Update on Mechanism and Catalytic Regulation in the NO Synthases

Nitric-oxide synthases (NOSs, EC 1.14.13.39) 1The abbreviations used are: NOS, nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; NOHA, Nω-hydroxy-l-arginine; H4B, (6R)-5,6,7,8-tetrahydro-l-biopterin. oxidize l-arginine to nitric oxide (NO) and are interesting for several reasons. They are present in many life forms (1Ghosh D.K. Salerno J.C. Front. Biosci. 2003; 8: 193-209Crossref PubMed Scopus (120) Google Scholar, 2Guo F.Q. Okamoto M. Crawford N.M. Science. 2003; 302: 100-103Crossref PubMed Scopus (721) Google Scholar), their gene regulation is complex (3Newton D.C. Bevan S.C. Choi S. Robb G.B. Millar A. Wang Y. Marsden P.A. J. Biol. Chem. 2003; 278: 636-644Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), they are the only flavoheme enzymes that utilize tetrahydrobiopterin (H4B) as a redox cofactor, and their electron transfer reactions are regulated by a Ca2+-binding protein (calmodulin). In the past 5 years, crystal structures of NOS heme (oxygenase) domains and bacterial NOS-like proteins have shown how Arg, heme, and H4B bind in the active site (4Crane 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, 5Bird L.E. Ren J. Zhang J. Foxwell N. Hawkins A.R. Charles I.G. Stammers D.K. Structure (Lond.). 2002; 10: 1687-1696Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Reviews are available on NOS biochemistry (6Mansuy D. Boucher J.L. Drug Metab. Rev. 2002; 34: 593-606Crossref PubMed Scopus (30) Google Scholar), regulation (7Li H. Wallerath T. Munzel T. Forstermann U. Nitric Oxide. 2002; 7: 149-164Crossref PubMed Scopus (188) Google Scholar, 8Roman L.J. Martasek P. Masters B.S. Chem. Rev. 2002; 102: 1179-1190Crossref PubMed Scopus (177) Google Scholar), protein-protein interactions (9Nedvetsky P.I. Sessa W.C. Schmidt H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16510-16512Crossref PubMed Scopus (66) Google Scholar), and post-translational modifications (10Greif D.M. Kou R. Michel T. Biochemistry. 2002; 41: 15845-15853Crossref PubMed Scopus (91) Google Scholar). This minireview updates the NO biosynthetic mechanism and describes a global catalytic model that highlights the role of NO as an intrinsic regulator. NOS is one of few heme-containing enzymes that make NO (11Rudolf M. Einsle O. Neese F. Kroneck P.M. Biochem. Soc. Trans. 2002; 30: 649-653Crossref PubMed Google Scholar, 12Hendrich M.P. Upadhyay A.K. Riga J. Arciero D.M. Hooper A.B. Biochemistry. 2002; 41: 4603-4611Crossref PubMed Scopus (34) Google Scholar). NOS hydroxylates a guanidino nitrogen of Arg and then oxidizes the Nω-hydroxy-l-arginine intermediate (NOHA) to NO and l-citrulline (Scheme 1). The NOS flavoprotein domain first provides an electron (derived from NADPH) to the ferric heme (Fig. 1). This is the slowest step of the biosynthetic reaction and enables formation of a ferric heme-superoxy species (I) in the Arg or NOHA reactions (13Wei C-C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 14Ledbetter A.P. McMillan K. Roman L.J. Masters B.S. Dawson J.H. Sono M. Biochemistry. 1999; 38: 8014-8021Crossref PubMed Scopus (56) Google Scholar). Species I is not reactive toward Arg but may (15Huang H. Hah J.M. Silverman R.B. J. Am. Chem. Soc. 2001; 123: 2674-2676Crossref PubMed Scopus (63) Google Scholar) or may not (13Wei C-C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) be reactive toward NOHA. Rates for many of the individual binding and electron transfer steps are known (16Santolini J. Adak S. Curran C.M.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Species I can receive an electron from H4B (18Wei C.C. Wang Z.Q. Arvai A.S. Hemann C. Hille R. Getzoff E.D. Stuehr D.J. Biochemistry. 2003; 42: 1969-1977Crossref PubMed Scopus (54) Google Scholar) or from the flavoprotein domain when H4B is absent (19Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (169) Google Scholar). The H4B electron transfer is the second slowest step in the biosynthetic reaction. Its kinetics is influenced by surrounding protein residues and by the pterin structure itself (18Wei C.C. Wang Z.Q. Arvai A.S. Hemann C. Hille R. Getzoff E.D. Stuehr D.J. Biochemistry. 2003; 42: 1969-1977Crossref PubMed Scopus (54) Google Scholar, 20Wang Z.Q. Wei C.-C. Stuehr D.J. J. Biol. Chem. 2002; 277: 12830-12837Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Timely electron transfer from H4B prevents superoxide release (Fig. 1). H4B may also donate an electron in the NOHA reaction (13Wei C-C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 21Hurshman A.R. Krebs C. Edmondson D.E. Marletta M.A. Biochemistry. 2003; 42: 13287-13303Crossref PubMed Scopus (52) Google Scholar), and in that case the radical is reduced back to H4B by a downstream reaction intermediate. Further discussion of H4B redox function in NOS is available (22Sorlie M. Gorren A.C. Marchal S. Shimizu T. Lange R. Andersson K.K. Mayer B. J. Biol. Chem. 2003; 278: 48602-48610Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 23Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (167) Google Scholar). The heme-peroxo species (II) has only been observed in NOS at cryogenic temperature (24Davydov R. Ledbetter-Rogers A. Martasek P. Larukhin M. Sono M. Dawson J.H. Masters B.S. Hoffman B.M. Biochemistry. 2002; 41: 10375-10381Crossref PubMed Scopus (108) Google Scholar). Species II may become protonated and lose water to form a heme iron-oxo species (III) that hydroxylates Arg or may react directly with NOHA (Fig. 1). The reactivity of NOS species III or a related species has been studied (25Porasuphatana S. Tsai P. Pou S. Rosen G.M. Biochim. Biophys. Acta. 2001; 1526: 95-104Crossref PubMed Scopus (12) Google Scholar). Importantly, the first observed product of NOS catalysis is a ferric heme-NO complex and not free NO (13Wei C-C. Wang Z.Q. Hemann C. Hille R. Stuehr D.J. J. Biol. Chem. 2003; 278: 46668-46673Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 26Negrerie M. Berka V. Vos M.H. Liebl U. Lambry J.C. Tsai A.L. Martin J.L. J. Biol. Chem. 1999; 274: 24694-24702Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 27Scheele J.S. Bruner E. Kharitonov V.G. Martasek P. Roman L.J. Masters B.S. Sharma V.S. Magde D. J. Biol. Chem. 1999; 274: 13105-13110Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) (Fig. 1).Fig. 1Model for NO biosynthesis. See text for details.View Large Image Figure ViewerDownload (PPT) As noted, practically all NO binds to the NOS ferric heme before exiting the enzyme. In isolation, this is just an example of how product release can limit enzyme catalysis. However, the attached flavoprotein domain of NOS provides an alternative path for the ferric heme-NO complex to return to the initial ferric state (Fig. 2A). The flavoprotein can reduce the ferric heme-NO complex to the ferrous heme-NO species, which releases NO very slowly (17Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 27Scheele J.S. Bruner E. Kharitonov V.G. Martasek P. Roman L.J. Masters B.S. Sharma V.S. Magde D. J. Biol. Chem. 1999; 274: 13105-13110Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and so reacts instead with O2 to regenerate ferric enzyme. Consequently after NO biosynthesis is finished two different cycles compete; NO dissociation from the ferric heme (kd) is part of a “productive cycle” that releases NO and is essential for NOS bioactivity. Conversely, reduction of the ferric heme-NO complex (kr′) channels the enzyme into a “futile cycle” that ultimately generates nitrate in place of NO. NOS futile cycling is also influenced by the rate at which O2 reacts with the ferrous heme-NO species (kox in Fig. 2). Together, the productive and futile cycles create a global kinetic mechanism for NOS catalysis. Thus, to synthesize NO is good but not sufficient; a NOS must also control partitioning between both cycles by balancing heme reduction (kr, kr′) 2kr and kr′ were found to be equivalent (16Santolini J. Adak S. Curran C.M.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). and NO dissociation (kd) if it is to release the NO that it makes. Computer simulations of a more detailed global kinetic mechanism 3The global kinetic model can be run on a desktop computer using programs like Mathcad. The detailed model is available free from the authors. have been run using individual rate measurements available in the literature (16Santolini J. Adak S. Curran C.M.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and these accurately model the pre-steady-state and steady-state behaviors of the mammalian endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) NOS isozymes. Some fundamental concepts are described below. Each NOS Distributes Differently during Catalysis—Values of the three kinetic parameters (kr, kox, kd) differ significantly among NOSs (Table I), and this causes each NOS to distribute differently during steady-state NO synthesis. The enzyme distributions in Fig. 2B were derived from computer simulations of the global kinetic mechanism and mimic distributions estimated from actual experiments (17Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 28Abu-Soud H.M. Ichimori K. Presta A. Stuehr D.J. J. Biol. Chem. 2000; 275: 17349-17357Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 29Abu-Soud H.M. Ichimori K. Nakazawa H. Stuehr D.J. Biochemistry. 2001; 40: 6876-6881Crossref PubMed Scopus (64) Google Scholar). NOSs distribute into five main forms during steady-state NO synthesis, namely the ferric, ferrous, ferrous-O2 (or ferric-superoxy), ferric-NO, and ferrous-NO forms. For nNOS, a fast kr, kr′ relative to kd and kox causes it to exist predominantly as a ferrous-NO species. For eNOS the situation is reversed; a slow kr relative to kd or kox causes it to exist predominantly as a ferric species. For iNOS, its moderately fast kr and fast kox create an enzyme distribution that is between the two extremes.Table IKinetic values for NOSs and selected heme proteinsProteinkrkdkoxApparent KmO2Refs.s-1s-1s-1μmnNOS3-450.235016Santolini J. Adak S. Curran C.M.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 30Abu-Soud H.M. Wang J. Rousseau D.L. Fukuto J.M. Ignarro L.J. Stuehr D.J. J. Biol. Chem. 1995; 270: 22997-23006Abstract Full Text Full Text PDF PubMed Scopus (201) Google ScholariNOS0.9-1.52313017Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 29Abu-Soud H.M. Ichimori K. Nakazawa H. Stuehr D.J. Biochemistry. 2001; 40: 6876-6881Crossref PubMed Scopus (64) Google ScholareNOS0.130.6417Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 28Abu-Soud H.M. Ichimori K. Presta A. Stuehr D.J. J. Biol. Chem. 2000; 275: 17349-17357Abstract Full Text Full Text PDF PubMed Scopus (115) Google ScholarW409F nNOS1.851.3NA16Santolini J. Adak S. Curran C.M.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 32Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 17434-17439Abstract Full Text Full Text PDF PubMed Scopus (54) Google ScholarS1412D nNOS5.45NA738aCalculated from a simulation of the global kinetic model.16Santolini J. Adak S. Curran C.M.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 31Adak S. Santolini J. Tikunova S. Wang Q. Johnson J.D. Stuehr D.J. J. Biol. Chem. 2001; 276: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (105) Google ScholarCyt P450BM399 (15 °C)NANANA42Ost T.W. Clark J. Mowat C.G. Miles C.S. Walkinshaw M.D. Reid G.A. Chapman S.K. Daff S. J. Am. Chem. Soc. 2003; 125: 15010-15020Crossref PubMed Scopus (98) Google ScholarMammalian Cyt P450NANANA4-10 (10 °C)43Jones D.P. Mason H.S. J. Biol. Chem. 1978; 253: 4874-4880Abstract Full Text PDF PubMed Google ScholarFlavohemoglobin150 (37 °C)200-4000 (20 °C)12 (10 °C)60-90 (20 °C)44Gardner A.M. Martin L.A. Gardner P.R. Dou Y. Olson J.S. J. Biol. Chem. 2000; 275: 12581-12589Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 45Gardner P.R. Gardner A.M. Martin L.A. Dou Y. Li T. Olson J.S. Zhu H. Riggs A.F. J. Biol. Chem. 2000; 275: 31581-31587Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 46Hausladen A. Gow A. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10108-10112Crossref PubMed Scopus (141) Google ScholarMyoglobinNA4010-3 (37 °C)NA47Cooper C.E. Biochim. Biophys. Acta. 1999; 1411: 290-309Crossref PubMed Scopus (463) Google Scholar, 48Moller J.K. Skibsted L.H. Chem. Rev. 2002; 102: 1167-1178Crossref PubMed Scopus (193) Google Scholara Calculated from a simulation of the global kinetic model. Open table in a new tab Knowing the distribution pattern helps to understand NOS catalytic behavior. For example, the specific activities of the three mammalian NOSs are of rank order iNOS ≥ nNOS > eNOS, with nNOS activity being 4 times that of eNOS. However, if one considers their actual rates of NO biosynthesis (the speed at which each NOS makes one NO) it is clear from their kr values (Table I) that nNOS is at least twice as fast as iNOS and about 30 times faster than eNOS. This discrepancy is explained by the global kinetic model and the different enzyme distribution pattern of each NOS in the steady state (17Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). NOSs Have an Optimal Rate of Heme Reduction—With sufficient substrates the rate of NO biosynthesis is simply limited by kr (Fig. 1). However, the relationship between kr and the rate of NO release is modified by ferric heme-NO complex formation at the end of each catalytic event. The global kinetic model predicts that NOS will increase its NO release as a function of kr, but at a certain point the NO release will reach a maximum and then fall despite the enzyme working faster and faster (Fig. 3). The bell-shaped curves can be rationalized by considering how kr impacts the ferric heme-NO product complex. Increasing kr speeds its formation but also partitions more of it into the futile cycle (kr′), which NO release the productive and futile cycling and nitrate (Fig. 3). The relationship between kr and the rate of release is also influenced by the kd and kox values of each The of each NOS on its is by an in Fig. that nNOS has a kr, eNOS and iNOS have The of NO to the NOS heme at NO This binding from the reaction of NO with the ferric heme in the heme NO binding is when the ferric form of a NOS during steady-state catalysis for eNOS and In this NO binding the of the ferric heme-NO species and the enzyme distribution pattern (Fig. to of the ferrous heme-NO species and so more NOS the futile which the NO release Thus, NOS activity can differ when it is in the or of an NO The of NO has been for the three NOS H.M. Ichimori K. Presta A. Stuehr D.J. J. Biol. Chem. 2000; 275: 17349-17357Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 29Abu-Soud H.M. Ichimori K. Nakazawa H. Stuehr D.J. Biochemistry. 2001; 40: 6876-6881Crossref PubMed Scopus (64) Google Scholar, 30Abu-Soud H.M. Wang J. Rousseau D.L. Fukuto J.M. Ignarro L.J. Stuehr D.J. J. Biol. Chem. 1995; 270: 22997-23006Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), and interesting can For example, at about NO iNOS at and NO (Fig. This enables iNOS to a steady NO in the it release of NO (17Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 29Abu-Soud H.M. Ichimori K. Nakazawa H. Stuehr D.J. Biochemistry. 2001; 40: 6876-6881Crossref PubMed Scopus (64) Google Scholar). NO the fast kox of iNOS enables it to be an NO at the of its NO biosynthesis activity (Fig. NOS activity on of kr, kox, and this in two of NOS catalytic that only be in the of the global kinetic model and the three kinetic the of eNOS and nNOS a for in their L.J. Martasek P. Masters B.S. Chem. Rev. 2002; 102: 1179-1190Crossref PubMed Scopus (177) Google Scholar, P.I. Sessa W.C. Schmidt H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16510-16512Crossref PubMed Scopus (66) Google Scholar). at this (or point to a increase in eNOS However, point in nNOS its NO release rate by S. Santolini J. Tikunova S. Wang Q. Johnson J.D. Stuehr D.J. J. Biol. Chem. 2001; 276: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). that nNOS has a faster kr than (Table I) and a faster NO biosynthesis. were used to slow kr in This its NO biosynthesis but its NO release rate S. Santolini J. Tikunova S. Wang Q. Johnson J.D. Stuehr D.J. J. Biol. Chem. 2001; 276: 1244-1252Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). This the kr in Conversely, kr in eNOS by the mechanism increase its NO release its kr is (Fig. 3). the of NOSs a forms a with the heme (Fig. or for in nNOS enzymes NO release rates were times than nNOS S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 17434-17439Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The have a kr with but have a faster kox (Table this makes their NO biosynthesis than the kox causes enzyme to the futile cycle during the steady state (Fig. This on enzyme distribution their kr and enables the to have NO release rates than NOSs Have Heme flavoheme enzymes have faster kr values with the NOSs (Table I), that NOSs are a slow kr is kr will more NOS into the futile cycle and ultimately it into an NO (Fig. 3). However, the slow kr makes it for NOS to to (19Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (169) Google Scholar, S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). This is of the second electron to the heme must be for the enzyme to the species that will react with Arg or NOHA before of species I (Fig. 1). NOS this by H4B as a of the second H4B the second electron about times faster than can the NOS and this is sufficient to superoxide release from the heme and so Z.Q. Wei C.-C. Stuehr D.J. J. Biol. Chem. 2002; 277: 12830-12837Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 23Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (167) Google Scholar). Thus, heme-NO binding a kinetic on NOS heme reduction that impacts its NOS this by using two of a slow electron transfer from the flavoprotein to ferrous heme-NO formation and futile and a fast reduction by H4B at the step in its The O2 of values differ among the NOSs and in are than for related (Table This is NOS with O2 in two the ferrous enzyme binds O2 during NO and the ferrous heme-NO species reacts with O2 in the futile cycle interactions to the The of kox if the ferrous heme-NO species in the steady This situation for nNOS but not for eNOS, and so the of eNOS is and the O2 of its NO biosynthetic reaction. The of nNOS for nNOS activity in and M.A. PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar), it is intrinsic to the enzyme. NO binding to the ferric heme also the of eNOS and iNOS H.M. Ichimori K. Presta A. Stuehr D.J. J. Biol. Chem. 2000; 275: 17349-17357Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 29Abu-Soud H.M. Ichimori K. Nakazawa H. Stuehr D.J. Biochemistry. 2001; 40: 6876-6881Crossref PubMed Scopus (64) Google Scholar). may be for eNOS to increase a of iNOS how it may in the T.W. Stuehr D.J. S.C. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). to NO are to increase their activities by different For example, kr only makes for eNOS and iNOS they have a eNOS like and (9Nedvetsky P.I. Sessa W.C. Schmidt H.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16510-16512Crossref PubMed Scopus (66) Google Scholar, D.M. Kou R. Michel T. Biochemistry. 2002; 41: 15845-15853Crossref PubMed Scopus (91) Google Scholar) function this For nNOS a be to increase its kox, as by the nNOS exist to so in can be are in of NO biosynthesis. For example, the species that react with Arg or NOHA have not been This will that the rate of the second electron transfer be the reactive species be using for A.R. Crane B.R. Dawson J.H. 2002; PubMed Scopus Google Scholar, D.E. Biochemistry. PubMed Scopus Google Scholar). The global kinetic mechanism its values for the three kinetic parameters differ among NOSs the the kinetic parameters of NOSs to futile cycling instead of NO NOSs may be this Z.Q. Wei C.C. Sharma M. K. Crane B.R. Stuehr D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is the of the three kinetic only that the NOS flavoprotein kr S. Stuehr D. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, P.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) the domain kox and kd (17Santolini J. Meade A.L. Stuehr D.J. J. Biol. Chem. 2001; 276: 48887-48898Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The in kox and kd values among heme proteins (Table I) that interesting to be NOS function in and may also be to its productive and futile catalytic as the of the futile cycle product on the mechanism of the ferrous heme-NO complex and be the will be interesting to how NOS product which to the and kinetic parameters of a NOS, with its