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Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology. Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown. We therefore cloned, expressed, and purified a NOS-like protein fromBacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions. bsNOS was dimeric, bound l-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite froml-Arg when incubated with NADPH and a mammalian NOS reductase domain. Stopped-flow analysis showed that ferrous bsNOS reacted with O2 to form a transient heme Fe(II)O2 species in the presence of either Arg or the reaction intermediate N-hydroxy-l-arginine. In the latter case, disappearance of the Fe(II)O2 species was kinetically and quantitatively coupled to formation of a transient heme Fe(III)NO product, which then dissociated to form ferric bsNOS. This behavior mirrors mammalian NOS enzymes and unambiguously shows that bsNOS can generate NO. NO formation required a bound tetrahydropteridine, and the kinetic effects of this cofactor were consistent with it donating an electron to the Fe(II)O2intermediate during the reaction. Dissociation of the heme Fe(III)NO product was much slower in bsNOS than in mammalian NOS. This constrains allowable rates of ferric heme reduction by a protein redox partner and underscores the utility of using a tetrahydropteridine electron donor in bsNOS. Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology. Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown. We therefore cloned, expressed, and purified a NOS-like protein fromBacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions. bsNOS was dimeric, bound l-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite froml-Arg when incubated with NADPH and a mammalian NOS reductase domain. Stopped-flow analysis showed that ferrous bsNOS reacted with O2 to form a transient heme Fe(II)O2 species in the presence of either Arg or the reaction intermediate N-hydroxy-l-arginine. In the latter case, disappearance of the Fe(II)O2 species was kinetically and quantitatively coupled to formation of a transient heme Fe(III)NO product, which then dissociated to form ferric bsNOS. This behavior mirrors mammalian NOS enzymes and unambiguously shows that bsNOS can generate NO. NO formation required a bound tetrahydropteridine, and the kinetic effects of this cofactor were consistent with it donating an electron to the Fe(II)O2intermediate during the reaction. Dissociation of the heme Fe(III)NO product was much slower in bsNOS than in mammalian NOS. This constrains allowable rates of ferric heme reduction by a protein redox partner and underscores the utility of using a tetrahydropteridine electron donor in bsNOS. nitric-oxide synthase calmodulin nNOS oxygenase domain nNOS reductase domain B. subtilis NO synthase (6R)-5,6,7,8-tetrahydro-l-biopterin tetrahydrofolate nitric oxide Nω-hydroxy-l-arginine D. radiodurans NO synthase iNOS oxygenase domain 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid dithiothreitol Nitric-oxide synthases (NOSs, EC1.14.13.39)1 are present in insects, mollusks, parasites, fungi, slime molds, and bacteria (1Muller U. Prog. Neurobiol. 1997; 51: 363-381Crossref PubMed Scopus (184) Google Scholar, 2Klesig D.F. Durner J. Noad R. Navarre D.A. Wendehenne D. Kumar D. Zhou J.M. Shah J. Zhang S. Kachroo P. et al.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8849-8855Crossref PubMed Scopus (588) Google Scholar, 3Golderer G. Werner E.R. Leitner S. Grobner P. Werner-Felmayer G. Gen. Dev. 2001; 15: 1299-1309Crossref PubMed Scopus (71) Google Scholar, 4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar). Their amino acid sequences and activities are similar to the mammalian NOSs, suggesting that the mammalian gene came from lower species through evolution. The mammalian NOSs catalyze the oxidation of l-arginine (Arg) to citrulline and NO, withN-hydroxy-l-arginine (NOHA) formed as an enzyme-bound intermediate (5Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). All mammalian NOSs are bi-domain proteins comprised of an N-terminal oxygenase domain (NOSoxy) that binds protoporphyrin IX (heme), 6R-tetrahydrobiopterin (H4B), and Arg, and a C-terminal flavoprotein domain (NOSred), linked together by a calmodulin (CaM) binding sequence (5Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). NOS flavoprotein domains are similar to NADPH-cytochrome P450 reductase and related electron transfer flavoproteins (5Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar) and function to provide NADPH-derived electrons to the ferric heme for O2activation during NO synthesis. Recent genome sequencing revealed that NOS-like proteins exist in many prokaryotes including Deinococcus radiodurans, Bacillus subtilis, Bacillus halodurans, Bacillus anthracis, 2Preliminary sequence data were obtained from The Institute for Genomic Research website at www.tigr.org. andStaphylococcus aureus2 (6Takami H. Nakasone K. Takaki Y. Maeno G. Sasaki R. Masui N. Fuji F. Hirama C. Nakamura Y. Ogasawara N. Kuhara S. Hirikoshi K. Nucleic Acids Res. 2000; 28: 4317-4331Crossref PubMed Scopus (452) Google Scholar, 7Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. Boriss R. Boursier L. Brans A. Braun M. Brignell S.C. Nature. 1997; 390: 249-256Crossref PubMed Scopus (3157) Google Scholar, 8White O. Eisen J.A. Heidelberg J.F. Hickey E.K. Peterson J.D. Dodson R.J. Haft D.H. Gwinn M.L. Nelson W.C. Richardson D.L. Science. 1999; 286: 1571-1577Crossref PubMed Scopus (808) Google Scholar). We recently sequenced, cloned, purified, and characterized D. radiodurans NOS-like protein (deiNOS) whose sequence is 34% identical and 52% conserved to the oxygenase domain of mammalian nitric-oxide synthases (NOSoxy). Purified deiNOS was dimeric, bound substrate Arg and cofactor H4B, and had a normal heme environment, despite its missing N-terminal structures that in NOSoxy bind Zn2+, the dihydroxypropyl side chain of H4B, and help form an active dimer in mammalian NOS (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar). The deiNOS heme accepted electrons from a separate mammalian NOS reductase and generated nitrite from Arg or NOHA in reactions stimulated by H4B. However, the oxyhemoglobin assay failed to show that deiNOS synthesized NO under these circumstances. Therefore, fundamental questions remain regarding the exact nature of the nitrogen oxide product formed by prokaryotic NOS-like proteins. To help address this issue we characterized the NOS-like protein fromB. subtilis (bsNOS). The results establish its Arg and H4B binding, product formation in multiple turnover reactions, nature and kinetics of heme transitions during Arg or NOHA oxidation under single turnover conditions, and unambiguously show that it produces NO as a product. All regents and materials were obtained from Sigma or sources reported previously (9Adak 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). The NOS gene of B. subtilis(ATCC) was amplified by PCR from genomic DNA. PCR primers generated aNdeI site before the 5′ start codon and a BamHI site after the 3′ stop codon and the amplified fragment cloned into a pET15B expression vector. B. subtilis NOS DNA in pET15B vector transformed into Escherichia coli strain BL21 (DE3) for protein expression. bsNOS had a His6 tag attached to its N terminus to aid purification. Proteins were overexpressed in E. coli strain BL21 (DE3) and purified by using chromatography on Ni2+-nitrilotriacetic acid resin for bsNOS and 2′,5′-ADP-Sepharose for nNOSred as described earlier (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar). Arg binding affinity was studied at 25 °C by perturbation difference spectroscopy using 10 mmimidazole according to methods described previously (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar). The steady state activities in the reconstituted system containing bsNOS and nNOSred in a 1:1.5 molar ratio were determined at 25 °C using spectroscopic and high performance liquid chromatography fluorometric assays that were described previously in detail (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar). Apparent Km values were determined by double-reciprocal analysis of the NADPH-dependent nitrite formation against various concentrations of H4B or THF in a reconstitution system containing nNOSred and bsNOS. Oxygen binding spectra were recorded in a stopped-flow instrument equipped with a rapid scanning diode array device (Hi-Tech MG-6000) designed to collect 96 complete spectra within 144 ms. Rapid scanning experiments involved mixing anaerobic solutions containing dithionite-reduced bsNOS, 40 mm EPPS buffer, pH 7.6, 0.5 mm DTT, 150 μm NaCl, and 1 mm Arg or NOHA with air-saturated buffer solutions at 10 °C in the presence or absence of 100 μm H4B or THF. Formation and decay of the Fe(II)O2 complexes were followed at 410 or 440 nm (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Signal-to-noise ratios were improved by averaging 10 individual traces. The diode-array data were then fit to different reaction models by a Specfit program from Hi-tech Ltd. to obtain the calculated number of species, their individual spectra, the concentration of each speciesversus time, and rate constants for each transition. We identified a 1-kb DNA segment that coded for a 369-amino acid bsNOS protein that has 35% identity and 49% conservation with deiNOS and 42% identity and 55% conservation with mouse iNOSoxy. The similarities among the primary sequences of bsNOS, deiNOS, and mouse iNOSoxy are shown in Fig.1A. Structural elements that make up the iNOS catalytic core are well conserved in bsNOS. This includes residues that contact the heme, bind the pteridine ring of H4B, and position substrate Arg. Like deiNOS, a notable similarity is that bsNOS is missing an extended portion of N-terminal sequence found in the mammalian enzymes. In mammalian NOSs this region codes for an N-terminal hook, a Zn2+ binding site, and contains residues that participate in forming the dimer interface and in binding the dihydroxypropyl side chain of H4B (Fig.1A). bsNOS is a heme protein similar to mammalian NOSs that contains residues to generate Arg and H4B binding purified bsNOS in a at a of identical to its calculated in a that bsNOS was in its form bsNOS is a despite its missing N-terminal elements that structures of mammalian NOS proteins B.R. R.J. Ghosh Ghosh S. Tainer J.A. Stuehr D.J. Getzoff E.D. J. 1999; PubMed Scopus Google Scholar). among amino in the interface and the of the N-terminal elements on dimer bsNOS therefore for residues and of obtained Arg or H4B binding are shown in bsNOS showed at and its heme bound to form a species identical to mammalian NOS S. Stuehr D.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). H4B a that generated a at of Arg the to to that Arg and the bsNOS heme in a high show that bsNOS binds H4B and Arg and has a similar heme with mammalian NOS. We Arg of to the Arg binding affinity of bsNOS. of Arg to bsNOS, was a that Arg a complete of The for Arg in presence of 10 mm and μm H4B was by double-reciprocal analysis and was μm in bsNOS, as with 10 μm and μm in deiNOS and We that Arg binding affinity of bsNOS is similar to mammalian NOS, consistent with its containing a conserved for high affinity Arg binding in mammalian activities and cofactor or substrate of bsNOS, deiNOS and for for was in reconstitution assays containing Arg, H4B, and nNOSred as described under Apparent Km values were determined by double-reciprocal analysis of the NADPH-dependent nitrite formation against various concentrations of H4B or THF. Apparent for Arg was determined by of during Arg in a was in reconstitution assays containing Arg, H4B, and nNOSred as described under Apparent Km values were determined by double-reciprocal analysis of the NADPH-dependent nitrite formation against various concentrations of H4B or THF. Apparent for Arg was determined by of during Arg NO by NOSs electron transfer reductase and oxygenase domains in a NOS bsNOS an attached reductase its heme can electrons from a separate donor The Bacillus genome contains electron transfer including an and NADPH that is similar to the mammalian P450 reductase T.A. 2001; PubMed Google Scholar). We therefore its catalytic in a reconstitution system that bsNOS with purified nNOSred a reductase that a binding site (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar). were in the presence or absence of H4B and using Arg or NOHA as We nitrite and citrulline in an assay as reported earlier (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar). formation from Arg or NOHA was and in reconstitution reactions that H4B and bsNOS and citrulline activities of bsNOS were similar to at oxygenase proteins or had under of the assay results that bsNOS can NADPH-derived electrons from nNOSred to Arg or NOHA to citrulline and nitrite in a reaction. the reconstitution system we determined an for H4B of 100 nm for bsNOS 10 nm for bsNOS affinity H4B is than deiNOS and that of or mammalian NOSs whose values nm to 1 THF is a tetrahydropteridine that can catalytic activities of deiNOS (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar). The bsNOS was found to bind THF with an Km of which is lower than that of deiNOS bsNOS from mammalian NOS in of THF in of H4B to The primary of bsNOS is consistent with its behavior H4B and THF. residues that the H4B ring in mammalian NOSs and position it the heme are conserved in bsNOS. we that deiNOS had affinity H4B to the absence of N-terminal residues that in NOSs bind the side chain of H4B B.R. R.J. Ghosh Ghosh S. Tainer J.A. Stuehr D.J. Getzoff E.D. J. 1999; PubMed Scopus Google Scholar). However, bsNOS this N-terminal region but has affinity similar to mammalian NOS. This that H4B affinity of deiNOS to for the absence of and elements in In case, the missing N-terminal region bsNOS to bind which contains a in of the side chain of H4B. NO formation of a transient Fe(II)O2 intermediate is for (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, C. R. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We stopped-flow spectroscopy to and kinetic of the Fe(II)O2 intermediate in bsNOS and whether H4B or THF its anaerobic of dithionite-reduced bsNOS containing Arg with or H4B was with air-saturated buffer at 10 The ferrous species a at nm in both This species within after mixing into a transient species with at nm in both The transient species to ferric that a at nm and at nm bsNOS formed a transient Fe(II)O2intermediate that is similar to that of deiNOS or (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The formation and decay kinetics of the Fe(II)O2intermediate were determined by at or 440 nm The of at these was as but with identical kinetics as found previously for deiNOS or (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). during or disappearance was described by a single in suggesting both transitions are of Fe(II)O2 formation and decay in the presence of Arg with or H4B are in The rate of was slower in both bsNOS and deiNOS with at the O2 concentration and this rate was by H4B in consistent with H4B O2 binding kinetics in NOSs (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The rate of in bsNOS was with H4B to bsNOS this rate by H4B in bsNOS as it in this kinetic has linked to reduction of Fe(II)O2 by H4B in mammalian NOS S. Stuehr D.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, C. R. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google results that H4B can an identical redox function in rates of Fe(II)O2 formation and disappearance in bsNOS, deiNOS, and under various were from were from Arg were from were from Arg were at 10 °C as described under The values are the obtained with or were from H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google in a were at 10 °C as described under The values are the obtained with or To formation and of the Fe(II)O2 species in the of NO we stopped-flow spectroscopy to heme transitions that during oxidation of NOHA in a single turnover reaction. of ferrous bsNOS with H4B and NOHA was with air-saturated buffer at 10 analysis of the data showed that it fit to a reaction to to to D. The calculated spectra for species are shown in The ferrous a at consistent with the of ferrous mammalian NOS under identical (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google S. L. Stuehr D.J. 2000; PubMed Scopus Google Scholar). has at and consistent with the of the Fe(II)O2 intermediate at 10 °C in presence of H4B and NOHA (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, S. L. Stuehr D.J. 2000; PubMed Scopus Google Scholar). has at and identical to the of the of deiNOS and S. L. Stuehr D.J. 2000; PubMed Scopus Google Scholar). has an at and it as ferric bsNOS. shows the concentrations of species during the reaction. The of ferric NOHA oxidation in the single turnover reaction S. L. Stuehr D.J. 2000; PubMed Scopus Google Scholar). shows that formed NO with the bsNOS ferric heme in a before the active site, during NO by mammalian NOS S. L. Stuehr D.J. 2000; PubMed Scopus Google Scholar). The results a for NOHA oxidation by bsNOS shown in heme binds O2 in the to generate a Fe(II)O2 transient This intermediate then to a ferric as an product. This for the NOHA reaction. NO then from heme to generate ferric bsNOS. The that the product was ferric than ferrous that bsNOS NO than which have generated a ferrous product (4Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer T.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, S. Stuehr D.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In a NOHA reaction H4B we or citrulline an identical Fe(II)O2 intermediate was generated H4B is for NO by bsNOS in the single turnover as is for mammalian NOSoxy enzymes S. Stuehr D.J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In mammalian NOSs, H4B is as an electron donor C. R. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google C. 1999; PubMed Scopus Google Scholar) and was recently shown to provide an electron to the heme Fe(II)O2 intermediate during Arg in a single turnover reaction C. R. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In that electron transfer from H4B is for the disappearance of the Fe(II)O2 intermediate (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The similar behavior of bsNOS regarding H4B binding and kinetic effects that a pteridine an identical redox function during its the B. subtilis genome contains enzymes to H4B from its F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. Bessieres P. Bolotin A. Borchert S. Boriss R. Boursier L. Brans A. Braun M. Brignell S.C. Nature. 1997; 390: 249-256Crossref PubMed Scopus (3157) Google Scholar). the of is that NO by bsNOS through the as in mammalian NOS and a tetrahydropteridine to provide the electron required for O2 NOHA oxidation by bsNOS the heme transitions with O2 binding were slower but within the of rates we have in mammalian NOSoxy reactions under the (10Abu-Soud H.M. Gachhui R. Raushel F.M. Stuehr D.J. J. Biol. Chem. 1997; 272: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, C. R. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. L. Stuehr D.J. 2000; PubMed Scopus Google Scholar). In the Fe(III)NO in bsNOS was slower than in mammalian which has from to at 10 °C S. L. Stuehr D.J. 2000; PubMed Scopus Google Scholar, Stuehr D.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). This difference is it the rate for NO from bsNOS during steady state NO synthesis. it constrains the rate of ferric heme reduction to remain or the is to NO. This is when the rate of ferric heme reduction NO a of the Fe(III)NO product to a ferrous species of NO S. J. S. J.D. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Dissociation of NO from the ferrous is E. P. D. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google the with O2 to generate or S. J. S. J.D. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). kinetic analysis that bsNOS either have a NO during steady state to its Fe(III)NO or generate in of NO ferric heme reduction as by its redox partner The on heme reduction rate by Fe(III)NO bsNOS from its Fe(II)O2 formation to substrate it a electron than the during O2 that the bsNOS Fe(II)O2intermediate is Arg or NOHA it However, its Fe(II)O2 intermediate is as by a decay rate of in the for bsNOS to substrate oxidation of the Fe(II)O2 intermediate to an electron at a rate than high rates NO from bsNOS to its NO as bsNOS this by using H4B, or a similar donor as a of the to the at rates that its decay by a tetrahydropteridine provide the electron during O2 bsNOS can its to substrate oxidation and a ferric heme reduction rate that is to for NO In NO by a prokaryotic NOS-like protein is but a number of related to the of a prokaryotic NOS, the redox product is generated during steady state expression of these proteins in the and exist that kinetic parameters of the as in M. D. D. Res. 1997; PubMed Scopus Google Scholar). provide of NOS and the of the NOS We McDonald for of for and of the Stuehr for and
Adak et al. (Wed,) studied this question.