were rapid-mixed at 10 °C with air-saturated buffer to start the reactions. Subsequent heme transformations were followed by stopped-flow rapid-scanning spectroscopy. Rates were calculated by Specfit global analysis of diode array spectral data. Values are the means ± S.D. with number of independent experiments in parentheses involving 8–15 individual reactions/experiment. WT, wild type; ND, not determined. In the first reaction of NO synthesis (Arg hydroxylation), H4B speeds FeIIO2 disappearance relative to an H2B-containing enzyme, because only H4B can donate an electron to the FeIIO2 intermediate (29.Wei 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 (131) Google Scholar, 30.Wang Z.Q. Wei C.C. Ghosh S. Meade A.L. Hemann C. Hille R. Stuehr D.J. Biochemistry. 2001; 40: 12819-12825Crossref PubMed Scopus (41) Google Scholar). To determine whether H4B accelerates FeIIO2disappearance in the second reaction of NO synthesis, we ran NOHA single turnover reactions using H2B-saturated iNOSoxy. Spectral data best fit to an A → B → C transition in this case, representing formation of the FeIIO2intermediate and its transition to ferric enzyme. Spectral features of the FeIIO2 intermediate that formed in iNOSoxy

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