Exploring the Electron Transfer Properties of Neuronal Nitric-oxide Synthase by Reversal of the FMN Redox Potential
2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês
10.1074/jbc.m806949200
ISSN1083-351X
AutoresHuiying Li, Aditi Das, Hiruy Sibhatu, J. Jamal, Stephen G. Sligar, T.L. Poulos,
Tópico(s)Electron Spin Resonance Studies
ResumoIn nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give ΔG810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. ΔG810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of ΔG810 is insensitive to calmodulin binding. In addition, calmodulin binding to ΔG810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in ΔG810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity. In nitric-oxide synthase (NOS) the FMN can exist as the fully oxidized (ox), the one-electron reduced semiquinone (sq), or the two-electron fully reduced hydroquinone (hq). In NOS and microsomal cytochrome P450 reductase the sq/hq redox potential is lower than that of the ox/sq couple, and hence it is the hq form of FMN that delivers electrons to the heme. Like NOS, cytochrome P450BM3 has the FAD/FMN reductase fused to the C-terminal end of the heme domain, but in P450BM3 the ox/sq and sq/hq redox couples are reversed, so it is the sq that transfers electrons to the heme. This difference is due to an extra Gly residue found in the FMN binding loop in NOS compared with P450BM3. We have deleted residue Gly-810 from the FMN binding loop in neuronal NOS (nNOS) to give ΔG810 so that the shorter binding loop mimics that in cytochrome P450BM3. As expected, the ox/sq redox potential now is lower than the sq/hq couple. ΔG810 exhibits lower NO synthase activity but normal levels of cytochrome c reductase activity. However, unlike the wild-type enzyme, the cytochrome c reductase activity of ΔG810 is insensitive to calmodulin binding. In addition, calmodulin binding to ΔG810 does not result in a large increase in FMN fluorescence as in wild-type nNOS. These results indicate that the FMN domain in ΔG810 is locked in a unique conformation that is no longer sensitive to calmodulin binding and resembles the "on" output state of the calmodulin-bound wild-type nNOS with respect to the cytochrome c reduction activity. Flavin-containing (FMN or FAD) enzymes catalyze a wide range of reactions and can be classified, according to their functions and reactivity with molecular oxygen, into oxidases, monooxygenases, dehydrogenases, oxidoreductases, and electron transferases (1.Massey V. J. Biol. Chem. 1994; 269: 22459-22462Abstract Full Text PDF PubMed Google Scholar, 2.Massey V. Biochem. Soc. Trans. 2000; 28: 283-296Crossref PubMed Google Scholar). The versatility of flavoprotein-catalyzed reactions is attributed to the rich chemistry of the flavin isoalloxazine ring system. Free flavin can exist in three different redox states: oxidized (ox), 3The abbreviations used are: ox, oxidized; sq, semiquinone; hq, hydroquinone; NOS, nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; CaM, calmodulin; CPR, microsomal cytochrome P450 reductase; SOD, superoxide dismutase. one-electron reduced semiquinoid (sq), and two-electron reduced hydroquinoid (hq) species (3.Ghisla S. Massey V. Biochem. J. 1986; 239: 1-12Crossref PubMed Scopus (172) Google Scholar, 4.Mayhew S.G. Eur. J. Biochem. 1999; 265: 698-702Crossref PubMed Scopus (117) Google Scholar), as shown in Fig. 1. The semiquinone radical can have two forms depending on whether or not the N5 atom is protonated (5.Draper R.D. Ingraham L.L. Arch. Biochem. Biophys. 1968; 125: 802-808Crossref PubMed Scopus (257) Google Scholar). The anionic semiquinone is red, whereas the neutral semiquinone is blue, each with its own distinct UV-visible absorption features (6.Massey V. Palmer G. Biochemistry. 1966; 5: 3181-3189Crossref PubMed Scopus (349) Google Scholar). The negative charge on the anionic form of the semiquinone or hydroquinone is localized on the N1–C2=O group (3.Ghisla S. Massey V. Biochem. J. 1986; 239: 1-12Crossref PubMed Scopus (172) Google Scholar). The redox potentials of the ox/sq and sq/hq couples are -314 mV and -124 mV, respectively, for free FMN (7.Anderson R.F. Biochim. Biophys. Acta. 1983; 722: 158-162Crossref PubMed Scopus (115) Google Scholar), although the FMN redox potentials and the pKa of N5 can vary dramatically from one flavoprotein to another. It is the variations of flavin-protein interactions in different flavoproteins that give rise to the versatility of flavin redox properties tailored to the specific chemical reaction catalyzed by the particular flavoenzyme (2.Massey V. Biochem. Soc. Trans. 2000; 28: 283-296Crossref PubMed Google Scholar). Before being identified as a heme-containing enzyme (8.McMillan K. Bredt D.S. Hirsch D.J. Snyder S.H. Clark J.E. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11141-11145Crossref PubMed Scopus (373) Google Scholar, 9.White K.A. Marletta M.A. Biochemistry. 1992; 31: 6627-6631Crossref PubMed Scopus (596) Google Scholar, 10.Stuehr D.J. Ikeda-Saito M. J. Biol. Chem. 1992; 267: 20547-20550Abstract Full Text PDF PubMed Google Scholar), nitric-oxide synthase (NOS) was first recognized as a flavoprotein (11.Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2240) Google Scholar, 12.Hevel J.M. White K.A. Marletta M.A. J. Biol. Chem. 1991; 266: 22789-22791Abstract Full Text PDF PubMed Google Scholar). The C-terminal domain of rat neuronal NOS shares high sequence identity with microsomal cytochrome P450 reductase (CPR) that also contains one molecule each of FMN and FAD. The catalytic center, heme group, and a nearby cofactor, tetrahydrobiopterin, reside in the N-terminal domain. The biosynthesis of nitric oxide (NO) is carried out by binding of the substrate, l-arginine (l-Arg), on the distal side of heme where one of the guanidino nitrogen atoms of l-Arg is oxidized to give NO in a two-step reaction with Nω-hydroxy-l-arginine as an intermediate and citrulline as a byproduct (13.Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 14.Raman C.S. Martasek P. Masters B.S.S. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. Academic Press, San Diego, CA2000: 293-339Google Scholar). Reaction 1 requires molecular oxygen and NADPH-supplied reducing equivalents. Similar to CPR, electron flow in the NOS reductase domain starts from NADPH, through FAD to FMN. The FMN in NOS forms an air-stable, blue neutral semiquinone. Only the lower potential hydroquinone of FMN is capable of transferring electrons to heme. In addition, electron transfer in NOS is regulated by the binding of calmodulin (CaM) to a linker peptide between the heme- and flavin-containing domains. The activity of both endothelial NOS and neuronal NOS (nNOS) is, therefore, regulated by the Ca2+-CaM binding to the enzyme (15.Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (407) Google Scholar, 16.Matsuoka A. Stuehr D.J. Olson J.S. Clark P. Ikeda-Saito M. J. Biol. Chem. 1994; 269: 20335-20339Abstract Full Text PDF PubMed Google Scholar). NOS has its two functional domains fused into a single polypeptide. This domain architecture is similar to that seen in cytochrome P450BM3, a well characterized bacterial P450 system isolated from Bacillus megaterium (17.Narhi L.O. Fulco A.J. J. Biol. Chem. 1987; 262: 6683-6690Abstract Full Text PDF PubMed Google Scholar). Although the C-terminal reductase domain of P450BM3 highly resembles the mammalian microsomal CPR, its FMN exhibits very different redox properties. Based on kinetic and anaerobic redox titration studies, Sevrioukova et al. (18.Sevrioukova I. Shaffer C. Ballou D.P. Peterson J.A. Biochemistry. 1996; 35: 7058-7068Crossref PubMed Scopus (54) Google Scholar) determined that the one-electron reduced FMN semiquinone in P450BM3 is a transient, red anionic form rather than the air-stable, blue neutral radical seen in mammalian CPR. The FMN hydroquinone is the more stable, high potential species, thus incapable of reducing the heme. The red anionic FMN semiquinone instead plays the role of the lower potential species donating electrons to the P450 heme. The presence of the anionic flavin semiquinone is also detected by EPR (19.Murataliev M.B. Klein M. Fulco A. Feyereisen R. Biochemistry. 1997; 36: 8401-8412Crossref PubMed Scopus (56) Google Scholar). Redox potential measurements (20.Hanley S.C. Ost T.W. Daff S. Biochem. Biophys. Res. Commun. 2004; 325: 1418-1423Crossref PubMed Scopus (31) Google Scholar) confirmed that the ox/sq is indeed the lower potential couple at -240 mV compared with -160 mV for the sq/hq couple. The reversal of FMN redox properties of P450BM3 compared with NOS and CPR was expected to derive from some major differences in the FMN binding environment between P450BM3 and mammalian CPR. The crystal structure of P450BM3 heme/FMN bidomain (21.Sevrioukova I.F. Li H. Zhang H. Peterson J.A. Poulos T.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1863-1868Crossref PubMed Scopus (469) Google Scholar) shed some light on this puzzle, as shown in Fig. 2. In P450BM3 the FMN binding loop is one-residue shorter with the backbone amide of Asn-537 donating a hydrogen bond to N5 of FMN. In flavodoxins and microsomal CPR a carbonyl in the longer loop can accept a H-bond from the protonated N5 of FMN upon reduction thereby stabilizing the blue neutral semiquinone. This carbonyl becomes available through a reduction-dependent peptide flip within the loop consistently observed in several flavodoxins from various organisms (22.Hoover D.M. Drennan C.L. Metzger A.L. Osborne C. Weber C.H. Pattridge K.A. Ludwig M.L. J. Mol. Biol. 1999; 294: 725-743Crossref PubMed Scopus (62) Google Scholar, 23.Ludwig M.L. Pattridge K.A. Metzger A.L. Dixon M.M. Eren M. Feng Y. Swenson R.P. Biochemistry. 1997; 36: 1259-1280Crossref PubMed Scopus (160) Google Scholar, 24.Watt W. Tulinsky A. Swenson R.P. Watenpaugh K.D. J. Mol. Biol. 1991; 218: 195-208Crossref PubMed Scopus (186) Google Scholar). The shorter loop in P450BM3 makes this peptide flip very unlikely. Thus the proton from the protein amide nitrogen would remain in place in the semiquinoid state, which decreases the pKa of the FMN N5 (8.5 in free FMN, Fig. 1) making the protonation of N5 difficult. The FMN-binding environment in nNOS resembles that in mammalian CPR, because the carbonyl oxygen atom of Gly-810 in the longer FMN binding loop can accept a H-bond from the N5 of FMN (Fig. 2). When the structure of the FMN binding loop in P450BM3 is superimposed onto that of nNOS the similarities of both the amino acid compositions and their backbone conformations are apparent even though the loop is one residue shorter in P450BM3. To test whether the redox behavior of FMN in nNOS can mimic that in P450BM3 by making a shorter FMN binding loop, we have made the Gly-810 deletion mutant of nNOS (ΔG810) in three constructs: full-length, heme/FMN bidomain, and FMN domain. The redox potentials of FMN have been determined with both the heme/FMN bidomain and the isolated FMN domain (with CaM bound). The enzymatic activities of NO synthesis, NADPH oxidation, and cytochrome c reduction of the full-length mutant were compared with the wild-type protein. Materials—The QuikChange mutagenesis kit was ordered from Stratagene. Hemoglobin A0 (reduced), cytochrome c (horse heart), catalase, and superoxide dismutase (SOD) were purchased from Sigma. Redox mediators, benzyl viologen, 2-hydroxy-1,4-naphthoquinone, and anthraquinone 2-sulfonate were from Sigma-Aldrich. All the other biochemical reagents were either from Fisher, VWR, or Calbiochem. Protein Mutagenesis, Expression, and Purification—Both the full-length rat nNOS (residues 1–1429) (25.Li H. Shimizu H. Flinspach M. Jamal J. Yang W. Xian M. Cai T. Wen E.Z. Jia Q. Wang P.G. Poulos T.L. Biochemistry. 2002; 41: 13868-13875Crossref PubMed Scopus (131) Google Scholar) and the heme/FMN bidomain (residues 299–955) (26.Li H. Igarashi J. Jamal J. Yang W. Poulos T.L. J. Biol. Inorg. Chem. 2006; 11: 753-768Crossref PubMed Scopus (62) Google Scholar) constructs have an N-terminal 6-His tag as described previously. The construct of the FMN domain consisting of residues 720–955 was cloned into the same pCWori vector through the NdeI and XbaI sites, thus it also has an N-terminal 6-His tag before the CaM binding motif. The ΔG810 mutant of nNOS was cloned in Escherichia coli strain DH5α using the QuikChange Mutagenesis Kit from Stratagene. The wild-type full-length, heme/FMN bidomain, and FMN domain proteins were used as the templates for PCR to generate the deletion for the three different constructs, respectively. The Gly-810 deletion was confirmed by DNA sequencing of the plasmids. The protein expression protocol for the full-length mutant is identical to that for the wild-type protein (25.Li H. Shimizu H. Flinspach M. Jamal J. Yang W. Xian M. Cai T. Wen E.Z. Jia Q. Wang P.G. Poulos T.L. Biochemistry. 2002; 41: 13868-13875Crossref PubMed Scopus (131) Google Scholar) using BL21(DE3) as the expression host. However, the mutant protein yields of the heme/FMN bidomain and the FMN domain were found to be higher when they were co-expressed with CaM in E. coli strain JM109 with the cell growth conditions similar to that described for the expression of the bidomain in BL21(DE3) (26.Li H. Igarashi J. Jamal J. Yang W. Poulos T.L. J. Biol. Inorg. Chem. 2006; 11: 753-768Crossref PubMed Scopus (62) Google Scholar). The purification protocols reported for the wild-type full-length (25.Li H. Shimizu H. Flinspach M. Jamal J. Yang W. Xian M. Cai T. Wen E.Z. Jia Q. Wang P.G. Poulos T.L. Biochemistry. 2002; 41: 13868-13875Crossref PubMed Scopus (131) Google Scholar) and bidomain (26.Li H. Igarashi J. Jamal J. Yang W. Poulos T.L. J. Biol. Inorg. Chem. 2006; 11: 753-768Crossref PubMed Scopus (62) Google Scholar) proteins were adopted for the mutant proteins. The FMN domain protein was purified through three column steps: Ni-Sepharose, HiTrap Q anion exchange, and Superdex 75 gel filtration columns (GE Healthcare). The buffer for equilibrating the Ni-Sepharose column was 50 mm sodium phosphate, pH 7.8, 10% glycerol, 5 mm 2-mercaptoethanol, 0.5 mm CaCl2, 0.25 mm phenylmethylsulfonyl fluoride, 2 μm FMN, and 200 mm NaCl. The same buffer plus 1 μg/ml each of pepstatin A and leupeptin was used for cell resuspension before the cell rupture through a microfluidizer at a pressure of 18,000 p.s.i. The cell-free extract obtained after a 1-h ultracentrifugation at 100,000 × g was loaded onto the nickel column. The wild-type FMN domain protein bound to the nickel column often showed a blue color due to its air-stable semiquinone, whereas the ΔG810 mutant FMN domain exhibited a bright orange color. After sample loading the column was washed with 5 bed volumes of the same buffer containing 20 mm imidazole. The protein elution was achieved by 10 bed volumes of 20–150 mm imidazole gradient. The peak fractions were often in good purity and, once being concentrated to a small volume, could be directly loaded onto a S75 column. Some side fractions did need to be further purified through an anion exchange column prior to the S75 column. One or two 5-ml HiTrap Q columns was(were) equilibrated with 50 mm sodium phosphate, pH 7.8, 10% glycerol, 5 mm 2-mercaptoethanol, 0.5 mm CaCl2, 2 μm FMN, and 0.25 mm phenylmethylsulfonyl fluoride. Fractions from the nickel column were diluted 2-fold with the salt-free phosphate buffer before loading onto the Q column using a peristaltic pump. After sample loading the Q column was connected to an AKTA system (GE Healthcare), being further washed with 5 bed volumes of phosphate buffer containing 100 mm NaCl before the protein elution with 20 bed volumes of 100–350 mm NaCl gradient. The same sodium phosphate buffer with 200 mm NaCl was the running buffer for the Superdex 75 column (2.6 × 30 cm). The flow rate was set at 1 ml/min, and the fraction size was 1 ml. The colored fractions with an absorbance ratio A280 nm/A456 (A280 nm/A468 for the mutant) of < 5.0 were pooled and concentrated, stored at -80 °C. The concentration of FMN domain protein was estimated using an extinction coefficient of 9.8 mm-1 cm-1 (18.Sevrioukova I. Shaffer C. Ballou D.P. Peterson J.A. Biochemistry. 1996; 35: 7058-7068Crossref PubMed Scopus (54) Google Scholar) at 456 nm for the wild-type and 468 nm for the mutant. Calmodulin—The human CaM expression plasmid, pACYC/trc-hCaM, was a generous gift from Dr. Paul Ortiz de Montellano's laboratory at the University of California at San Francisco. The E. coli BL21(DE3) cells were transformed with the plasmid and plated on LB agar containing 35 μg/ml chloramphenicol. A single colony was used to inoculate 5 ml of LB overnight culture. The large scale terrific broth cultures with chloramphenicol were inoculated with a small overnight starter (1:500) and grown at 37 °C with 220 rpm agitation until the A600 nm reached ∼0.8. The protein expression was induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside and the incubation continued for another 20 h at 30 °C and 100 rpm. Cells were harvested by centrifugation at 6000 rpm for 10 min and then washed twice with 50 mm Tris, pH 7.5, 100 mm NaCl before storage at -80 °C. The protocol for phenyl-Sepharose chromatography was modified from the one in the literature (27.Putkey J.A. Slaughter G.R. Means A.R. J. Biol. Chem. 1985; 260: 4704-4712Abstract Full Text PDF PubMed Google Scholar). The cell pastes were resuspended with 50 mm Tris, pH 7.8, 1 mm dithiothreitol, 2 mm CaCl2, 100 mm NaCl, and 0.25 mm phenylmethylsulfonyl fluoride and lysed by microfluidizer. The cell-free extract was obtained by ultracentrifugation at 100,000 × g for 1 h and loaded onto a small phenyl-Sepharose column (2.6 × 4.0 cm, GE Healthcare) pre-equilibrated with the same Tris buffer. The column was washed with 100 ml of Tris buffer containing 500 mm NaCl. The protein was then eluted with 100 ml of CaCl2-free Tris buffer containing 5 mm EGTA. The fractions with strong UV absorption at 280 nm were pooled and concentrated. The excess EGTA was removed by passing the purified CaM through a 10-DG de-salting column (Bio-Rad). The concentration of CaM was estimated using an extinction coefficient of 2.98 mm-1 cm-1 at 280 nm based on the chromophore content (28.Pace C. Stankovich M. Biochemistry. 1986; 25: 2516-2522Crossref PubMed Scopus (13) Google Scholar). The homemade CaM was as efficient as the commercial protein purchased from Sigma in supporting NOS activity. Spectro-potentiometric Titrations—Redox titrations were carried out in a cuvette that was assembled in an anaerobic glove box (COY Laboratory Products, Inc., Grass Lake, MI). The cuvette was sealed with an air tight septum through which the Ag/AgCl reference electrode, the gold working electrode, and gas-tight Hamilton syringe were inserted. A small magnetic stir bar was placed at the bottom of the cuvette to mix the reagents. The temperature was maintained at ∼25 °C. The titration buffer (50 mm Tris, pH 7.5, 10% glycerol, 5 mm 2-mercaptoethanol, 1 mm CaCl2, 100 mm NaCl) was made anaerobic by flushing with ultrapure argon while stirring. The final experimental volume was 1.3 ml with an optical density = 1.0 near the main FMN visible band (near 450 nm). The following typical redox mediators were used: benzyl viologen (-374 mV), 2-hydroxy-1,4-naphthoquinone (-145 mV), and anthraquinone 2-sulfonate (-230 mV) to a final concentration of 2 μm each. All the potentials here are reported against standard hydrogen electrode. The protein and mediator mixtures were deoxygenated under the flow of argon gas for several minutes. The protein was reduced by addition of a small excess of anaerobically prepared sodium dithionite solution (concentration was determined using a molar absorption coefficient ϵ315 = 8.05 mm-1 cm-1). The reduced protein spectrum was recorded to confirm complete reduction. The redox titration was done using Dutton's method (29.Dutton P.L. Methods Enzymol. 1978; 54: 411-435Crossref PubMed Scopus (743) Google Scholar). A small aliquot of oxidant/reductant was added, and the solution was stirred until equilibration (stabilization of the potential) was reached (∼15–20 min), and then the spectrum (350–800 nm) was recorded using a Cary 300 UV-visible spectrophotometer. The titration was continued until the sample solution was maximally oxidized by ferricyanide. The reverse electrochemical titration was done with dithionite as the reductant. The electrochemical potential was monitored using an Orion pH/mV meter (Model SA 720) coupled to a gold electrode and an Ag/AgCl reference electrode from Bioanalytical Systems, Inc. The gold electrode was modified using 4,4′-dithiodipyridine. The electrode system was calibrated using the ferrous-ferric ammonium sulfate couple (+675 mV versus standard hydrogen electrode). The observed potential was obtained relative to the Ag/AgCl reference electrode. Hence, they were corrected (using the calibration data for the ferrous-ferric ammonium sulfate solution) to values relative to the standard hydrogen electrode. Analysis of Absorbance versus Potential Data—Data analysis was done using Origin (OriginLab). For the FMN domain of the wild-type nNOS, the absorbance changes at wavelengths 456 nm and 590 nm were plotted against the measured potential of the gold electrode. 456 nm and 590 nm are the absorption maximum of the oxidized flavin and semiquinone, respectively. For the ΔG810 mutant FMN domain, there is no neutral semiquinone peak at 590 nm. Hence, the absorbance changes at 391, 408, and 468 nm were plotted against the measured potential and fit to the modified Nernst equation (Equation 1). This equation is for a two electron redox process where a, b, and c are the absorbance values of oxidized flavin, flavin semiquinone, and hydroquinone, respectively. E is the potential at the working electrode, and E1 and E2 are the midpoint potentials of the oxidized/semiquinone (ox/sq) and the semiquinone/hydroquinone (sq/hq) redox couples. All these five variables are determined by least-squares fitting. A=a+b×10E-E159+c×10E-E159×10E-E2591+10E-E159+10E-E159×10E-E259(Eq. 1) For the ΔG810 bidomain, which has both the FMN and heme domain, the absorbance changes at wavelengths 553 nm, 650 nm (heme components show maximum spectral change), 478 nm, and 501 nm (approximate FMN absorption maxima) were plotted against the measured potential and fit to another modified Nernst equation (Equation 2) for a three-electron redox process where one is an independent redox couple. In the Equation 2, a, b, and c are the absorbance values of oxidized flavin, flavin semiquinone, and hydroquinone, respectively, and d and f are the absorbance values of oxidized heme and reduced heme. E is the potential at the working electrode and E1, E2, and E3 are the midpoint potentials of the ox/sq, sq/hq, and heme redox couples. All these eight variables are determined by least-square fitting. A=a+b×10E-E159+c×10E-E159×10E-E2591+10E-E159+10E-E159×10E-E259+d×10E-E359+f10E-E359+1(Eq. 2) FMN Reduction Monitored by Stopped Flow—The reduction of FMN in the FMN domain of ΔG810 was monitored under anaerobic conditions using an SX.18MV-R stopped-flow spectrophotometer (Applied Photophysics Ltd.). The 100 mm Tris/HCl buffers (100 mm NaCl) at three pH values, 7.0, 8.0, and 9.0, were degassed by alternating between evacuation and purging with pure argon. The air-tight 2.5-ml syringes were assembled inside a COY glove box. Syringe A was filled with nNOS FMN domain and syringe B with dithionite solution (concentration of dithionite was calibrated by cytochrome c reduction immediately before usage). Each syringe was then connected to a three-way stopcock so that another 5-ml syringe filled with Tris buffer can be connected to each sample syringe. Two pairs of syringes were then brought out of the glove box and attached to the stopped flow apparatus. The optical cell and drive syringes were flushed with the degassed buffer before experiments. The spectral changes as a function of time upon rapid mixing of 11 μm FMN domain protein and ∼300 μm dithionite were monitored by the photodiode array detector by obtaining 400–500 spectra for 1000 s in the range of 350–700 nm. The baseline was set with the buffer containing dithionite only. Single wave-length kinetic traces (50 s per trace) were obtained by mixing 18.5 μm FMN domain and ∼1 mm dithionite at 391 nm for the formation of red anionic FMN semiquinone and its further reduction to hydroquinone, and at 468 nm for the entire 2-electron reduction process. The pseudo first order rate constants were extracted by fitting of the single wavelength scan curves with a single or double exponential equation using the Igor Pro program. Steady-state Enzymatic Activity Assays—All steady-state enzymatic activity assays were performed at room temperature on a Cary 3E spectrophotometer (Varian) in an absorbance versus time kinetic scanning mode. The heme protein content of the full-length nNOS was determined using an extinction coefficient of 75 mm-1 cm-1 for the absorbance difference ΔA444–490 nm when enzyme was reduced by dithionite and with CO bound (10.Stuehr D.J. Ikeda-Saito M. J. Biol. Chem. 1992; 267: 20547-20550Abstract Full Text PDF PubMed Google Scholar). Turnover numbers are expressed as nanomoles of product formed/min/nmol of heme enzyme. Enzyme concentration was adjusted to maintain a linear absorbance change in the first 2–3 min of the reaction. The NO synthesis activities for both the wild-type and ΔG810 mutant were measured using the hemoglobin capture assay (31.Murphy M.E. Noack E. Methods Enzymol. 1994; 233: 240-250Crossref PubMed Scopus (347) Google Scholar). The absorption increase at 401 nm was monitored within 1 min at room temperature using an extinction coefficient of 38 mm-1 cm-1 to estimate the amount of methemoglobin generated. The buffer components for the assay include 25 mm potassium phosphate, pH 7.5, 100 mm KCl, 10 μm oxy-hemoglobin, 10 units/ml catalase, 10 units/ml SOD, 5 μm FMN, 5 μm FAD, 25 μm l-Arg, 10 μm tetrahydrobiopterin, 10 μg/ml CaM, 0.5 mm CaCl2, 100 μm NADPH. 1 μg of the wild-type nNOS or 5 μg of mutant was added last to start the reaction. The NADPH oxidation assay was performed in the same phosphate buffer with the exception that 100 μm l-Arg and 250 μm NADPH were used and hemoglobin was omitted. The absorption decrease at 340 nm was tracked at room temperature for 1 min using 6.2 mm-1 cm-1 as the extinction coefficient. 5 μg of wild-type or 10 μg of mutant enzyme was used for each reaction to improve the signal to noise ratio. The cytochrome c reduction assay was conducted with 40 μm cytochrome c in the same phosphate buffer except that hemoglobin, catalase, SOD, l-Arg, and tetrahydrobiopterin were omitted. The absorbance increase at 550 nm was monitored for 1 min with 21 mm-1 cm-1 as the extinction coefficient. To achieve a stable linear trace only 0.1 μg of wild-type or mutant enzyme was needed for each reaction. Fluorescence—Fluorescence measurements of both the wild-type and mutant full-length enzymes were carried out at room temperature on a Hitachi F-4500 fluorescence spectrophotometer. The flavin (mainly FMN) fluorescence signal was excited at 450 nm and recorded from 470 to 650 nm. Two buffer systems were tested, 50 mm HEPES, pH 7.5, or 25 mm potassium phosphate, pH 7.5. The changes of fluorescence intensity with 6.5 μm (wild-type) or 6.0 μm (ΔG810) of enzymes in a 100-μl cuvette were recorded twice within 3–4 min. The effects of CaM binding and removal to the fluorescence intensity change were monitored by adding 20 μm CaM plus 0.5 mm CaCl2 and then 10 mm EGTA, respectively. The total volume change was kept at <5%. The changes in fluorescence intensity were found to be ionic strength-dependent. The 25 mm potassium phosphate, pH 7.5, with 100 mm KCl was found to be a better buffer with which the fluorescence signals were more stable with time. Titration of nNOS Wild-type FMN Domain—The redox titrations were done using Dutton's method (29.Dutton P.L. Methods Enzymol. 1978; 54: 411-435Crossref PubMed Scopus (743) Google Scholar). The proteins were initially reduced and then oxidized using small aliquots of potassium ferricyanide and then reduced with dithionite. After each addition of oxidant/reductant, equilibration was confirmed by observing a stable potential at the electrode. No hysteresis was observed during the oxidative and reductive cycle of the redox titrations. A representative spectrum of the redox titration of the nNOS wild-type FMN domain is given in Fig. 3A. The protein was completely soluble and stable during the redox titrations. The spectrum of the fully oxidized FMN domain of the wild-type has a maximum at 456 nm. When reduction proceeds from the oxidized to the semiquinone form, the intensity of the band at 590 nm increases while that at 456 nm decreases. As reduction proceeds from the semiquinone to the hydroquinone peaks at 456 and 590 nm disappear. The absor
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