Update on Mechanism and Catalytic Regulation in the NO Synthases

Abstract

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 tran