Surface Charge Interactions of the FMN Module Govern Catalysis by Nitric-oxide Synthase
2006; Elsevier BV; Volume: 281; Issue: 48 Linguagem: Inglês
10.1074/jbc.m606129200
ISSN1083-351X
AutoresKoustubh Panda, Mohammad Mahfuzul Haque, Elsa D. Garcin, Deborah Durra, Elizabeth D. Getzoff, Dennis J. Stuehr,
Tópico(s)Analytical Chemistry and Sensors
ResumoThe FMN module of nitric-oxide synthase (NOS) plays a pivotal role by transferring NADPH-derived electrons to the enzyme heme for use in oxygen activation. The process may involve a swinging mechanism in which the same face of the FMN module accepts and provides electrons during catalysis. Crystal structure shows that this face of the FMN module is electronegative, whereas the complementary interacting surface is electropositive, implying that charge interactions enable function. We used site-directed mutagenesis to investigate the roles of six electronegative surface residues of the FMN module in electron transfer and catalysis in neuronal NOS. Results are interpreted in light of crystal structures of NOS and related flavoproteins. Neutralizing or reversing the negative charge of each residue altered the NO synthesis, NADPH oxidase, and cytochrome c reductase activities of neuronal NOS and also altered heme reduction. The largest effects occurred at the NOS-specific charged residue Glu762. Together, the results suggest that electrostatic interactions of the FMN module help to regulate electron transfer and to minimize flavin autoxidation and the generation of reactive oxygen species during NOS catalysis. The FMN module of nitric-oxide synthase (NOS) plays a pivotal role by transferring NADPH-derived electrons to the enzyme heme for use in oxygen activation. The process may involve a swinging mechanism in which the same face of the FMN module accepts and provides electrons during catalysis. Crystal structure shows that this face of the FMN module is electronegative, whereas the complementary interacting surface is electropositive, implying that charge interactions enable function. We used site-directed mutagenesis to investigate the roles of six electronegative surface residues of the FMN module in electron transfer and catalysis in neuronal NOS. Results are interpreted in light of crystal structures of NOS and related flavoproteins. Neutralizing or reversing the negative charge of each residue altered the NO synthesis, NADPH oxidase, and cytochrome c reductase activities of neuronal NOS and also altered heme reduction. The largest effects occurred at the NOS-specific charged residue Glu762. Together, the results suggest that electrostatic interactions of the FMN module help to regulate electron transfer and to minimize flavin autoxidation and the generation of reactive oxygen species during NOS catalysis. Nitric oxide is an important biological signal molecule produced in animals by three nitric-oxide synthase (NOS) 3The abbreviations used are: NOS, nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; CaM, calmodulin; nNOSr, neuronal nitric-oxide synthase flavoprotein domain; FNR, ferredoxin-NADP+ reductase; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; l-Arg, l-Arginine. isozymes: inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 2MacMicking J. Xie Q.W. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Crossref PubMed Scopus (3501) Google Scholar). NOS enzymes have unique characteristics, and their protein structure-function relationships are of current interest. All mammalian NOS enzymes are comprised of an N-terminal oxygenase domain and a C-terminal flavoprotein domain, with a calmodulin (CaM)-binding site connecting the two domains (3Masters B.S. McMillan K. Sheta E.A. Nishimura J.S. Roman L.J. Martásek P. FASEB J. 1996; 10: 552-558Crossref PubMed Scopus (196) Google Scholar). During NO synthesis, the flavoprotein domain transfers NADPH-derived electrons through its FAD and FMN cofactors to a heme located in the oxygenase domain. This enables NOS to catalyze heme-dependent oxygen activation and a stepwise conversion of l-Arg to NO and citrulline (4Andrew P.J. Mayer B. Cardiovasc. Res. 1999; 43: 521-531Crossref PubMed Scopus (588) Google Scholar, 5Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (167) Google Scholar). CaM binding to NOS activates NO synthesis by triggering electron transfer from the FMN to the heme (6Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (398) Google Scholar). CaM also relieves repression of NOS flavoprotein electron transfer to external acceptors like cytochrome c (7Gachhui 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, 9Newman E. Spratt D.E. Mosher J. Cheyne B. Montgomery H.J. Wilson D.L. Weinberg J.B. Smith S.M. Salerno J.C. Ghosh D.K. Guillemette J.G. J. Biol. Chem. 2004; 279: 33547-33557Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). How these processes occur is mostly unknown. The nNOS flavoprotein domain (nNOSr) is similar to dual-flavin oxidoreductases like cytochrome P450 reductase (10Wang M. Roberts D.L. Paschke R. Shea T.M. Siler Masters B.S. Kim J.J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (671) Google Scholar), novel reductase-1 (11Finn R.D. Basran J. Roitel O. Wolf C.R. Munro A.W. Paine M.J. Scrutton N.S. Eur. J. Biochem. 2003; 270: 1164-1175Crossref PubMed Scopus (42) Google Scholar), and methionine synthase reductase (12Olteanu H. Wolthers K.R. Munro A.W. Scrutton N.S. Banerjee R. Biochemistry. 2004; 43: 1988-1997Crossref PubMed Scopus (43) Google Scholar) and to the flavoprotein domain of bacterial cytochrome P450BM3 (13Munro A.W. Daff S. Coggins J.R. Lindsay J.G. Chapman S.K. Eur. J. Biochem. 1996; 239: 403-409Crossref PubMed Scopus (114) Google Scholar, 14Sevrioukova I. Shaffer C. Ballou D.P. Peterson J.A. Biochemistry. 1996; 35: 7058-7068Crossref PubMed Scopus (54) Google Scholar). All are comprised of a ferredoxin-NADP+ reductase (FNR) module that contains FAD and that binds NADPH, a connecting subdomain that is inserted into the FNR module, and an FMN module. All transfer NADPH-derived electrons to native hemeprotein acceptors (NOS and cytochrome P450BM3) or to artificial hemeprotein acceptors like cytochrome c. In nNOS, the electron transfer process appears to involve a conformational equilibrium of the FMN module (9Newman E. Spratt D.E. Mosher J. Cheyne B. Montgomery H.J. Wilson D.L. Weinberg J.B. Smith S.M. Salerno J.C. Ghosh D.K. Guillemette J.G. J. Biol. Chem. 2004; 279: 33547-33557Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 15Garcin E.D. Bruns C.M. Lloyd S.J. Hosfield D.J. Tiso M. Gachhui R. Stuehr D.J. Tainer J.A. Getzoff E.D. J. Biol. Chem. 2004; 279: 37918-37927Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 16Konas D.W. Zhu K. Sharma M. Aulak K.S. Brudvig G.W. Stuehr D.J. J. Biol. Chem. 2004; 279: 35412-35425Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), which is illustrated in Fig. 1. The FMN module must dock with the FNR module in an "FMN-shielded" conformation to receive electrons from FAD. Thereafter, the FMN module must swing away by means of a flexible linker to populate an "FMN-deshielded" conformation to interact with electron acceptors like the NOS oxygenase domain and cytochrome c. The crystal structure of nNOSr (15Garcin E.D. Bruns C.M. Lloyd S.J. Hosfield D.J. Tiso M. Gachhui R. Stuehr D.J. Tainer J.A. Getzoff E.D. J. Biol. Chem. 2004; 279: 37918-37927Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) depicts the FMN module docked against the FNR module in the electron-accepting, FMN-shielded conformation (Fig. 2). A similar arrangement is observed in the crystal structure of cytochrome P450 reductase (10Wang M. Roberts D.L. Paschke R. Shea T.M. Siler Masters B.S. Kim J.J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (671) Google Scholar), suggesting that a conformational switching mechanism is likely to operate in related flavoproteins.FIGURE 2Structure of nNOSr. The ribbon diagram shows the FMN module (yellow) docked against the FNR module (pink) in the FMN-shielded conformation, with bound cofactors FMN, FAD, and NADP shown in ball-and-stick representation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The conformational switching model implies that electron transfer into and out of the FMN module is mutually exclusive and probably subject to complex control. How any flavoprotein controls this process is largely unclear (17Grunau A. Paine M.J. Ladbury J.E. Gutierrez A. Biochemistry. 2006; 45: 1421-1434Crossref PubMed Scopus (42) Google Scholar). For nNOS, the equilibrium between the FMN-shielded and FMN-deshielded conformations appears to have an intrinsic set point that can be influenced by several factors. For example, CaM binding shifts the equilibrium toward the FMN-deshielded conformation (16Konas D.W. Zhu K. Sharma M. Aulak K.S. Brudvig G.W. Stuehr D.J. J. Biol. Chem. 2004; 279: 35412-35425Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 18Craig D.H. Chapman S.K. Daff S. J. Biol. Chem. 2002; 277: 33987-33994Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), whereas the FMN-shielded conformation is stabilized by residues in or near a C-terminal tail regulatory element in conjunction with bound NADPH (15Garcin E.D. Bruns C.M. Lloyd S.J. Hosfield D.J. Tiso M. Gachhui R. Stuehr D.J. Tainer J.A. Getzoff E.D. J. Biol. Chem. 2004; 279: 37918-37927Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 16Konas D.W. Zhu K. Sharma M. Aulak K.S. Brudvig G.W. Stuehr D.J. J. Biol. Chem. 2004; 279: 35412-35425Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 18Craig D.H. Chapman S.K. Daff S. J. Biol. Chem. 2002; 277: 33987-33994Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 19Tiso M. Konas D.W. Panda K. Garcin E.D. Sharma M. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 2005; 280: 39208-39219Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Shifting the conformational equilibrium of the FMN module in nNOS directly impacts its cytochrome c reductase activity and, in some cases, alters the susceptibility of its reduced flavins to autoxidation (16Konas D.W. Zhu K. Sharma M. Aulak K.S. Brudvig G.W. Stuehr D.J. J. Biol. Chem. 2004; 279: 35412-35425Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 19Tiso M. Konas D.W. Panda K. Garcin E.D. Sharma M. Getzoff E.D. Stuehr D.J. J. Biol. Chem. 2005; 280: 39208-39219Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Variables that may control FMN module function in nNOS and related flavoproteins include surface hydrophobic and electrostatic contacts, hinge length and flexibility, and thermodynamic stability of reduced flavin states (15Garcin E.D. Bruns C.M. Lloyd S.J. Hosfield D.J. Tiso M. Gachhui R. Stuehr D.J. Tainer J.A. Getzoff E.D. J. Biol. Chem. 2004; 279: 37918-37927Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 20Govindaraj S. Poulos T.L. Biochemistry. 1995; 34: 11221-11226Crossref PubMed Scopus (37) Google Scholar, 25Zhang J. Martásek P. Paschke R. Shea T. Siler Masters B.S. Kim J.J.P. J. Biol. Chem. 2001; 276: 37506-37513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Charged residues at the FMN module interface are prominent in the crystal structure of nNOSr (15Garcin E.D. Bruns C.M. Lloyd S.J. Hosfield D.J. Tiso M. Gachhui R. Stuehr D.J. Tainer J.A. Getzoff E.D. J. Biol. Chem. 2004; 279: 37918-37927Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) and may be important for function as judged by experiments demonstrating salt effects on nNOS catalysis (26Ortiz de Nishida C.R. Montellano P.R. J. Biol. Chem. 2001; 276: 20116-20124Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 27Schrammel A. Gorren A.C. Stuehr D.J. Schmidt K. Mayer B. Biochim. Biophys. Acta. 1998; 1387: 257-263Crossref PubMed Scopus (20) Google Scholar). Six acidic residues create an electronegative patch on the surface of the FMN module in nNOSr (Fig. 3A). This electronegative patch interacts with a complementary electropositive patch on the surface of the FNR module (Fig. 3B). A closer view of how these residues interact is shown in Fig. 3C. Similar electrostatic complementation is present to varying degrees in related flavoproteins, suggesting that it may be a general means to control subdomain interactions. NOS oxygenase domains also contain an electropositive surface patch (Fig. 3D) in an area considered to be a potential docking site for the FMN module (28Crane 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). Thus, electrostatic interactions could conceivably regulate both the electron import and export reactions of the FMN module during NOS catalysis. We therefore employed point mutagenesis to neutralize or reverse the charges of the six acidic residues that create the electronegative patch on the FMN module, along with the interactions of one residue in the complementary electropositive surface patch of the FNR module, as listed in Table 1. The results obtained with these mutants support a role for electrostatic surface interactions in controlling electron transfer, O2 reduction, and catalysis in nNOS and reveal which residues are the most important.TABLE 1Mutations used in the studynNOS mutationsFMN moduleGlu762 to Asn, Arg, or AlaGlu789 to Asn, Arg, or AlaGlu790 to Asn, Arg, or AlaAsp813 to Asn, Arg, or AlaGlu816 to Asn, Arg, or AlaGlu819 to Asn, Arg, or AlaFNR moduleArg1229 to Asn, Glu, or Ala Open table in a new tab Reagents—All reagents and materials were obtained from Sigma or sources reported previously (29Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 31Panda K. Adak S. Konas D. Sharma M. Stuehr D.J. J. Biol. Chem. 2004; 279: 18323-18333Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Molecular Biology—Wild-type and mutant nNOS proteins containing a His6 tag attached to their N termini were overexpressed in Escherichia coli strain BL21(DE3) using a modified pCWori vector as described (32Ghosh D.K. Wu C. Pitters E. Moloney M. Werner E.R. Mayer B. Stuehr D.J. Biochemistry. 1997; 36: 10609-10619Crossref PubMed Scopus (154) Google Scholar). Restriction digestions, cloning, and bacterial growth was performed using standard procedures. Transformations were done using a TransformAid bacterial transformation kit (Fermentas). Oligonucleotides used to construct site-directed mutants in nNOS were obtained from Integrated DNA Technologies (Coralville, IA) and are listed in supplemental Table S1. Site-directed mutagenesis was done using a QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). Incorporated mutations were confirmed by DNA sequencing at the Cleveland Clinic Molecular Biotechnology Core. Expression and Purification of nNOS Proteins—All proteins were purified in the presence of (6R)-tetrahydrobiopterin and l-Arg as described previously (29Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 33Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2001; 276: 23246-23252Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The ferrous heme-CO adduct absorbing at 444 nm was used to measure hemeprotein content with an extinction coefficient of ϵ444 = 74 mm-1 cm-1 (A444-A500). Measurement of Flavin Content—Bound FAD and FMN were released from nNOS proteins by heat denaturation of the enzyme (95 °C for 5 min in the dark) in sealed and light-protected tubes. The samples were then cooled to 4 °C and filtered to remove denatured protein. Filtrates were injected onto an Alltech Partisil ODS-3 column (250 × 4.6 mm, 5 μm) and subjected to binary gradient elution with 25 mm phosphate buffer (pH 5.8) and 100% acetonitrile at a flow rate of 1 ml/min. FAD and FMN had retention times of 4.1 and 7.6 min, respectively, and the peaks were completely resolved. Flavins were detected by fluorescence emission (λex = 460 nm and λem = 530 nm) and quantitated against freshly prepared FAD and FMN standards. NO Synthesis, NADPH Oxidation, and Cytochrome c Reduction—NO synthesis activity was determined using the spectrophotometric oxyhemoglobin assay (29Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 30Adak S. Santolini J. Tikunova S. Wang Q. Johnson J.D. Stuehr D.J. J. Biol. Chem. 2001; 276: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 33Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2001; 276: 23246-23252Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Cuvettes contained 0.2 μm nNOS, 40 mm EPPS (pH 7.6), 150 mm NaCl, 0.3 mm dithiothreitol, 4 μm FAD, 4 μm FMN, 10 μm (6R)-tetrahydrobiopterin, 10 mm l-Arg, 1 mg/ml bovine serum albumin, 0.8 mm Ca2+, 0.6 mm EDTA, 0.9 μm CaM, 100 units/ml catalase, 25 units/ml superoxide dismutase, and 5 μm oxyhemoglobin. The reaction was initiated with 300 μm NADPH in a total reaction volume of 500 μl and was run for 3 min at 25 °C. The NO-mediated conversion of oxyhemoglobin to methemoglobin was monitored at 401 nm and converted to a rate of NO synthesis using a difference extinction coefficient of ϵ401 = 38 mm-1 cm-1. NADPH oxidation rates were similarly measured at 340 nm in the presence of oxyhemoglobin under identical conditions, and the rate of NADPH oxidation was calculated using an extinction coefficient of ϵ340 = 6.2 mm-1 cm-1. Cytochrome c reductase activity was determined at 550 nm (ϵ550 = 21 mm-1 cm-1) using an assay mixture containing 40 mm EPPS (pH 7.6), 150 mm NaCl, 4 μm FAD, 4 μm FMN, 0.1 mg/ml bovine serum albumin, 10 μg/ml CaM, 0.6 mm EDTA, 10 units/ml catalase, 25 units/ml superoxide dismutase, and 0.1 mm cytochrome c. In the cytochrome c reductase assays, the concentration of added NaCl in the buffer was either 0 or 150 mm as indicated. The reaction was initiated by addition of 0.1 mm NADPH. Heme Reduction—The kinetics of heme reduction were analyzed at 10 °C as described previously (30Adak S. Santolini J. Tikunova S. Wang Q. Johnson J.D. Stuehr D.J. J. Biol. Chem. 2001; 276: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) using a stopped-flow apparatus and diode array detector (Hi-Tech Ltd. Model SF-61) equipped for anaerobic analysis. Ferric heme reduction was followed by formation of the ferrous heme-CO complex at 444 nm. Reactions were initiated by rapidly mixing an anaerobic, buffered, CO-saturated solution containing either 50 μm NADPH or 5 mm CaCl2 with an anaerobic, buffered, CO-saturated solution containing wild-type or mutant nNOS (5 μm), 100 mm EPPS (pH 7.6), 100 mm NaCl, 10 μm (6R)-tetrahydrobiopterin, 5 mm l-Arg, 0.3 mm dithiothreitol, 4 μm CaM, and either 1 mm Ca2+ when triggered with NADPH or 50 μm NADPH when triggered with Ca2+. Signal-to-noise ratios were improved by averaging 8-10 individual mixing experiments. The time course of the absorbance change was fit to single or multiple exponential equations using a nonlinear least-square method provided by Hi-Tech Ltd. Overall Properties of the FMN Module Mutants—In general, the mutations incorporated at the interface of the FNR/FMN modules did not alter protein expression or content of bound FAD, FMN, and heme (data not shown). The only mutant with aberrant properties was R1229E nNOS, which bound poorly to the 2′,5′-ADP affinity column during its purification. Because of this, we did not utilize R1229E nNOS in this study. However, the properties of the other mutants indicated they could be used to evaluate roles for the charged surface residues in nNOS function. Cytochrome c Reductase Activity in the Absence or Presence of Bound CaM—The cytochrome c reductase activity of NOS is repressed in the CaM-free state, and the repression is relieved upon CaM binding (34Stuehr D.J. Ghosh S. Mayer B. Nitric Oxide. Springer-Verlag, Berlin2000: 33-70Google Scholar). We compared the steady-state cytochrome c reductase activities of each mutant in the absence and presence of bound CaM. All assays contained superoxide dismutase to ensure we detected only superoxide-independent cytochrome c reductase activity, i.e. that which involves only a direct electron transfer from the FMN hydroquinone (FMNH2) of nNOS to cytochrome c (8Klatt P. Heinzel B. John M. Kastner M. Bohme E. Mayer B. J. Biol. Chem. 1992; 267: 11374-11378Abstract Full Text PDF PubMed Google Scholar, 18Craig D.H. Chapman S.K. Daff S. J. Biol. Chem. 2002; 277: 33987-33994Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Under CaM-free conditions, most of the mutants had higher cytochrome c reductase activities relative to wild-type nNOS (Fig. 4, left panel). The most prominent increases were in the Glu762 and Glu816 mutants. The relative effect of charge neutralization versus charge reversal depended on the acidic surface residue being considered. For example, at Glu762, the rank order of mutational effect was Asn > Ala > Arg, whereas for Glu816, the rank order was Arg > Ala > Asn. Our results show that the electronegative patch residues of the FMN module (particularly Glu762 and Glu816) help to repress the cytochrome c reductase activity of nNOS in the CaM-free state. To investigate the importance of charge interaction of each of the six acidic residues, we determined how the two charge-neutralizing mutations (Ala and Asn) at each residue might impact the salt effect on nNOS cytochrome c reductase activity. In CaM-free nNOS, a change in salt concentration from 0 to 250 mm is known to double the activity (35Roman L.J. Martásek P. Miller R.T. de la Harris D.E. Garza M.A. Shea T.M. Kim J.J.P. Siler Masters B.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), consistent with charge interactions playing a role in repression of the activity. In our hands, the cytochrome c reductase activity of CaM-free wild-type nNOS increased by 150 ± 8% (n = 3) in going from 0 to 150 mm added NaCl. The charge-neutralizing mutations resulted in a lesser salt effect in at least 7 of 12 cases (supplemental Table S2). A lesser salt effect was observed for both charge-neutralizing mutations at Glu762 and Asp813 and was observed for at least one of the two charge-neutralizing mutations at Glu790, Glu816, and Glu819, which together represent five of the six charged FMN surface residues that we studied. Charge neutralization did not clearly impact the salt effect in only one case, Glu789 (supplemental Table S2). These results suggest that the charge interactions of five of the six acidic surface residues of the FMN module contribute to the overall salt effect on the cytochrome c reductase activity of CaM-free nNOS. The cytochrome c reductase activities of the CaM-bound mutants are shown in Fig. 4 (right panel). Each FMN domain mutant had an increase in activity relative to the CaM-free condition, indicating that they all still responded to CaM. Most of the mutants achieved activities that were similar (±20%) to those of CaM-bound nNOS. The exceptions were the E762A, E762N, and D813N mutants, whose activities were 130%, 180%, and 160%, respectively, that of CaM-bound wild-type nNOS, and the E762R, E819R, and D813R mutants, whose activities were about 65% that of CaM-bound wild-type nNOS. The latter three mutants had normal Km values for cytochrome c (data not shown), suggesting that their lower activities were not due to an impaired interaction with cytochrome c. Together, the data indicate that charge neutralization at Glu762, Asp813, and possibly Glu819 increases the activity of CaM-bound nNOS, whereas charge reversal at these same sites diminishes activity. Fig. 4 also reports the cytochrome c reductase activities of the Arg1229 mutants. The CaM-free R1229N and R1229A mutants had only slightly higher activities compared with CaM-free wild-type nNOS, suggesting that Arg1229 is not so important for repressing activity in the CaM-free state. However, the activities of the CaM-bound R1229A and R1229N mutants were only 20% that of CaM-bound wild-type nNOS, indicating that Arg1229 enables nNOS to increase activity in response to CaM. NADPH Oxidase Activity of the CaM-free Mutants—Almost all of the CaM-free mutants oxidized NADPH at rates that exceeded wild-type nNOS (Fig. 5), despite their not synthesizing detectable NO under this circumstance (data not shown). The NADPH oxidation rates of the CaM-free E762N, E762A, and E816R mutants were highest and were 12-18 times faster than the wild-type value. The NADPH oxidation rates of the CaM-free R1229N and R1229A mutants were also four to seven times faster than that of wild-type nNOS (Fig. 5). Because the NADPH oxidase activity of CaM-free nNOS is directly linked to the autoxidation rate of its reduced flavins (35Roman L.J. Martásek P. Miller R.T. de la Harris D.E. Garza M.A. Shea T.M. Kim J.J.P. Siler Masters B.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 36Stuehr D. Pou S. Rosen G.M. J. Biol. Chem. 2001; 276: 14533-14536Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), our results establish that electronegative charged residues at the FNR/FMN module interface are required to minimize flavin autoxidation and the consequent generation of reduced oxygen species by nNOS. NO Synthesis and NADPH Oxidation by the CaM-bound Mutants—Eleven of the 18 FMN surface mutants had a 30% or greater reduction in their NO synthesis activities relative to the wild-type activity (Fig. 6, left panel). The mutants with the lowest NO synthesis activities were, in decreasing order, E762R and E816A (40% of the wild-type activity), E816R and E819A (32% of the wild-type activity), and E762A (21% of the wild-type activity). Substitution of three of the residues (Glu762, Glu816, and Glu819) with Ala or Arg diminished NO synthesis activity, whereas substitution with Asn either had little effect or, in one case (E762N), doubled the NO synthesis activity relative to the wild-type activity. The NO synthesis activities of the complementary FNR surface mutants R1229N and R1229A were 23% and 34% that of wild-type nNOS, respectively. Thus, charged residues at the FNR/FMN module interface are important for maintaining NO synthesis activity in nNOS. Fig. 6 (right panel) reports the NADPH oxidation rates during NO synthesis from l-Arg by the mutants and wild-type nNOS. Several mutants had higher rates of NADPH oxidation despite their lower rates of NO synthesis. Wild-type nNOS oxidized 2.0 molecules of NADPH/molecule of NO formed, which is close to the theoretical minimum of 1.5 (1Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 4Andrew P.J. Mayer B. Cardiovasc. Res. 1999; 43: 521-531Crossref PubMed Scopus (588) Google Scholar). The E819A, E819R, and E762A mutants had NADPH:NO stoichiometric ratios of greater than 3.5, indicating an NADPH consumption significantly beyond that required for NO synthesis. The NADPH oxidation rates of the R1229N and R1229A FNR surface mutants were also in excess of that required for NO synthesis. Thus, the electrostatic charge of these residues appears to be important for coupling NADPH consumption to NO synthesis. Rates of Heme Reduction—To examine mutational effects on electron transfer, we measured the rate of heme reduction in single turnover reactions that were run in a stopped-flow spectrophotometer at 10 °C. Reactions were initiated by mixing CaM-bound enzymes with excess NADPH under a N2/CO atmosphere. For comparison, we also ran reactions in which heme reduction was triggered by mixing Ca2+ and CaM with each NADPH-reduced enzyme. The rates of heme reduction were determined by following the absorbance increase at 444 nm versus time, which tracked formation of the ferrous heme-CO complex. As shown in Fig. 7, all of the mutants had slower heme reduction rates compared with wild-type nNOS, consistent with many of them having lower NO synthesis activities. In some cases, the heme reduction rate did not correspond well with the NO synthesis rate. This was most prominent for the Glu762 mutants, which had slower than expected rates of heme reduction and, to a lesser extent, for the Glu816 and Glu790 mutants. The heme reduction rates for the R1229A and R1229N FNR surface mutants were also slower than that of wild-type nNOS, consistent with their lower NO synthesis activities. Our results indicate that the electronegative surface patch of the FMN module plays a significant role in controlling electron transfer and catalysis in nNOS. Mutations that neutralized or reversed the electronegative charge of six surface residues on the FMN module altered cytochrome c reduction, flavin autoxidation, heme reduction, and NO synthesis by nNOS. In general, the largest effects were seen in the Glu762 mutants, followed by the Glu816, Glu819, and Asp813 mutants and then the others (Glu790 and Glu789), with the exact rank order often depending on whether CaM was bound or not and what aspect of catalysis was being considered. Structure-Function Relationships—The six acidic residues are only partly conserved among NOS enzymes and related flavoproteins (Fig. 8). Remarkably, Glu762 is conserved among NOS enzymes but is replaced by neutral residues in related flavoproteins. Thus, Glu762 appears to be a
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