Structural Basis for Isozyme-specific Regulation of Electron Transfer in Nitric-oxide Synthase
Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2+/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg1400, and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme. Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2+/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg1400, and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; cNOS, constitutive NOS; eNOS, nNOS, and iNOS, endothelial, neuronal, and inducible NOS, respectively; NOSox and NOSred, oxygenase and reductase modules, respectively; CaM, calmodulin; CYPOR, cytochrome P450 reductase; FNR, ferredoxin NADP-reductase; CD, connecting domain; CT, C-terminal tail; AH, autoinhibitory helix. acts in key physiological processes including neurotransmission, blood pressure regulation, and the immune response (reviewed in Ref. 1Schmidt H. Walter U. Cell. 1994; 78: 919-925Abstract Full Text PDF PubMed Scopus (1498) Google Scholar). NO is transient, small, and easily diffusible; its availability is solely regulated at the synthesis level by the nitric-oxide synthase (NOS) enzymes (EC 1.14.13.39). In mammals, two constitutively expressed NOS isozymes (cNOSs), endothelial (eNOS) and neuronal NOS (nNOS), are Ca2+-responsive and control basal NO levels, whereas the Ca2+-insensitive inducible NOS (iNOS) is expressed in response to specific cytokines or bacterial products (2Mayer B. Hemmens B. Trends Biochem. Sci. 1997; 22: 477-481Abstract Full Text PDF PubMed Scopus (511) Google Scholar). Deregulation of NO synthesis is associated with diverse human pathologies including immune-type diabetes, stroke, inflammatory bowel disease, rheumatoid arthritis, hypertension, arteriosclerosis, and infection susceptibility (3Chiueh C.C. Hong J.-S. Leong S.K. Ann. N. Y. Acad. Sci. 2002; 962: 1-437Crossref PubMed Scopus (64) Google Scholar). Each chain of a NOS homodimer (Fig. 1a) contains an N-terminal catalytic oxygenase module (NOSox) and a C-terminal electron-supplying reductase module (NOSred) linked by a 32-residue Ca2+/calmodulin (CaM) binding region (4Abu-Soud H.M. Loftus M. Stuehr D.J. Biochemistry. 1995; 34: 11167-11175Crossref PubMed Scopus (97) Google Scholar). In NOSox, the unique winged β-sheet fold (5Crane B.R. Arvai A.S. Gachhui R. Wu C. Ghosh D.K. Getzoff E.D. Stuehr D.J. Tainer J.A. Science. 1997; 278: 425-431Crossref PubMed Scopus (337) Google Scholar) binds substrate l-arginine and heme; two (6R)-5,6,7,8-tetrahydro-l-biopterin cofactors and a structural zinc ion stabilize the dimer interface (6Crane 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, 7Crane B.R. Rosenfeld R.J. Arvai A.S. Ghosh D.K. Ghosh S. Tainer J.A. Stuehr D.J. Getzoff E.D. EMBO J. 1999; 18: 6271-6281Crossref PubMed Scopus (100) Google Scholar, 8Raman C.S. Li H. Martasek P. Kral V. Masters B.S.S. Poulos T.L. Cell. 1998; 95: 939-950Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 9Fischmann T.O. Hruza A. Niu X.D. Fossetta J.D. Lunn C.A. Dolphin E. Prongay A.J. Reichert P. Lundell D.J. Narula S.K. Weber P.C. Nat. Struct. Biol. 1999; 6: 233-242Crossref PubMed Scopus (409) Google Scholar). Extensive crystallographic studies of NOSox in complex with substrate, intermediates, and inhibitors in the three isozymes have considerably advanced our understanding of the structural chemistry underlying NOS activity (10Alderton W.K. Cooper C.E. Knowles R.G. Biochem. J. 2001; 357: 593-615Crossref PubMed Scopus (3273) Google Scholar, 11Li 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, 12Rosenfeld R.J. Garcin E.D. Panda K. Andersson G. Åberg A. Wallace A.V. Morris G.M. Olson A.J. Stuehr D.J. Tainer J.A. Getzoff E.D. Biochemistry. 2002; 41: 13915-13925Crossref PubMed Scopus (57) Google Scholar). NOSox accepts electrons from NOSred to catalyze the sequential monooxygenation of l-arginine into N-hydroxyarginine, then citrulline and NO. NOSred belongs to a large protein family that includes NADPH-dependent cytochrome P450 reductase (CYPOR), sulfite reductase flavoprotein and novel reductase 1. These reductases share a conserved organization of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH)-binding domains (13Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (668) Google Scholar, 14Gruez A. Pignol D. Zeghouf M. Coves J. Fontecave M. Ferrer J.-L. Fontecilla-Camps J.-C. J. Mol. Biol. 2000; 299: 199-212Crossref PubMed Scopus (82) Google Scholar, 15Paine M.J. Garner A.P. Powell D. Sibbald J. Sales M. Pratt N. Smith T. Tew D.G. Wolf C.R. J. Biol. Chem. 2000; 275: 1471-1478Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The FMN-binding domain is homologous to small electron-carrier flavodoxins (16Watenpaugh K.D. Sieker L.C. Jensen L.H. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 3857-3860Crossref PubMed Scopus (176) Google Scholar), whereas the FAD- and NADPH-binding domains associate into a “FNR-like unit,” related to ferredoxin-NADP+ reductase (17Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (462) Google Scholar). An α-helical connecting domain (CD) orients the flanking FMN- and FAD-binding domains to align the two flavins (13Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (668) Google Scholar). Electron transfer proceeds from NADPH to FAD to FMN to heme (18Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). is Martasek P. T. Masters B.S.S. Biochem. 1999; PubMed Scopus Google Scholar), in from NOSred of to NOSox of the and is by by binding U. Wu C. Abu-Soud H.M. J. Ghosh D.K. Stuehr D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). NOSred is to control NO (reviewed in Ref. Martasek P. Masters B.S.S. Chem. 2002; PubMed Scopus Google Scholar). In a C-terminal tail electron In the a autoinhibitory in the FMN-binding domain that with binding and and electron of of these the in the of the is to to the by with the G.M. C.R. J. Biol. Chem. 278: Full Text Full Text PDF PubMed Scopus Google Scholar). The structure of a of NOSred J. Martasek P. Paschke R. Shea T. Masters B.S.S. Kim J.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), the FMN-binding domain and the C-terminal the conserved but address current questions NOS including the unique NOS electron Here, we address these questions by and the structure of a fully reductase dimer of These into the that a autoinhibitory NADPH, the C-terminal tail and its phosphorylation to NOS with these structural results, suggest a for in the FMN domain large scale to electrons between the two NOS and expressed in R. A. Abu-Soud H.M. R. G. Ghosh D.K. Stuehr D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and in and (18Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). of and at (18Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). The of the flavins in the crystal by a and and with a at in the at in an the and for at The structure by by and to current and of and and for are in the with and for the is at with and for and structure of the for in of of for the is at with and for and structure of the for in a new NOSred and the for NOSred we the crystallographic structure of a fully module at (Fig. used the structure of (13Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (668) Google Scholar), its FMN a for and the FMN domain into the electron of and the with and of and and with of the in the and of the The structure FMN binding domain connecting domain and with a and a FAD binding domain and and NADPH binding domain (Fig. in our crystallographic structure (Fig. and in by is a for and the NADPH-binding binds at of the β-sheet (Fig. in related reductases (13Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (668) Google Scholar, 14Gruez A. Pignol D. Zeghouf M. Coves J. Fontecave M. Ferrer J.-L. Fontecilla-Camps J.-C. J. Mol. Biol. 2000; 299: 199-212Crossref PubMed Scopus (82) Google Scholar, P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (462) Google Scholar). The is (Fig. with the FAD and in the two its In the (Fig. binds in of the dimer with and to conserved and two The phosphate of are by an of including from the C-terminal tail (Fig. The from the of to the of are is conserved in eNOS, but (Fig. and phosphate NADPH from of the C-terminal tail to its in NADPH binding and NOS The protein of electron transfer to cytochrome in the of and with the (Fig. The binding and with a structural for the of and residue in and of NOS and accepts electrons from NADPH and to binds in an (Fig. at of the fold of the FAD-binding domain (Fig. in related (17Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (462) Google Scholar). The FMN is into the FMN-binding domain (16Watenpaugh K.D. Sieker L.C. Jensen L.H. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 3857-3860Crossref PubMed Scopus (176) Google Scholar), of a β-sheet by (Fig. Extensive of and and for (13Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (668) Google Scholar), stabilize In the structure (Fig. the flavins FMN is a to the chain of J. Mol. Biol. PubMed Scopus Google and is FMN FAD a to chain and FAD to a conserved to for in P.A. Paschke R. Kim J.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). These structural that the FMN is the with the of the of NOS in the FAD we used of the flavins in the and the of the at that is (Fig. Our structural and studies that the structure from of the FMN and the FAD or a of to of the S.K. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (97) Google Scholar) catalytic The and of the FMN interface is by the of the FMN-binding domain into the (Fig. by the NADPH, and connecting domains to the the of interface a of the FMN- and FAD-binding domains a between and are conserved in (Fig. and of the the autoinhibitory of the FMN domain the FMN- and NADPH-binding domains the the the FMN- and FAD-binding domains, in CYPOR, to align the of the two flavin cofactors (Fig. In the of the cofactors are (Fig. electron transfer from FAD to FMN in electron-accepting position. The FMN is in of electrons from FMN to the heme or electron The two FMN-binding domains of the two NOSred to an the interface of the (Fig. These are by crystal in NOSred is from electron for of the CD, including the the FMN domain to the CD, and the the The two FMN-binding domains have crystallographic The of the FMN domains NOSred fully NOSred structure the structure of the NOSred J. Martasek P. Paschke R. Shea T. Masters B.S.S. Kim J.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) to the FMN-binding domain and the dimer but key of three unique the FMN domain autoinhibitory the C-terminal and its phosphorylation The FMN domain autoinhibitory contains an into a between FMN-binding domain and and NADPH-binding domain (Fig. from these and with the autoinhibitory the by to the the region of the autoinhibitory is by N-terminal and C-terminal the to the FMN these and C-terminal and The C-terminal tail contains an a the the flavins from (Fig. C-terminal tail from the of the NADPH-binding domain and a at by an is by the phosphorylation and C-terminal tail are to the FMN- and NADPH-binding domains with and and with of to the FMN domain into its electron-accepting (Fig. Extensive with FMN-binding domain and the C-terminal the of the C-terminal the of is FMN-binding domain in and conserved and structure a for NOS by of structural and studies that is (Fig. is in with studies that NOSox D.K. Wu C. E. M. B. Stuehr D.J. Biochemistry. 1997; PubMed Scopus Google Scholar). The dimer interface is with studies H. R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) that dimer in and but in The the to and of the dimer interface (Fig. is for NOS activity S. Stuehr D.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the NOS of and FMN for Electron NOSred structure unique into the of electron transfer to is the Martasek P. T. Masters B.S.S. Biochem. 1999; PubMed Scopus Google Scholar) in NO synthesis by NOS. The for the FMN is between the FMN-binding domain and the (Fig. an the electrons from FMN to of NOSox and NOSred the of the FMN but of the FMN-binding domain a of the FMN-binding domain in reductase is between the two but by with (Fig. in P.A. Paschke R. Kim J.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and sulfite reductase flavoprotein A. Pignol D. Zeghouf M. Coves J. Fontecave M. Ferrer J.-L. Fontecilla-Camps J.-C. J. Mol. Biol. 2000; 299: 199-212Crossref PubMed Scopus (82) Google Scholar) the electron for the FMN domain is in binding or of that are to the FMN-binding domain (18Adak S. Ghosh S. Abu-Soud H.M. Stuehr D.J. J. Biol. Chem. 1999; 274: 22313-22320Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, R. K. Shea T.M. Masters B.S.S. Nitric 1997; PubMed Scopus Google Scholar). These support a for electron transfer in the of the FMN-binding domain is a key of and a of family of structurally for electron transfer from FMN to heme An structure of NOS of in with our a for the the we used to the of its the NOSox and NOSred modules, and a complex M. Arvai A.S. Tainer J.A. Getzoff E.D. EMBO J. 22: PubMed Scopus Google Scholar). and the NOSox module and complex with to the NOSred module to the by the from NOSox to NOSred the of with the of NOSox and the of NOSred at of the the large between these (Fig. to to is with the of the α-helical of in the NOSox is the NOSred (Fig. an that is easily with the of the The of is with that region is to Smith D. D.K. V. S. A. Biochem. J. PubMed Scopus Google Scholar). In family of the and the NOSox and NOSred (Fig. The transfer of electrons in from NOSred to NOSox the dimer U. Wu C. Abu-Soud H.M. J. Ghosh D.K. Stuehr D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) of the two a In our the between the FMN and heme cofactors is is C.C. C.C. 1999; PubMed Scopus Google Scholar) for electron the structural and that the entire FMN domain a by swinging and between its two The region a for motion and with the of region in related cytochrome Li H. H. J.A. Poulos T.L. Proc. Natl. Acad. Sci. U. S. 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Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) residue the of the heme and of the FMN-binding in electron from NOSred to in the electron-accepting the FMN-binding domain and have (Fig. and (Fig. FMN R.G. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar) the FMN to with its The of the NOSox heme (Fig. and the NOSred FMN-binding domains (Fig. of NOS dimer are for a motion of the FMN domain from FAD to the These are with the swinging FMN domain in the FMN-binding domain the the of the FMN to with the and In the electron-accepting and and the (Fig. for the FMN domain electron transfer is C.C. C.C. 1999; PubMed Scopus Google Scholar). The FAD to FMN is and the FMN to heme is in the electron-accepting and that sequential of the FMN domain with by are to electrons from NOSred to Electron in the and of key of the the NOSred structure novel into the mechanisms that control the electron transfer from NADPH to the flavins and the electron transfer from FMN to The NOSred structure the of the FMN domain autoinhibitory unique to The N-terminal of including the phosphorylation into an Three conserved and by two and a new to the AH, striking similarities with the canonical binding J. 1997; PubMed Scopus Google Scholar). is that the contains an residue in (Fig. studies the of a C-terminal of in the and suggest that the autoinhibitory with binding by with the K. B. A.J. Smith Martasek P. Masters B.S.S. P. Q. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the and its in the structure between the FMN- and NADPH-binding domains, we two for its in NOS binds and acts a that binding to the of the with the FMN- and NADPH-binding domains to the electron-accepting of the FMN of to the and and the FMN domain for electron These mechanisms binding of the to CaM, C.R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and an for by NADPH, Arg1400, and the C-terminal the NOSred structure and the is from with the FAD by an chain to (13Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (668) Google Scholar, 14Gruez A. Pignol D. Zeghouf M. Coves J. Fontecave M. Ferrer J.-L. Fontecilla-Camps J.-C. J. Mol. Biol. 2000; 299: 199-212Crossref PubMed Scopus (82) Google Scholar, P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-66Crossref PubMed Scopus (462) Google Scholar). The of the are from that in the structure J. Martasek P. Paschke R. Shea T. Masters B.S.S. Kim J.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), of the of the C-terminal studies P.A. Paschke R. Kim J.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and S. M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar) the of for and the that residue acts in with the C-terminal tail in to repress electron NADPH in a for transfer to we the of and the P.A. Paschke R. Kim J.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). (Fig. in nNOS, structural for electron transfer of and the of the C-terminal tail of the chain (Fig. the of NOS isozymes (Fig. we two of the C-terminal the N-terminal is conserved in the and with and the C-terminal is in in and in and in the that of the two the NO synthesis activity of P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), whereas the and the NO Martasek P. Shea T.M. Kim J.J. Masters B.S.S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In a α-helical whereas is In to Martasek P. Shea T.M. Kim J.J. Masters B.S.S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), between the FAD and FMN but in electron suggest that the FMN domain its to the is with the P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) that with The in the structure to the phosphate from the FMN- and the NADPH-binding domains (Fig. for NADPH between the NADPH and conserved in in related reductases P.A. Paschke R. Kim J.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, C.C. P.A. Sci. PubMed Scopus Google Scholar). In the in the NADPH binding and to between its and residue and with the a in the for NADPH with E. D. E. D. and D. J. studies that NADPH binding to NOS the FMN domain and electron transfer S.K. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Our a structural that in the of CaM, the between NADPH and the residue in to the C-terminal and repress NOS by locking the FMN domain into the electron-accepting position. with the in NADPH, the and of electron transfer from NADPH to cytochrome in the of of activity of is that of (Fig. for that the or electron-accepting of the FMN domain with S. M. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, S. Biochem. PubMed Scopus Google Scholar), the structural and studies suggest that NADPH, the and the C-terminal tail synergistically to repress NOS activity in the of for in structure an to the of the phosphorylation of NOS a C-terminal phosphorylation In the is at the of the helix, in a at the of NOSred (Fig. The by the phosphate or the S. J. S. Wang Q. J.D. Stuehr D.J. J. Biol. 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Our an phosphorylation of a residue large that NOS. current understanding of NOS and regulation and for new of The crystallographic structure of the dimer the and for the FMN-binding domains in electron-accepting key of including the autoinhibitory the C-terminal and its phosphorylation The striking of the autoinhibitory with canonical that Our structural and mutagenesis a new isozyme-specific residue in the of NOS the structure the of the by NADPH, Arg1400, and the the FMN- and NADPH-binding domains and the FMN-binding domain into its electron-accepting position. the of the the phosphorylation is to from the FMN-binding In of results, we the of the NOSox and NOSred and a complex to the In NOS and in related our a swinging electron large scale of the two FMN domains, between electron-accepting and In NOS, the two of the FMN are a for the electron transfer In our that binding and phosphorylation NO synthesis by the C-terminal the FMN-binding domain to electron in understanding the structural chemistry of NOSox the striking of the three NOS a for the of isozyme-specific inhibitors for the of including stroke, rheumatoid arthritis, or The structural and mutagenesis studies isozyme-specific in electron transfer for NO and suggest an that the electron-accepting of the FMN-binding domain the to or NO synthesis in an isozyme-specific by or electron B. and A. Arvai for the of C. D. for in and D. S. for of the P. and G. and A. Åberg for of the and D. P. and M. for of at the a by of the of of Science. The is by the of of and and by the of for and the of with