The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

Abstract

Bacterial nitric-oxide synthase (NOS)-like proteins are believed to be genuine NOSs. As for cytochromes P450 (CYPs), NOS-proximal ligand is a thiolate that exerts a push effect crucial for the process of dioxygen activation. Unlike CYPs, this catalytic electron donation seems controlled by a hydrogen bond (H-bond) interaction between the thiolate ligand and a vicinal tryptophan. Variations of the strength of this H-bond could provide a direct way to tune the stability along with the electronic and structural properties of NOS. We generated five different mutations of bsNOS Trp66, which can modulate this proximal H-bond. We investigated the effects of these mutations on different NOS complexes (FeIII, FeIICO, and FeIINO), using a combination of UV-visible absorption, EPR, FTIR, and resonance Raman spectroscopies. Our results indicate that (i) the proximal H-bond modulation can selectively decrease or increase the electron donating properties of the proximal thiolate, (ii) this modulation controls the σ-competition between distal and proximal ligands, (iii) this H-bond controls the stability of various NOS intermediates, and (iv) a fine tuning of the electron donation by the proximal ligand is required to allow at the same time oxygen activation and to prevent uncoupling reactions. Bacterial nitric-oxide synthase (NOS)-like proteins are believed to be genuine NOSs. As for cytochromes P450 (CYPs), NOS-proximal ligand is a thiolate that exerts a push effect crucial for the process of dioxygen activation. Unlike CYPs, this catalytic electron donation seems controlled by a hydrogen bond (H-bond) interaction between the thiolate ligand and a vicinal tryptophan. Variations of the strength of this H-bond could provide a direct way to tune the stability along with the electronic and structural properties of NOS. We generated five different mutations of bsNOS Trp66, which can modulate this proximal H-bond. We investigated the effects of these mutations on different NOS complexes (FeIII, FeIICO, and FeIINO), using a combination of UV-visible absorption, EPR, FTIR, and resonance Raman spectroscopies. Our results indicate that (i) the proximal H-bond modulation can selectively decrease or increase the electron donating properties of the proximal thiolate, (ii) this modulation controls the σ-competition between distal and proximal ligands, (iii) this H-bond controls the stability of various NOS intermediates, and (iv) a fine tuning of the electron donation by the proximal ligand is required to allow at the same time oxygen activation and to prevent uncoupling reactions. IntroductionNitrogen monoxide (NO) is a well described radical molecule (1Hughes M.N. Methods Enzymol. 2008; 436: 3-19Crossref PubMed Scopus (112) Google Scholar) that has been shown to exert major physiological functions in mammals, ranging from signaling processes to cytotoxic activities (2Ignarro L.J. J. Physiol. Pharmacol. 2002; 53: 503-514PubMed Google Scholar, 3Bogdan C. Nat. Immunol. 2001; 2: 907-916Crossref PubMed Scopus (2532) Google Scholar, 4Bredt D.S. J. Cell Sci. 2003; 116: 9-15Crossref PubMed Scopus (128) Google Scholar, 5Pacher P. Beckman J.S. Liaudet L. Physiol. Rev. 2007; 87: 315-424Crossref PubMed Scopus (4514) Google Scholar). It is exclusively synthesized by a family of enzymes named nitric-oxide synthases (NOSs) 2The abbreviations used are: NOS, nitric-oxide synthase; bacNOS, bacterial NOS-like protein; eNOS, endothelial nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; mNOS, mammalian nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; bsNOS, NOS-like protein isolated from B. subtilis; saNOS, NOS-like protein isolated from S. aureus; ATR, attenuated total reflection; FeIINO, ferrous heme-nitrosyl complex; FeIICO, ferrous heme-carbon monoxide complex; FeIIO2, ferrous heme-oxygen complex; FeIIINO, ferric heme-nitrosyl complex; XO, either NO, CO, or O2; HS-5c and LS-6c, high spin pentacoordinated and low spin hexacoordinated, respectively; KPi, inorganic phosphate buffer; P420, thiolate-free coordination of the heme; mT, millitesla(s); H-bond, hydrogen bond; H4B, BH4, tetrahydrobiopterin, (6R)-5,6,7,8-tetrahydro-l-biopterin. that have been cloned and characterized in the early 1990s (6Sessa W.C. J. Cell Sci. 2004; 117: 2427-2429Crossref PubMed Scopus (453) Google Scholar, 7Lowenstein C.J. Padalko E. J. Cell Sci. 2004; 117: 2865-2867Crossref PubMed Scopus (191) Google Scholar, 8Mungrue I.N. Bredt D.S. J. Cell Sci. 2004; 117: 2627-2629Crossref PubMed Scopus (86) Google Scholar, 9Ghosh D.K. Salerno J.C. Front. Biosci. 2003; 8: d193-d209Crossref PubMed Scopus (118) Google Scholar). With the emergence of efficient DNA sequencing techniques, the accessibility of an increasing number of genomes led to an unprecedented quest for new NOSs in other organisms. Although no NOSs have been found so far in plants and yeasts (10Moreau M. Lee G.I. Wang Y. Crane B.R. Klessig D.F. J. Biol. Chem. 2008; 283: 32957-32967Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 11Zemojtel T. Fröhlich A. Palmieri M.C. Kolanczyk M. Mikula I. Wyrwicz L.S. Wanker E.E. Mundlos S. Vingron M. Martasek P. Durner J. Trends Plant Sci. 2006; 11 (author reply 526–528): 524-525Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar), random BLAST analyses of several bacterial genomes led to the discovery of new NOS-like proteins, mostly in Gram-positive bacteria (12Sudhamsu J. Crane B.R. Trends Microbiol. 2009; 17: 212-218Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). These proteins correspond to the partially truncated oxygenase domain of mammalian NOSs (mNOSs). Because of the dominant structure-function approach, the first works on bacterial NOS-like proteins (bacNOSs) aimed at highlighting the similarities between mammalian and bacterial NOSs. Indeed, the crystallographic structures of NOSs from Bacillus subtilis (bsNOS (13Pant K. Bilwes A.M. Adak S. Stuehr D.J. Crane B.R. Biochemistry. 2002; 41: 11071-11079Crossref PubMed Scopus (120) Google Scholar)), Staphylococcus aureus (saNOS (14Bird L.E. Ren J. Zhang J. Foxwell N. Hawkins A.R. Charles I.G. Stammers D.K. Structure. 2002; 10: 1687-1696Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar)), or Geobacillus stearothermophilus (15Sudhamsu J. Crane B.R. J. Biol. Chem. 2006; 281: 9623-9632Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) were perfectly superimposable to the three-dimensional structure of mNOSs with the exception of a portion of the N-terminal region involved in BH4 binding and in the formation of the zinc-tetrathiolate complex (12Sudhamsu J. Crane B.R. Trends Microbiol. 2009; 17: 212-218Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In addition, the first enzymological experiments suggested that bacNOSs, similarly to mNOSs, had the capacity to catalyze oxygen activation and to metabolize l-Arg, the natural substrate of mNOSs, into citrulline and NO (16Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Bilwes A.M. K. Aulak K.S. Crane B.R. Stuehr D.J. Sci. 2002; PubMed Scopus Google Scholar). an of genuine NO in has been investigated and a of first on the of suggested a to the of a of the of a on a J. Crane B.R. 2004; PubMed Scopus Google Scholar, B. Crane B.R. Chem. Biol. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). to to the NOS, which to with the and to Crane B.R. Sci. 2004; PubMed Scopus Google Scholar, T. Crane B.R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). different and I. E. Sci. PubMed Scopus Google Scholar, K. I. E. Y. L.E. E. Sci. 2008; PubMed Scopus Google Scholar, I. K. M. E. 2009; PubMed Scopus Google Scholar) that could in a of functions to interaction I. E. Sci. PubMed Scopus Google Scholar), and K. I. E. Y. L.E. E. Sci. 2008; PubMed Scopus Google Scholar), or I. K. M. E. 2009; PubMed Scopus Google Scholar). Crane suggested that NOS could in the processes of bacteria to a M. J. L. Y. Crane B.R. Sci. 2009; PubMed Scopus Google Scholar). Because NO is used by the a that NO is the molecule to a the In that bsNOS is to in NO and A. Stuehr D.J. J. J. PubMed Scopus Google Scholar). the the in of genuine nitric-oxide with mNOSs, the catalytic of has been the results so far between mNOSs and of are an electron of a I. M. Wang L. Stuehr D.J. E. J. Biol. Chem. 2008; 283: Full Text Full Text PDF PubMed Scopus Google Scholar) prevent an efficient first electron have been to NO Crane B.R. Stuehr D.J. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). has been so far for the of a in the electron natural is synthesized by these and BH4 and increase the of of the complex of several (16Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Biochemistry. 2009; PubMed Scopus Google Scholar, M. K. Crane B.R. Stuehr D.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), no radical has been in so is the NO of the bsNOS Indeed, the in the seems to a NO M. K. Crane B.R. Stuehr D.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), which is for NO D.J. J. Wang Adak S. J. Biol. Chem. 2004; 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). Indeed, the NO by inducible NOS and into a NO Stuehr D.J. J. PubMed Scopus Google Scholar). the of by bsNOS, in with of the distal that to a of NO or other the catalytic which in could to reactions. these could the of oxygenase this mNOSs is to the oxygen activation described for cytochromes P450 M. Chem. Rev. PubMed Scopus Google Scholar, B. S. Chem. Rev. 2004; PubMed Scopus Google Scholar, I.G. I. Chem. Rev. PubMed Scopus Google Scholar, Rev. 2007; PubMed Scopus Google Scholar). It is characterized by processes that allow the bond the effects J. Biol. Chem. Scopus Google Scholar). effect the distal H-bond that the required for oxygen activation for NOS, resonance Raman of M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. 2004; 87: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) and bsNOS M. J. 2007; PubMed Scopus Google Scholar, J. M. Stuehr D.J. Biochemistry. 2006; PubMed Scopus Google Scholar) complexes no in the distal of and mNOSs M. Stuehr D.J. J. Chem. 2007; PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar, B. Wang J. Stuehr D.J. Biochemistry. PubMed Scopus Google Scholar). have been between the structures of complexes of bsNOS K. Crane B.R. Biochemistry. 2006; PubMed Scopus Google Scholar) and neuronal NOS T. M. Biochemistry. 2009; PubMed Scopus Google Scholar, J. J. J. Biol. Chem. 2006; PubMed Scopus Google Scholar) in the of and l-Arg, the In the of the distal structure is mNOSs between and a in the We that the proximal bond for bsNOS for mNOSs, a modulation of the electron donation by the thiolate ligand J. M. Stuehr D.J. Biochemistry. 2006; PubMed Scopus Google Scholar). seems to be to Unlike the of NOS-proximal thiolate is in a H-bond interaction with the of the of the vicinal of this H-bond and the of this H-bond for the of the of S. Stuehr D.J. J. 2001; PubMed Scopus Google Scholar, S. Wang Stuehr D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of this H-bond, in the of the of iNOS, results in a oxygen activation process J. A. Wang C. S. Stuehr D.J. J. Biol. Chem. 2008; 283: Full Text Full Text PDF PubMed Scopus Google Scholar). are the same has been suggested by and J. S. M. J. 2009; PubMed Scopus Google Scholar), that the of the H-bond in to proximal a of the modulation of the interaction between the proximal ligand and the vicinal for We the effects of several mutations on the of bsNOS by using a combination of resonance and spectroscopies. Our results that the modulation of this interaction the electron donation of the thiolate ligand the electronic structure of the seems to indicate that the of NOS major and is controlled by the same proximal H-bond in bacterial and mammalian of bsNOS is in an H-bond with the proximal ligand that is believed to tune the properties of the NOS bond J. S. M. J. 2009; PubMed Scopus Google Scholar). We for the first time a of five different of this that the of the proximal (i) the and described for S. C. Wang Crane B.R. Stuehr D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and for and J. S. M. J. 2009; PubMed Scopus Google Scholar), correspond to a of the (ii) the described for J. A. Wang C. S. Stuehr D.J. J. Biol. Chem. 2008; 283: Full Text Full Text PDF PubMed Scopus Google Scholar), is to a of the (iii) the and H-bond and of the effects of these different mutations on bsNOS structural and electronic properties allow a of the way this H-bond NOS structure and of the between and complex is used a to the of on structure and C. M. Y. Stuehr D.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Chem. 116: Scopus Google Scholar, J. PubMed Scopus (191) Google Scholar). resonance Raman and allow the of and of these are to the electronic properties of the proximal ligand and to between and the distal J. Chem. 116: Scopus Google Scholar, J. PubMed Scopus (191) Google Scholar). of the distal of the ligand in in the donation from the to the of to the well between the of the and M. J. PubMed Scopus Google Scholar). is a between the proximal and distal for to the of the Variations in the electronic properties of the proximal ligand this and the the thiolate of NOS and cytochromes low and proximal ligand the of or to and results that the mutations in the an increase in the and for the the and are characterized by and in the these a in the of the to the of the proximal ligand that the the electron donating of the proximal and are characterized by electron donation with the of the is characterized by electron donation with a of the this using the are for the first time to a NOS protein the increase and the decrease of this σ-competition and to these to the of the interaction in which the proximal ligand is of H-bond in bsNOS in σ-competition has effects on the properties of bsNOS by the effects of the on and stability and and We a of pentacoordinated of these complexes for and bsNOS to a complex a proximal seems same is in the of the of for bsNOS is the for for and for and a of and for no H-bond with the the for J. S. M. J. 2009; PubMed Scopus Google Scholar), can be from Our that the H-bond interaction controls the stability of bsNOS FeIINO, FeIICO, and that other structural to bsNOS Indeed, binding of a distal or NO, the complex is and into pentacoordinated the of the proximal thiolate with and this decrease in stability from the of that NOS the between the and the the a of the by the of the of the ferric a of the protein and a of the of on the of in the properties of the proximal thiolate to exert an on the electronic structure of bsNOS the of the is for and and for bsNOS proximal These are by the of the of and complexes and to a by the of the low spin of bsNOS electronic properties can be by of the of bsNOS, is for for and between on and on the other is for the of the HS-5c Indeed, the is for the for and and same are for is for for and bsNOS effects of the of the proximal bond on the electronic structure of complexes are to the of the for this of a of the proximal H-bond in the of the electronic on the spin on the NO seems for and the hydrogen bond strength on the proximal thiolate ligand is These results that in the properties of the proximal ligand the electronic structure of and of the H-bond on of NOS electronic structure by the in the proximal H-bond are believed to catalytic Indeed, the of and increase for D.J. J. Wang Adak S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. Wang Crane B.R. Stuehr D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of this H-bond in the of a decrease of and activation that allow the of new J. A. Wang C. S. Stuehr D.J. J. Biol. Chem. 2008; 283: Full Text Full Text PDF PubMed Scopus Google of these results that bacterial NOSs the same the PubMed Scopus Google Scholar), to tune the to the proximal ligand electron electron donation for the of NOS and mostly to uncoupling processes and and oxygen electron donation for the to an of complexes S. J. 2002; PubMed Scopus Google Scholar). of of by the and this the an synthase which to of the other with that of bsNOS could from uncoupling to a electron on the a of the of the H-bond in the of NOS structural and electronic We the effects of and of this H-bond on FeIICO, and for the first time a between the proximal H-bond the properties of the proximal and the stability and of NOS. Our indicate that the of the H-bond the electron donation properties of the proximal which in an σ-competition on the to the of FeIINO, FeIICO, and is the are by a that the proximal H-bond and the These results and for the of J. S. M. J. 2009; PubMed Scopus Google Scholar), and M. Adak S. Stuehr D.J. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Google Scholar, M. Adak S. T. M. Stuehr D.J. Biochemistry. 2003; PubMed Scopus Google Scholar) and the crucial of this proximal H-bond in the stability of NOS results that bacterial NOSs are genuine that to the electron donation from the thiolate to distal ligands, and NO, and to the same the proximal H-bond to tune and catalytic could in the interaction between NOS and other ligands, of the of this H-bond in the of dioxygen of or of activation could the of mammalian and bacterial NOSs. IntroductionNitrogen monoxide (NO) is a well described radical molecule (1Hughes M.N. Methods Enzymol. 2008; 436: 3-19Crossref PubMed Scopus (112) Google Scholar) that has been shown to exert major physiological functions in mammals, ranging from signaling processes to cytotoxic activities (2Ignarro L.J. J. Physiol. Pharmacol. 2002; 53: 503-514PubMed Google Scholar, 3Bogdan C. Nat. Immunol. 2001; 2: 907-916Crossref PubMed Scopus (2532) Google Scholar, 4Bredt D.S. J. Cell Sci. 2003; 116: 9-15Crossref PubMed Scopus (128) Google Scholar, 5Pacher P. Beckman J.S. Liaudet L. Physiol. Rev. 2007; 87: 315-424Crossref PubMed Scopus (4514) Google Scholar). It is exclusively synthesized by a family of enzymes named nitric-oxide synthases (NOSs) 2The abbreviations used are: NOS, nitric-oxide synthase; bacNOS, bacterial NOS-like protein; eNOS, endothelial nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; mNOS, mammalian nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; bsNOS, NOS-like protein isolated from B. subtilis; saNOS, NOS-like protein isolated from S. aureus; ATR, attenuated total reflection; FeIINO, ferrous heme-nitrosyl complex; FeIICO, ferrous heme-carbon monoxide complex; FeIIO2, ferrous heme-oxygen complex; FeIIINO, ferric heme-nitrosyl complex; XO, either NO, CO, or O2; HS-5c and LS-6c, high spin pentacoordinated and low spin hexacoordinated, respectively; KPi, inorganic phosphate buffer; P420, thiolate-free coordination of the heme; mT, millitesla(s); H-bond, hydrogen bond; H4B, BH4, tetrahydrobiopterin, (6R)-5,6,7,8-tetrahydro-l-biopterin. that have been cloned and characterized in the early 1990s (6Sessa W.C. J. Cell Sci. 2004; 117: 2427-2429Crossref PubMed Scopus (453) Google Scholar, 7Lowenstein C.J. Padalko E. J. Cell Sci. 2004; 117: 2865-2867Crossref PubMed Scopus (191) Google Scholar, 8Mungrue I.N. Bredt D.S. J. Cell Sci. 2004; 117: 2627-2629Crossref PubMed Scopus (86) Google Scholar, 9Ghosh D.K. Salerno J.C. Front. Biosci. 2003; 8: d193-d209Crossref PubMed Scopus (118) Google Scholar). With the emergence of efficient DNA sequencing techniques, the accessibility of an increasing number of genomes led to an unprecedented quest for new NOSs in other organisms. Although no NOSs have been found so far in plants and yeasts (10Moreau M. Lee G.I. Wang Y. Crane B.R. Klessig D.F. J. Biol. Chem. 2008; 283: 32957-32967Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 11Zemojtel T. Fröhlich A. Palmieri M.C. Kolanczyk M. Mikula I. Wyrwicz L.S. Wanker E.E. Mundlos S. Vingron M. Martasek P. Durner J. Trends Plant Sci. 2006; 11 (author reply 526–528): 524-525Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar), random BLAST analyses of several bacterial genomes led to the discovery of new NOS-like proteins, mostly in Gram-positive bacteria (12Sudhamsu J. Crane B.R. Trends Microbiol. 2009; 17: 212-218Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). These proteins correspond to the partially truncated oxygenase domain of mammalian NOSs (mNOSs). Because of the dominant structure-function approach, the first works on bacterial NOS-like proteins (bacNOSs) aimed at highlighting the similarities between mammalian and bacterial NOSs. Indeed, the crystallographic structures of NOSs from Bacillus subtilis (bsNOS (13Pant K. Bilwes A.M. Adak S. Stuehr D.J. Crane B.R. Biochemistry. 2002; 41: 11071-11079Crossref PubMed Scopus (120) Google Scholar)), Staphylococcus aureus (saNOS (14Bird L.E. Ren J. Zhang J. Foxwell N. Hawkins A.R. Charles I.G. Stammers D.K. Structure. 2002; 10: 1687-1696Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar)), or Geobacillus stearothermophilus (15Sudhamsu J. Crane B.R. J. Biol. Chem. 2006; 281: 9623-9632Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) were perfectly superimposable to the three-dimensional structure of mNOSs with the exception of a portion of the N-terminal region involved in BH4 binding and in the formation of the zinc-tetrathiolate complex (12Sudhamsu J. Crane B.R. Trends Microbiol. 2009; 17: 212-218Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In addition, the first enzymological experiments suggested that bacNOSs, similarly to mNOSs, had the capacity to catalyze oxygen activation and to metabolize l-Arg, the natural substrate of mNOSs, into citrulline and NO (16Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Bilwes A.M. K. Aulak K.S. Crane B.R. Stuehr D.J. Sci. 2002; PubMed Scopus Google Scholar). an of genuine NO in has been investigated and a of first on the of suggested a to the of a of the of a on a J. Crane B.R. 2004; PubMed Scopus Google Scholar, B. Crane B.R. Chem. Biol. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). to to the NOS, which to with the and to Crane B.R. Sci. 2004; PubMed Scopus Google Scholar, T. Crane B.R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). different and I. E. Sci. PubMed Scopus Google Scholar, K. I. E. Y. L.E. E. Sci. 2008; PubMed Scopus Google Scholar, I. K. M. E. 2009; PubMed Scopus Google Scholar) that could in a of functions to interaction I. E. Sci. PubMed Scopus Google Scholar), and K. I. E. Y. L.E. E. Sci. 2008; PubMed Scopus Google Scholar), or I. K. M. E. 2009; PubMed Scopus Google Scholar). Crane suggested that NOS could in the processes of bacteria to a M. J. L. Y. Crane B.R. Sci. 2009; PubMed Scopus Google Scholar). Because NO is used by the a that NO is the molecule to a the In that bsNOS is to in NO and A. Stuehr D.J. J. J. PubMed Scopus Google Scholar). the the in of genuine nitric-oxide with mNOSs, the catalytic of has been the results so far between mNOSs and of are an electron of a I. M. Wang L. Stuehr D.J. E. J. Biol. Chem. 2008; 283: Full Text Full Text PDF PubMed Scopus Google Scholar) prevent an efficient first electron have been to NO Crane B.R. Stuehr D.J. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). has been so far for the of a in the electron natural is synthesized by these and BH4 and increase the of of the complex of several (16Adak S. Aulak K.S. Stuehr D.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Biochemistry. 2009; PubMed Scopus Google Scholar, M. K. Crane B.R. Stuehr D.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), no radical has been in so is the NO of the bsNOS Indeed, the in the seems to a NO M. K. Crane B.R. Stuehr D.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), which is for NO D.J. J. Wang Adak S. J. Biol. Chem. 2004; 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). Indeed, the NO by inducible NOS and into a NO Stuehr D.J. J. PubMed Scopus Google Scholar). the of by bsNOS, in with of the distal that to a of NO or other the catalytic which in could to reactions. these could the of oxygenase this mNOSs is to the oxygen activation described for cytochromes P450 M. Chem. Rev. PubMed Scopus Google Scholar, B. S. Chem. Rev. 2004; PubMed Scopus Google Scholar, I.G. I. Chem. Rev. PubMed Scopus Google Scholar, Rev. 2007; PubMed Scopus Google Scholar). It is characterized by processes that allow the bond the effects J. Biol. Chem. Scopus Google Scholar). effect the distal H-bond that the required for oxygen activation for NOS, resonance Raman of M. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. 2004; 87: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) and bsNOS M. J. 2007; PubMed Scopus Google Scholar, J. M. Stuehr D.J. Biochemistry. 2006; PubMed Scopus Google Scholar) complexes no in the distal of and mNOSs M. Stuehr D.J. J. Chem. 2007; PubMed Scopus Google Scholar, M. J. PubMed Scopus Google Scholar, B. Wang J. Stuehr D.J. Biochemistry. PubMed Scopus Google Scholar). have been between the structures of complexes of bsNOS K. Crane B.R. Biochemistry. 2006; PubMed Scopus Google Scholar) and neuronal NOS T. M. Biochemistry. 2009; PubMed Scopus Google Scholar, J. J. J. Biol. Chem. 2006; PubMed Scopus Google Scholar) in the of and l-Arg, the In the of the distal structure is mNOSs between and a in the We that the proximal bond for bsNOS for mNOSs, a modulation of the electron donation by the thiolate ligand J. M. Stuehr D.J. Biochemistry. 2006; PubMed Scopus Google Scholar). seems to be to Unlike the of NOS-proximal thiolate is in a H-bond interaction with the of the of the vicinal of this H-bond and the of this H-bond for the of the of S. Stuehr D.J. J. 2001; PubMed Scopus Google Scholar, S. Wang Stuehr D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of this H-bond, in the of the of iNOS, results in a oxygen activation process J. A. Wang C. S. Stuehr D.J. J. Biol. Chem. 2008; 283: Full Text Full Text PDF PubMed Scopus Google Scholar). are the same has been suggested by and J. S. M. J. 2009; PubMed Scopus Google Scholar), that the of the H-bond in to proximal a of the modulation of the interaction between the proximal ligand and the vicinal for We the effects of several mutations on the of bsNOS by using a combination of resonance and spectroscopies. Our results that the modulation of this interaction the electron donation of the thiolate ligand the electronic structure of the seems to indicate that the of NOS major and is controlled by the same proximal H-bond in bacterial and mammalian NOSs.