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Calmodulin Activates Electron Transfer through Neuronal Nitric-oxide Synthase Reductase Domain by Releasing an NADPH-dependent Conformational Lock

2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês

10.1074/jbc.m203118200

ISSN

1083-351X

Autores

DANIEL H. CRAIG, Stephen K. Chapman, Simon Daff,

Tópico(s)

Electron Spin Resonance Studies

Resumo

Neuronal nitric-oxide synthase (nNOS) is activated by the Ca2+-dependent binding of calmodulin (CaM) to a characteristic polypeptide linker connecting the oxygenase and reductase domains. Calmodulin binding also activates the reductase domain of the enzyme, increasing the rate of reduction of external electron acceptors such as cytochrome c. Several unusual structural features appear to control this activation mechanism, including an autoinhibitory loop, a C-terminal extension, and kinase-dependent phosphorylation sites. Pre-steady state reduction and oxidation time courses for the nNOS reductase domain indicate that CaM binding triggers NADP+ release, which may exert control over steady-state turnover. In addition, the second order rate constant for cytochrome c reduction in the absence of CaM was found to be highly dependent on the presence of NADPH. It appears that NADPH induces a conformational change in the nNOS reductase domain, restricting access to the FMN by external electron acceptors. CaM binding reverses this effect, causing a 30-fold increase in the second order rate constant. The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties. In the full-length enzyme, NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis. Neuronal nitric-oxide synthase (nNOS) is activated by the Ca2+-dependent binding of calmodulin (CaM) to a characteristic polypeptide linker connecting the oxygenase and reductase domains. Calmodulin binding also activates the reductase domain of the enzyme, increasing the rate of reduction of external electron acceptors such as cytochrome c. Several unusual structural features appear to control this activation mechanism, including an autoinhibitory loop, a C-terminal extension, and kinase-dependent phosphorylation sites. Pre-steady state reduction and oxidation time courses for the nNOS reductase domain indicate that CaM binding triggers NADP+ release, which may exert control over steady-state turnover. In addition, the second order rate constant for cytochrome c reduction in the absence of CaM was found to be highly dependent on the presence of NADPH. It appears that NADPH induces a conformational change in the nNOS reductase domain, restricting access to the FMN by external electron acceptors. CaM binding reverses this effect, causing a 30-fold increase in the second order rate constant. The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties. In the full-length enzyme, NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis. nitric-oxide synthase(s) calmodulin nitric oxide iNOS, and nNOS, endothelial, inducible, and neuronal nitric-oxide synthases, respectively recombinant neuronal nitric-oxide synthase reductase domain Mammalian nitric-oxide synthases (NOS)1 are responsible for generating NO in a wide range of cell types during the immune system response and as part of numerous intercellular signaling mechanisms (1Mayer, B. (ed) (2000) Nitric Oxide: Handbook of Experimental Pharmacology,Vol. 143, Springer-Verlag, BerlinGoogle Scholar, 2Ignarro, L. J. (ed) (2000) Nitric Oxide: Biology and Pathobiology, AcademicPress, San DiegoGoogle Scholar, 3Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2001; 357: 593-615Crossref PubMed Scopus (3233) Google Scholar, 4Stuehr D.J. Biochim. Biophys. Acta. 1999; 1411: 217-230Crossref PubMed Scopus (805) Google Scholar). They are homodimeric and consist of a reductase domain, which binds FAD and FMN stoichiometrically, and an oxygenase domain, which contains a P450-like Cys-ligated heme and a tetrahydrobiopterin molecule. The oxygenase domain forms the main dimer interface, and tetrahydrobiopterin is an integral part of this. Crystal structures are available for several NOS oxygenase domain dimers (5Crane 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 (621) Google Scholar, 6Raman C.S., Li, H. Martásek P. Kral V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar) and for the FAD binding subdomain of neuronal NOS (nNOS) (7Zhang J. Martásek P. Paschke R. Shea T.M. Masters B.S.S. Kim J.P. J. Biol. Chem. 2001; 276: 37506-37513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The reductase domain closely resembles mammalian cytochrome P450 reductase (7Zhang J. Martásek P. Paschke R. Shea T.M. Masters B.S.S. Kim J.P. J. Biol. Chem. 2001; 276: 37506-37513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 8Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (661) Google Scholar, 9Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2164) Google Scholar, 10Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar, 11Lowenstein C.J. Glatt C.S. Bredt D.S. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6711-6715Crossref PubMed Scopus (621) Google Scholar) and similarly catalyzes NADPH dehydrogenation at the FAD site and electron transfer to the FMN. The oxygenase domain of one subunit accepts electrons from the reductase domain of the other subunit (12Siddhanta U. Presta A. Fan B.C. Wolan D. Rousseau D.L. Stuehr D.J. J. Biol. Chem. 1998; 273: 18950-18958Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 13Panda K. Ghosh S. Stuehr D.J. J. Biol. Chem. 2001; 276: 23349-23356Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 14Sagami I. Daff S. Shimizu T. J. Biol. Chem. 2001; 276: 30036-30042Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and generates NO from l-arginine via a unique two-step monooxygenation reaction (1Mayer, B. (ed) (2000) Nitric Oxide: Handbook of Experimental Pharmacology,Vol. 143, Springer-Verlag, BerlinGoogle Scholar, 2Ignarro, L. J. (ed) (2000) Nitric Oxide: Biology and Pathobiology, AcademicPress, San DiegoGoogle Scholar, 3Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2001; 357: 593-615Crossref PubMed Scopus (3233) Google Scholar, 4Stuehr D.J. Biochim. Biophys. Acta. 1999; 1411: 217-230Crossref PubMed Scopus (805) Google Scholar, 15Rosen G.M. Tsai P. Sovitj P. Chem. Rev. 2002; 102: 1191-1200Crossref PubMed Scopus (117) Google Scholar). The two domains are linked by a functional peptide of 20–25 amino acids which binds calmodulin (CaM) reversibly at elevated Ca2+ concentrations in the nNOS and endothelial NOS (eNOS) isoforms but irreversibly in the inducible isoform (iNOS). CaM binding activates nNOS and eNOS, providing them with a rapid response mechanism during their participation in signaling cascades. The inducible isoform, on the other hand, is regulated at the transcriptional level. CaM binding has been shown to control NO synthesis by activating electron transfer through the enzyme (16Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar, 17Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). This effect is manifested in both the reductase domain and the enzyme as a whole by increases in the rates of steady-state cytochrome c reduction and NOS heme reduction. The isolated nNOS reductase domain (nNOSrd) retains its CaM-dependent cytochrome c reductase activity in the absence of the heme domain (18Gachhui 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, 19Newton D.C. Montgomery H.J. Guillemette J.G. Arch. Biochem. Biophys. 1998; 359: 249-257Crossref PubMed Scopus (27) Google Scholar), whereas the isolated iNOSrd is not CaM-dependent (19Newton D.C. Montgomery H.J. Guillemette J.G. Arch. Biochem. Biophys. 1998; 359: 249-257Crossref PubMed Scopus (27) Google Scholar, 20Montgomery H.J. Romanov V. Guillemette J.G. J. Biol. Chem. 2000; 275: 5052-5058Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The three NOS isoforms show close sequence homology but can be readily identified by the presence, or absence, of several unusual isoform-specific inserts and extensions, which are responsible for defining their particular functional characteristics. Many of these regulatory control elements occur in the reductase domain (21Roman L.J. Martásek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1190Crossref PubMed Scopus (176) Google Scholar) and include an autoinhibitory loop in the FMN binding subdomain of 40–50 amino acids (20Montgomery H.J. Romanov V. Guillemette J.G. J. Biol. Chem. 2000; 275: 5052-5058Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 21Roman L.J. Martásek P. Masters B.S.S. Chem. Rev. 2002; 102: 1179-1190Crossref PubMed Scopus (176) Google Scholar, 22Salerno J.C. Harris D.E. Irizarry K. Patel B. Morales A.J. Smith S.M.E. Martásek P. Roman L.J. Masters B.S.S. Jones C.L. Weissman B.A. Lane P. Liu Q. Gross S.S. J. Biol. Chem. 1997; 272: 29769-29777Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 23Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1998; 273: 5566-5571Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 24Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 1999; 274: 14692-14698Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 25Daff S. Sagami I. Shimizu T. J. Biol. Chem. 1999; 274: 30589-30595Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 26Chen P.F. Wu K.K. J. Biol. Chem. 2000; 275: 13155-13163Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 27Nishida C.R. Ortiz de Montellano P.R. J. Biol. Chem. 2001; 276: 20116-20124Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), a C-terminal extension to the FAD binding subdomain of 20–40 amino-acids (28Roman L.J. Miller R.T., De la Garza M.A. Kim J.P. Masters B.S.S. J. Biol. Chem. 2000; 275: 21914-21919Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 29Roman L.J. Martásek P. Miller R.T. Harris D.E., De la Garza M.A. Shea T.M. Kim J.P. Masters B.S.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and kinase-dependent phosphorylation sites (30Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujin Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2211) Google Scholar, 31Dimmler S. Fleming I. Fissithaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3019) Google Scholar, 32Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriquez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (710) Google Scholar, 33Butt E. Bernhardt M. Smolenski A. Kotsonis P. Frohlich B.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 (253) Google Scholar, 34Hayashi Y. Nishio M. Naito Y. Yokokura H. Nimura Y. Hidaka H. Watanabe Y. J. Biol. Chem. 1999; 274: 20597-20602Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 35McCabe T.J. Fulton D. Roman L.J. Sessa W.C. J. Biol. Chem. 2000; 275: 6123-6128Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 36Adak S. Santolini J. Tikunova S. Wang Q. Johnson D. Stuehr D.J. J. Biol. Chem. 2001; 276: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). All influence the CaM-dependent activation mechanism by either altering the CaM binding affinity or by affecting enzyme activity directly. Mutation of these control elements (e.g. by deletion) activates the reductase domains of eNOS and nNOS in the absence of CaM (Table I) and in some cases triggers NO synthesis (25Daff S. Sagami I. Shimizu T. J. Biol. Chem. 1999; 274: 30589-30595Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 29Roman L.J. Martásek P. Miller R.T. Harris D.E., De la Garza M.A. Shea T.M. Kim J.P. Masters B.S.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The reduction potentials of the FAD and FMN cofactors of the nNOSrd are unperturbed by CaM binding (37Noble M.A. Munro A.W. Rivers S.L. Robledo L. Daff S.N. Yellowlees L.J. Shimizu T. Sagami I. Guillemette J.G. Chapman S.K. Biochemistry. 1999; 38: 16413-16418Crossref PubMed Scopus (119) Google Scholar), indicating that the activation mechanism relies on a large scale structural rearrangement. In this paper, we probe the effect of CaM binding on the isolated nNOSrd by following reduction by NADPH and oxidation by cytochrome c under pre-steady-state conditions. The results indicate that the CaM-free enzyme forms a conformationally locked complex with NADPH, which has poor electron transfer capabilities. The importance of the NADPH and NADP+dissociation rates to the CaM-dependent activation mechanism is discussed.Table ISteady-state cytochrome c reduction rates for wild type and mutants of rat nNOSCytochrome c reductase activity1-aMeasured under similar (although not identical) conditions.Ref.+CaM−CaMs−1Wild type nNOS596.025Daff S. Sagami I. Shimizu T. J. Biol. Chem. 1999; 274: 30589-30595Abstract Full Text Full Text PDF PubMed Scopus (118) Google ScholarnNOSrd604.520Montgomery H.J. Romanov V. Guillemette J.G. J. Biol. Chem. 2000; 275: 5052-5058Abstract Full Text Full Text PDF PubMed Scopus (63) Google ScholarS1412D mutant1501836Adak S. Santolini J. Tikunova S. Wang Q. Johnson D. Stuehr D.J. J. Biol. Chem. 2001; 276: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (103) Google ScholarΔ40 loop-deletion nNOS522225Daff S. Sagami I. Shimizu T. J. Biol. Chem. 1999; 274: 30589-30595Abstract Full Text Full Text PDF PubMed Scopus (118) Google ScholarnNOStr C-terminal truncation385929Roman L.J. Martásek P. Miller R.T. Harris D.E., De la Garza M.A. Shea T.M. Kim J.P. Masters B.S.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar1-a Measured under similar (although not identical) conditions. Open table in a new tab Recombinant nNOSrd was expressed in Escherichia coli strain JM109 (DE3) using plasmid pCRNNR as described previously (19Newton D.C. Montgomery H.J. Guillemette J.G. Arch. Biochem. Biophys. 1998; 359: 249-257Crossref PubMed Scopus (27) Google Scholar). This plasmid coexpresses the rat nNOSrd residues 695–1429 (including the CaM binding site) and synthetic bovine brain calmodulin. The enzyme was purified essentially as described previously, in 50 mm Tris-HCl, pH 7.5, 100 mmNaCl, 1 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, and 5% glycerol (buffer A) plus 2 mm CaCl2 on 2′,5′-ADP-agarose (Sigma). Bound CaM was removed by washing with buffer A containing 10 mmEGTA. The enzyme was eluted in buffer A plus 10 mmNADP+ and 2 mm CaCl2. The nNOSrd fractions were purified further on CaM-agarose (Sigma) and eluted in buffer A plus 10 mm EGTA. The enzyme was exchanged into 50 mm Tris-HCl, pH 7.5, 2 mm CaCl2, 100 mm NaCl, and 5% glycerol and stored at −80 °C. Enzyme concentrations were calculated from the absorbance at 457 nm of nNOSrd fully oxidized by potassium ferricyanide, using an extinction coefficient of 22,900 m−1 cm−1(38Matsuda H. Iyanagi T. Biochim. Biophys. Acta. 1999; 1473: 345-355Crossref PubMed Scopus (84) Google Scholar). Enzyme activity and CaM sensitivity were checked using the cytochrome c turnover assay (without FAD, FMN, superoxide dismutase, and catalase) described by Gachhui et al. (18Gachhui 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). Absorbance and kinetic measurements were made on a Shimadzu UV-1601 spectrophotometer. The CaM-rich 2′,5′-ADP-agarose wash fraction was made 10 mm with CaCl2 and then loaded onto a 200-ml phenyl-Sepharose column and washed with 800 ml of 50 mm Tris-HCl, pH 7.5, 1 mm CaCl2(buffer B), 1 liter of 50 mm Tris-HCl, pH 7.5, 1 mm CaCl2, 0.5 m NaCl, and 1 liter of buffer B. The CaM was eluted from the column in 50 mmTris-HCl, pH 7.5, 1 mm EDTA. CaM-containing fractions were identified by SDS-PAGE and pooled before being dialyzed in buffer B to remove EDTA. The CaM was freeze dried and stored at −80 °C. Analysis by the Bio-Rad protein assay showed that the freeze-dried residue was 70% protein by weight, whereas SDS-PAGE indicated that the protein was >90% CaM. CaM prepared in this manner was indistinguishable from the CaM supplied by Sigma when used in the CaM activation assay described above. Stopped-flow experiments were performed on an SX.18MV stopped-flow spectrophotometer (Applied Photophysics) at a temperature of 25 ± 1 °C contained within an anaerobic glove box (Belle Technology) to prevent the reaction of reduced nNOSrd with molecular oxygen. Oxygen levels were maintained below 5 ppm in a nitrogen atmosphere at all times. Data were analyzed using Origin 6.1 (Microcal). Averaged traces from four or more measurements were used for analysis. For all stopped-flow experiments, the buffer was 50 mm Tris-HCl, pH 7.5, 100 mmNaCl, supplemented with 2 mm CaCl2 when CaM was present, unless otherwise stated. Buffer was made anaerobic by bubbling with nitrogen for 2 h before being left to equilibrate in the anaerobic box overnight. NADPH, NADP+, NADH, NAD+, 2′,5′-ADP, cytochrome c (horse heart, type I; Sigma), and dithionite were brought into the anaerobic box in powder form and dissolved in anaerobic buffer. Concentrations were determined by absorption spectroscopy on a Cary 50 Biospectrophotometer within the anaerobic box using the extinction coefficients 29,500m−1 cm−1 at 550 nm (dithionite-reduced sample), 6,200 m−1cm−1 at 340 nm, 18,000 m−1cm−1 at 260 nm, 6,220 m−1cm−1 at 340 nm, 18,000 m−1cm−1at 260 nm, and 15,400 m−1cm−1 at 260 nm. Reduced nNOSrd was generated by titration with a concentrated solution of NADPH or dithionite until no further spectral change occurred. To produce one-electron-reduced enzyme, nNOSrd was incubated in the anaerobic box in a 1 mmsolution of dithiothreitol overnight. Enzyme (reduced or oxidized) was made anaerobic and free of excess reductant by passage down a 1.5 × 20-ml Sephadex G-25 (Sigma) size separation column immediately prior to use. CaM was brought into the box in a 3 mm solution containing 30 mm CaCl2 and added to the nNOSrd to achieve a 2:1 concentration ratio of CaM to nNOSrd. Concentrations of components of stopped-flow reaction mixtures are given as final concentrations after mixing. 2 μm cytochrome c was mixed with nNOSrd (NADPH- or dithionite-reduced) at concentrations of 5–40 μm with and without bound CaM and with and without a 10-fold excess of NADPH (incubated with the enzyme 10 min prior to use). Cytochrome c reduction was monitored at 550 nm, and pseudo-first order rate constants were calculated by fitting the resultant traces to single exponential functions. Second order rate constants were determined by plotting the pseudo-first order rate constants against enzyme concentration and performing linear regression analysis constrained to pass through the origin. Inhibition of cytochrome c reduction by substrates and substrate analogs was studied by mixing 2 μm cytochrome c with 10 μm dithionite-reduced nNOSrd, plus or minus bound CaM, in the presence of 5–1,000 μm NADPH, NADP+, NADH, NAD+, or 2′,5′ ADP. Rate constants for the reduction of cytochrome c were calculated from the change in absorbance at 550 nm as described above. Oxidation of the nNOSrd flavins by an excess of cytochrome c was conducted by mixing 2 μm enzyme (with and without bound CaM and reduced by either NADPH or dithionite) with 10–130 μmcytochrome c. Cytochrome c reduction was monitored at 550 nm. At low concentrations of cytochrome coxidation was slow, allowing the entire reaction course to be observed. These traces were used to calculate the number of electron equivalents removed from the nNOSrd sample, based on Δε550 = 22,640 for cytochrome c reduction. Faster reactions using higher cytochrome c concentrations, in which much of the absorbance change occurred in the stopped-flow dead time, were rescaled accordingly. Reduction of the flavins of nNOSrd by NADPH was achieved by mixing 5 μm one-electron-reduced enzyme (prepared with bound CaM; with and without 5 mmEGTA) with 200 μm NADPH. The change in absorbance at 458 nm was followed and the resultant traces fitted to double or triple exponential functions. To study the rate of electron transfer from NADPH through to cytochrome c, 10 μm one-electron-reduced nNOSrd (prepared with bound CaM) with 200 μm cytochrome c, with and without 5 mm EGTA, was mixed with 2 μm NADPH. The change in absorbance was monitored at 550 nm. The traces were fitted to a two-step consecutive reaction model in which both phases were fixed to have the same absorbance change. Electron transfer from nNOS or nNOSrd to cytochrome c occurs from the FMN hydroquinone (two-electron-reduced FMN) and is essentially unidirectional (25Daff S. Sagami I. Shimizu T. J. Biol. Chem. 1999; 274: 30589-30595Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 39Adak 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). A kinetic barrier prevents oxidation of the blue FMN semiquinone by cytochrome c despite the reaction being thermodynamically favorable (29Roman L.J. Martásek P. Miller R.T. Harris D.E., De la Garza M.A. Shea T.M. Kim J.P. Masters B.S.S. J. Biol. Chem. 2000; 275: 29225-29232Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 37Noble M.A. Munro A.W. Rivers S.L. Robledo L. Daff S.N. Yellowlees L.J. Shimizu T. Sagami I. Guillemette J.G. Chapman S.K. Biochemistry. 1999; 38: 16413-16418Crossref PubMed Scopus (119) Google Scholar, 44Miller R.T. Martásek P. Omura T. Masters B.S.S. Biochem. Biophys. Res. Commun. 1999; 265: 184-188Crossref PubMed Scopus (74) Google Scholar). Therefore, during catalytic turnover with NADPH and cytochrome c, the FMN alternates between the hydroquinone and semiquinone redox states, receiving electrons from FAD and passing them to cytochrome c, as in SchemeFS1. The rate of reduction of cytochrome c by an excess of reduced nNOSrd is dependent only on the rate of formation of the binary complex (and its dissociation constant) and the rate of the actual electron transfer from the FMN hydroquinone. Pseudo-first order rate constants for cytochrome c reduction were measured by mixing NADPH- or dithionite-reduced enzyme with a substoichiometric concentration of cytochrome c in a stopped-flow spectrophotometer. The rate constants, derived from fitting to single exponential functions, were linearly dependent on enzyme concentration (Fig.1). Second order rate constants for cytochrome c reduction were calculated in the presence and absence of NADPH and bound CaM by linear regression analysis (TableII). The rate constants are greatest for CaM-bound nNOSrd, indicating that the FMN is most accessible for reaction with cytochrome c in the CaM-bound enzyme. These values are independent of whether the enzyme was reduced initially by sodium dithionite (which was removed) or by NADPH (present in 10-fold excess). In the absence of CaM the second order rate constant for dithionite-reduced nNOSrd more than halves, suggesting that the FMN is less accessible. However, in the presence of NADPH this decrease is much more dramatic, more than 30-fold, indicating a large change in FMN accessibility. A similar effect was seen when the enzyme was initially reduced by dithionite, with the NADPH being added subsequently, which rules out the possibility that a combination of partial reduction and a flavin redox potential shift might be responsible. It appears, therefore, that NADPH binding strongly inhibits cytochrome creduction by CaM-free nNOSrd, whereas no inhibition occurs for the CaM-bound enzyme, even at much higher NADPH concentrations.Figure 1Determination of second order rate constants for pre-steady-state cytochrome c reduction by excess nNOSrd. Shown are pseudo-first order rate constants for the reduction of 2 μm cytochrome c on mixing with excess nNOSrd prereduced by excess dithionite (◯), in the presence of a 10-fold excess of NADPH (•), prereduced by excess dithionite and in the presence of a 10-fold excess of NADPH (♦), prereduced by excess dithionite with bound CaM and in the presence of 1 mmCa2+ (▵), or with bound CaM in the presence of 1 mm Ca2+ and a 10-fold excess of NADPH (▴). Rate constants were determined from the absorbance changes at 550 nm by fitting the resultant traces to single exponential functions. Data are shown fitted to straight lines through the origin, giving the second order rate constants (Table II).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IISecond order rate constants (k2nd) for reduction of cytochrome c by excess nNOSrd in the presence or absence of bound CaM (+/− CaM)Dithionite reduced2-aPrereduced by excess dithionite.NADPH2-bIn the presence of a 10-fold excess of NADPH.k2ndμm−1 s−1nNOSrd − CaM+15.4 ± 0.9nNOSrd + CaM+37.8 ± 0.4nNOSrd − CaM+1.1 ± 0.1nNOSrd + CaM+35.5 ± 1.9nNOSrd − CaM++2.2 ± 0.22-a Prereduced by excess dithionite.2-b In the presence of a 10-fold excess of NADPH. Open table in a new tab In Fig.2 the concentrations of NADPH and NADP+ are plotted against the pseudo-first order rate constant for cytochrome c reduction by 10 μmCaM-free nNOSrd (prereduced by dithionite). Data were fitted to single binding site noncompetitive inhibition models to determine values for percentage inhibition and KI (inhibition constants). These data, and parameters for NAD+, NADH, and 2′,5′-ADP, are shown in Table III. NADPH inhibited the reaction to the greatest extent (87%) and had a lowKI (2.7 μm), indicating tight binding. NADP+ inhibited the reaction in two phases, the first resulting in 46% inhibition caused by tight binding (2 μm), the second requiring much more NADP+ and possibly resulting from partial oxidation of the flavins by excess NADP+. This effect complicates the data, adding a degree of uncertainty to the values. NAD+ had a similar effect. NADH inhibited cytochrome c reduction much less than NADPH, and 2′,5′-ADP had no significant effect, consequently, no accurateKI values could be obtained for these. It appears, therefore, that the inhibition effect relies on the nicotinamide substituent and occurs whether this is reduced or oxidized. Surprisingly, the physiological substrate of the enzyme, NADPH, appears to be its most potent inhibitor. No inhibition effect was observed with CaM-bound nNOSrd with any of the ligands.Table IIINoncompetitive inhibition constants Ki for the action of substrate and substrate analog inhibitors on the pre-steady-state rate constants for reduction of 1 μm cytochrome c by 5 μm dithionite-reduced nNOSrd in the absence of CaM (Fig.2)Inhibitor% InhibitionKiμmNADPH872.6 ± 1.0NADP+43<2NADH17NAD+55<2ADP0Fitting was performed as described under "Experimental Procedures." Open table in a new tab Fitting was performed as described under "Experimental Procedures." The rates of cytochrome c reduction determined above are linearly dependent on the concentration of nNOSrd, suggesting that the actual rate of FMN to cytochrome c electron transfer is very rapid. In the case of CaM-bound nNOSrd, rates are measured above 600 s−1 and must occur beyond the resolution of the stopped-flow instrument. Mixing 10 μm reduced nNOSrd with 130 μm excess cytochrome c will therefore lead to rapid, successive electron transfers until the nNOSrd has been oxidized completely to its stable one-electron-reduced form. For example, the bimolecular reaction of 130 μm cytochromec with nNOSrd (the first electron transfer) will occur at 4,700 s−1, based on the second order rate constant determined above. The rates of subsequent electron transfer events will depend on the rate at which reduced cytochrome c dissociates and the rate at which the second electron arrives at the FMN. This series of reactions represents part of the catalytic cycle for cytochrome c reduction as illustrated in Scheme FS1. In the scheme, Steps 1–5 represent the nNOSrd catalytic cycle in terms of individual electron transfer events. The flavin oxidation experiment begins with Step 1a (the first electron transfer to cytochromec) and ends with Step 5. As can be seen, a total of three electron equivalents are transferred from nNOSrd to cytochromec. Fig. 3 shows stopped-flow time courses for complete nNOSrd oxidation by excess cytochromec using CaM-bound and CaM-free enzyme reduced by either sodium dithionite or NADPH. In Fig. 3 the absorbance change observed at 550 nm caused by cytochrome c reduction has been converted to molar reducing equivalents by determining the s

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