Characterization of the Reductase Domain of Rat Neuronal Nitric Oxide Synthase Generated in the Methylotrophic Yeast Pichia pastoris

Abstract

Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2′,5′-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself. Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2′,5′-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself. INTRODUCTIONNeuronal nitric oxide synthase (nNOS, NOS I) 1The abbreviations used are: nNOSneuronal nitric oxide synthaseDTTdithiothreitolNOnitric oxideDCPIPdichlorophenolindophenolMOPS4-morpholinepropanesulfonic acidBisTris(2-[bis(2-hy droxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. is one of three NOS isoforms found in animals (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2127) Google Scholar, 2Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 3Kerwin Jr., J.F. Lancaster Jr., J.R. Feldman P.L. J. Med. Chem. 1995; 38: 4343-4362Crossref PubMed Scopus (535) Google Scholar). The enzyme is expressed constitutively in different neuronal cell types and in skeletal muscle but is inactive in its native form and requires Ca2+-promoted calmodulin binding to activate its NO synthesis (4Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2163) Google Scholar). This property enables nNOS to participate in signal transduction cascades by generating NO only in response to increases in intracellular Ca2+ (5Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1533) Google Scholar) but may also lead to neuropathological conditions due to excessive NO production during the prolonged Ca2+ influx that occurs following reperfusion of ischemic tissues (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2127) Google Scholar, 5Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1533) Google Scholar, 6Dawson T.M. Dawson V.L. Neuroscientist. 1995; 1: 7-18Crossref Scopus (109) Google Scholar).The nNOS purified from rat brain is a bi-functional enzyme comprised of an N-terminal oxygenase domain that contains binding sites for iron protoporphyrin IX (heme), (6R,6S)-2-amino-4-hydroxy-6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropteridine, and L-arginine, and a C-terminal reductase domain that contains binding sites for FMN, FAD, and NADPH (4Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2163) Google Scholar, 7Sheta E.A. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar, 8Klatt P. Schmidt K. Brunner F. Mayer B. J. Biol. Chem. 1994; 269: 1674-1680Abstract Full Text PDF PubMed Google Scholar, 9McMillan K. Masters B.S.S. Biochemistry. 1995; 34: 3686-3693Crossref PubMed Scopus (168) Google Scholar). Sequence analysis indicates that the reductase domain of nNOS is structurally homologous to mammalian NADPH cytochrome P-450 reductase, the flavoprotein of Escherichia coli sulfite reductase, and the reductase domain of bacterial cytochrome P450BM3 (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2127) Google Scholar, 2Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). In these proteins, the flavins function to transfer NADPH-derived electrons to either an external heme protein acceptor or to an internal heme group located within the protein itself (7Sheta E.A. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar, 8Klatt P. Schmidt K. Brunner F. Mayer B. J. Biol. Chem. 1994; 269: 1674-1680Abstract Full Text PDF PubMed Google Scholar, 10Iyanagi T. Mason H.S. Biochemistry. 1973; 12: 2297-2308Crossref PubMed Scopus (279) Google Scholar, 11Ostrowski J. Barber M.J. Rueger D.C. Miller B.E. Siegel L.M. Kredich N.M. J. Biol. Chem. 1989; 264: 15796-15808Abstract Full Text PDF PubMed Google Scholar, 12Fulco A.J. Annu. Rev. Pharmacol. Toxicol. 1991; 31: 177-203Crossref PubMed Scopus (147) Google Scholar, 13Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar). Flavin-mediated electron transfer to the nNOS heme iron is thought to enable it to bind dioxygen and catalyze stepwise NO synthesis (2Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar).A variety of evidence indicates that calmodulin binds between the reductase and oxygenase domains of nNOS at a site represented by amino acids 725-745 (4Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2163) Google Scholar, 7Sheta E.A. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar, 14Vorher T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar). We recently reported that calmodulin binding to nNOS triggers interdomain electron transfer between the flavins and heme iron, providing a basis by which calmodulin may activate NO synthesis in this isoform (13Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar). Besides activating interdomain electron transfer to the heme iron, calmodulin binding to nNOS increases its rate of electron transfer to a variety of exogenous acceptors including cytochrome c, ferricyanide, dichlorophenolindophenol (DCPIP), and dioxygen (15Heinzel B. John M. Klatt P. Bohme E. Mayer B. Biochem. J. 1992; 281: 627-630Crossref PubMed Scopus (470) Google Scholar, 16Schmidt H.H.H.W. Smith R.M. Nakane M. Murad F. Biochemistry. 1992; 31: 3243-3248Crossref PubMed Scopus (146) Google Scholar). The mechanism(s) by which calmodulin activates nNOS to reduce these external acceptors is controversial, in that there is evidence both for and against interdomain electron transfer or superoxide production being involved in the reactions (15Heinzel B. John M. Klatt P. Bohme E. Mayer B. Biochem. J. 1992; 281: 627-630Crossref PubMed Scopus (470) Google Scholar, 17McMillan 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 (354) Google Scholar, 18Frey C. Narayanan K. McMillan K. Spack L. Gross S.S. Masters B.S. Griffith O.W. J. Biol. Chem. 1994; 269: 26083-26091Abstract Full Text PDF PubMed Google Scholar, 19Persechini A. McMillan K. Masters B.S.S. Biochemistry. 1995; 34: 15091-15095Crossref PubMed Scopus (62) Google Scholar, 20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar, 21Abu-Soud H.M. Feldman P.L. Clark P. Stuehr D.J. J. Biol. Chem. 1994; 269: 32318-32326Abstract Full Text PDF PubMed Google Scholar, 22Wolff D.J. Datto G.A. Samatovicz R.A. Tempsick R.A. J. Biol. Chem. 1993; 268: 9425-9429Abstract Full Text PDF PubMed Google Scholar). We recently showed that nNOS reductase activities toward cytochrome c or ferricyanide were increased to nearly the same extent when calmodulin bound to a heme-free form of nNOS, consistent with calmodulin increasing reduction of these acceptors by a mechanism that does not involve interdomain electron transfer to the nNOS heme iron (20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). Further analysis revealed that calmodulin binding also increases the rate by which NADPH reduces the nNOS flavins (20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar), suggesting that calmodulin may increase electron transfer rates to certain external acceptors by activating the nNOS reductase domain itself.To investigate further the function of the nNOS reductase domain and its possible activation by calmodulin, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) that contains the calmodulin binding site, using the eukaryotic Pichia a form of the nNOS reductase domain recently expressed in E. coli K. Masters B.S.S. Biochemistry. 1995; 34: 3686-3693Crossref PubMed Scopus (168) Google Scholar), it was found to that from native neuronal we the and of the recombinant reductase and calmodulin of the The with with native nNOS or with a form of the reductase protein that is a calmodulin binding that calmodulin binding fully activates the electron transfer of the nNOS reductase domain by a mechanism that is of the the oxygenase domain. calmodulin's activation of nNOS to a of function when it is to the three structurally flavin found in animals and overexpressed the rat nNOS reductase domain in yeast to its and to calmodulin electron transfer in nNOS. The recombinant reductase protein was to native nNOS in many including its flavin of a flavin semiquinone of flavin reduction by and rates of electron transfer to cytochrome c and ferricyanide in the or of bound the yeast to an reductase protein that fully the of the native enzyme with the of reduction or NO synthesis, consistent with these reactions the nNOS heme iron (20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar, 21Abu-Soud H.M. Feldman P.L. Clark P. Stuehr D.J. J. Biol. Chem. 1994; 269: 32318-32326Abstract Full Text PDF PubMed Google Scholar). protein from nNOS reductase that were expressed in E. coli K. Masters B.S.S. Biochemistry. 1995; 34: 3686-3693Crossref PubMed Scopus (168) Google Scholar) or by of full-length nNOS (7Sheta E.A. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar), which both a toward cytochrome c reduction native nNOS and either or a calmodulin showed that calmodulin binding activates an interdomain electron transfer between the nNOS flavins and heme iron (13Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar) and also increases nNOS reduction of exogenous electron acceptors such as cytochrome c and ferricyanide (15Heinzel B. John M. Klatt P. Bohme E. Mayer B. Biochem. J. 1992; 281: 627-630Crossref PubMed Scopus (470) Google Scholar, 16Schmidt H.H.H.W. Smith R.M. Nakane M. Murad F. Biochemistry. 1992; 31: 3243-3248Crossref PubMed Scopus (146) Google Scholar, 17McMillan 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 (354) Google Scholar, 20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). that calmodulin can fully activate nNOS reduction of cytochrome c and ferricyanide of electron transfer to the oxygenase domain. This is consistent with of the that showed reductase activities of native nNOS to that nNOS heme iron reduction H.M. Feldman P.L. Clark P. Stuehr D.J. J. Biol. Chem. 1994; 269: 32318-32326Abstract Full Text PDF PubMed Google Scholar, 22Wolff D.J. Datto G.A. Samatovicz R.A. Tempsick R.A. J. Biol. Chem. 1993; 268: 9425-9429Abstract Full Text PDF PubMed Google Scholar) and could in a native nNOS that is its heme iron (20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). also evidence against reduction as being an in reduction of cytochrome c. the rate of reduction by the or reductase protein indicated by NADPH is to to the rate of electron transfer to cytochrome c. we conclude that calmodulin a electron transfer from nNOS to these acceptors by activating the nNOS reductase domain of the nNOS reductase domain to its with of homologous NADPH-dependent isolated from or eukaryotic The nNOS reductase protein is to mammalian reductase T. Mason H.S. Biochemistry. 1973; 12: 2297-2308Crossref PubMed Scopus (279) Google Scholar, Masters Biochemistry. PubMed Scopus Google Scholar, T. Mason H.S. Biochemistry. PubMed Scopus Google Scholar) and the flavoprotein of sulfite reductase J. Barber M.J. Rueger D.C. Miller B.E. Siegel L.M. Kredich N.M. J. Biol. Chem. 1989; 264: 15796-15808Abstract Full Text PDF PubMed Google Scholar, M. J. M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) in and a flavin semiquinone of the nNOS reductase protein with NADPH showed that of the flavin semiquinone when of NADPH per of reductase was This that electron transfer can between a and fully reductase in to a of of the flavin semiquinone was possible conditions 1 per the protein not to be the protein of its semiquinone after to This is consistent with ferricyanide titration of the protein as isolated which indicates that only to of the reductase contains an the of the radical is and further reduction with NADPH the nNOS reductase protein to a but of its semiquinone as by of the that is to this reductase M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, B.S.S. R.A. Biochemistry. PubMed Scopus Google Scholar) and the flavoprotein of sulfite reductase M. J. M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), the nNOS reductase protein further reduction by NADPH after This is consistent with the of that between these three and that the of and flavin may be of the reductase activities for the nNOS reductase protein and the for the that the nNOS reductase is with to its rate of electron transfer to artificial only calmodulin binding the rates with the homologous or with which as purified contains bound calmodulin J. R.A. J. Med. 1992; PubMed Scopus Google Scholar). in this calmodulin binding to the of the nNOS reductase activate is a mechanism by which calmodulin with including calmodulin and B.E. 1992; Google Scholar, D.J. B.E. Scopus Google Scholar, B.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Scholar, J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). In these either the calmodulin binding domain or an site on the enzyme as a that of the to the site it is binding it is that calmodulin binding may a function to enable the reductase domain of nNOS to with the oxygenase domain and in electron transfer (2Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar), calmodulin does not nNOS to its (7Sheta E.A. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar, 13Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar, A. Stuehr D.J. Clark P. M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) or to cytochrome c (15Heinzel B. John M. Klatt P. Bohme E. Mayer B. Biochem. J. 1992; 281: 627-630Crossref PubMed Scopus (470) Google Scholar, 20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar), nNOS affinity for these is nearly in the or of bound with the protein that the calmodulin site may be with to cytochrome c, ferricyanide, and in its we reductase activities in the that the rates not between the and that calmodulin increase nNOS reductase activities by a mechanism a with its on the a that there is a interaction between nNOS and the of calmodulin that is for activating NO synthesis in the full-length enzyme J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). We an interaction with the is also involved in the activities of the nNOS reductase protein calmodulin S. and J. of a site on nNOS the calmodulin be consistent with the in the reductase domain to be associated with increased calmodulin we an increase in protein tryptophan and flavin that was to the when calmodulin bound to full-length nNOS. This that the increases in the native enzyme not due to a of the reductase domain to the oxygenase domain or of the flavins to the heme but due to within the reductase domain itself. from the that occurs when NOS to form a H.M. Stuehr D.J. Biochemistry. PubMed Scopus Google Scholar), which the oxygenase domains of the increase in flavin associated with calmodulin binding was not calmodulin with the that was may a that increases of bound flavins to This be consistent with that calmodulin binding to the reductase protein increases a interaction between its flavin semiquinone radical and and J. calmodulin binding to the nNOS reductase domain may a that up the domain as by a internal of protein tryptophan and in associated with an increased rate of electron influx into the flavins from an increased rate of electron transfer from the flavins to acceptors such as cytochrome c, and activation of interdomain electron transfer to the heme iron in the of full-length of calmodulin on the reductase domain of nNOS, it is which of the to calmodulin activating electron transfer to the heme iron and NO synthesis by full-length nNOS. calmodulin's increasing the rate of flavin reduction on NO synthesis the rate of flavin reduction could the rate of NO synthesis (20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). the this may be of the increased rate of cytochrome c calmodulin binding to its and does not to the reductase affinity for cytochrome c but may be in its interaction with and electron transfer to the nNOS oxygenase conclude that the reductase domain plays a role in the calmodulin response by both its and calmodulin This the nNOS reductase protein from the three structurally reductase, the flavoprotein of sulfite reductase, and cytochrome activities not by at reductase and sulfite through that may involve an interaction between a protein and a flavoprotein of the J. Biochem. 1991; PubMed Scopus Google Scholar, M. Scopus Google Scholar). with the nNOS reductase domain may can electron transfer in a variety of INTRODUCTIONNeuronal nitric oxide synthase (nNOS, NOS I) 1The abbreviations used are: nNOSneuronal nitric oxide synthaseDTTdithiothreitolNOnitric oxideDCPIPdichlorophenolindophenolMOPS4-morpholinepropanesulfonic acidBisTris(2-[bis(2-hy droxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. is one of three NOS isoforms found in animals (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2127) Google Scholar, 2Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar, 3Kerwin Jr., J.F. Lancaster Jr., J.R. Feldman P.L. J. Med. Chem. 1995; 38: 4343-4362Crossref PubMed Scopus (535) Google Scholar). The enzyme is expressed constitutively in different neuronal cell types and in skeletal muscle but is inactive in its native form and requires Ca2+-promoted calmodulin binding to activate its NO synthesis (4Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2163) Google Scholar). This property enables nNOS to participate in signal transduction cascades by generating NO only in response to increases in intracellular Ca2+ (5Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1533) Google Scholar) but may also lead to neuropathological conditions due to excessive NO production during the prolonged Ca2+ influx that occurs following reperfusion of ischemic tissues (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2127) Google Scholar, 5Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1533) Google Scholar, 6Dawson T.M. Dawson V.L. Neuroscientist. 1995; 1: 7-18Crossref Scopus (109) Google Scholar).The nNOS purified from rat brain is a bi-functional enzyme comprised of an N-terminal oxygenase domain that contains binding sites for iron protoporphyrin IX (heme), (6R,6S)-2-amino-4-hydroxy-6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropteridine, and L-arginine, and a C-terminal reductase domain that contains binding sites for FMN, FAD, and NADPH (4Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2163) Google Scholar, 7Sheta E.A. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar, 8Klatt P. Schmidt K. Brunner F. Mayer B. J. Biol. Chem. 1994; 269: 1674-1680Abstract Full Text PDF PubMed Google Scholar, 9McMillan K. Masters B.S.S. Biochemistry. 1995; 34: 3686-3693Crossref PubMed Scopus (168) Google Scholar). Sequence analysis indicates that the reductase domain of nNOS is structurally homologous to mammalian NADPH cytochrome P-450 reductase, the flavoprotein of Escherichia coli sulfite reductase, and the reductase domain of bacterial cytochrome P450BM3 (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2127) Google Scholar, 2Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). In these proteins, the flavins function to transfer NADPH-derived electrons to either an external heme protein acceptor or to an internal heme group located within the protein itself (7Sheta E.A. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar, 8Klatt P. Schmidt K. Brunner F. Mayer B. J. Biol. Chem. 1994; 269: 1674-1680Abstract Full Text PDF PubMed Google Scholar, 10Iyanagi T. Mason H.S. Biochemistry. 1973; 12: 2297-2308Crossref PubMed Scopus (279) Google Scholar, 11Ostrowski J. Barber M.J. Rueger D.C. Miller B.E. Siegel L.M. Kredich N.M. J. Biol. Chem. 1989; 264: 15796-15808Abstract Full Text PDF PubMed Google Scholar, 12Fulco A.J. Annu. Rev. Pharmacol. Toxicol. 1991; 31: 177-203Crossref PubMed Scopus (147) Google Scholar, 13Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar). Flavin-mediated electron transfer to the nNOS heme iron is thought to enable it to bind dioxygen and catalyze stepwise NO synthesis (2Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar).A variety of evidence indicates that calmodulin binds between the reductase and oxygenase domains of nNOS at a site represented by amino acids 725-745 (4Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2163) Google Scholar, 7Sheta E.A. McMillan K. Masters B.S.S. J. Biol. Chem. 1994; 269: 15147-15153Abstract Full Text PDF PubMed Google Scholar, 14Vorher T. Knopfel L. Hofmann F. Mollner S. Pfeuffer T. Carafoli E. Biochemistry. 1993; 32: 6081-6088Crossref PubMed Scopus (139) Google Scholar). We recently reported that calmodulin binding to nNOS triggers interdomain electron transfer between the flavins and heme iron, providing a basis by which calmodulin may activate NO synthesis in this isoform (13Abu-Soud H.M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10769-10772Crossref PubMed Scopus (393) Google Scholar). Besides activating interdomain electron transfer to the heme iron, calmodulin binding to nNOS increases its rate of electron transfer to a variety of exogenous acceptors including cytochrome c, ferricyanide, dichlorophenolindophenol (DCPIP), and dioxygen (15Heinzel B. John M. Klatt P. Bohme E. Mayer B. Biochem. J. 1992; 281: 627-630Crossref PubMed Scopus (470) Google Scholar, 16Schmidt H.H.H.W. Smith R.M. Nakane M. Murad F. Biochemistry. 1992; 31: 3243-3248Crossref PubMed Scopus (146) Google Scholar). The mechanism(s) by which calmodulin activates nNOS to reduce these external acceptors is controversial, in that there is evidence both for and against interdomain electron transfer or superoxide production being involved in the reactions (15Heinzel B. John M. Klatt P. Bohme E. Mayer B. Biochem. J. 1992; 281: 627-630Crossref PubMed Scopus (470) Google Scholar, 17McMillan 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 (354) Google Scholar, 18Frey C. Narayanan K. McMillan K. Spack L. Gross S.S. Masters B.S. Griffith O.W. J. Biol. Chem. 1994; 269: 26083-26091Abstract Full Text PDF PubMed Google Scholar, 19Persechini A. McMillan K. Masters B.S.S. Biochemistry. 1995; 34: 15091-15095Crossref PubMed Scopus (62) Google Scholar, 20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar, 21Abu-Soud H.M. Feldman P.L. Clark P. Stuehr D.J. J. Biol. Chem. 1994; 269: 32318-32326Abstract Full Text PDF PubMed Google Scholar, 22Wolff D.J. Datto G.A. Samatovicz R.A. Tempsick R.A. J. Biol. Chem. 1993; 268: 9425-9429Abstract Full Text PDF PubMed Google Scholar). We recently showed that nNOS reductase activities toward cytochrome c or ferricyanide were increased to nearly the same extent when calmodulin bound to a heme-free form of nNOS, consistent with calmodulin increasing reduction of these acceptors by a mechanism that does not involve interdomain electron transfer to the nNOS heme iron (20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar). Further analysis revealed that calmodulin binding also increases the rate by which NADPH reduces the nNOS flavins (20Abu-Soud H.M. Yoho L.L. Stuehr D.J. J. Biol. Chem. 1994; 269: 32047-32050Abstract Full Text PDF PubMed Google Scholar), suggesting that calmodulin may increase electron transfer rates to certain external acceptors by activating the nNOS reductase domain itself.To investigate further the function of the nNOS reductase domain and its possible activation by calmodulin, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) that contains the calmodulin binding site, using the eukaryotic Pichia a form of the nNOS reductase domain recently expressed in E. coli K. Masters B.S.S. Biochemistry. 1995; 34: 3686-3693Crossref PubMed Scopus (168) Google Scholar), it was found to that from native neuronal we the and of the recombinant reductase and calmodulin of the The with with native nNOS or with a form of the reductase protein that is a calmodulin binding that calmodulin binding fully activates the electron transfer of the nNOS reductase domain by a mechanism that is of the the oxygenase domain. calmodulin's activation of nNOS to a of function when it is to the three structurally flavin found in animals and