title: Dissecting structural and electronic effects in inducible nitric oxide synthase.
abstract: Nitric oxide synthases (NOSs) are haem-thiolate enzymes that catalyse the conversion of L-arginine (L-Arg) into NO and citrulline. Inducible NOS (iNOS) is responsible for delivery of NO in response to stressors during inflammation. The catalytic performance of iNOS is proposed to rely mainly on the haem midpoint potential and the ability of the substrate L-Arg to provide a hydrogen bond for oxygen activation (O-O scission). We present a study of native iNOS compared with iNOS-mesohaem, and investigate the formation of a low-spin ferric haem-aquo or -hydroxo species (P) in iNOS mutant W188H substituted with mesohaem. iNOS-mesohaem and W188H-mesohaem were stable and dimeric, and presented substrate-binding affinities comparable to those of their native counterparts. Single turnover reactions catalysed by iNOSoxy with L-Arg (first reaction step) or N-hydroxy-L-arginine (second reaction step) showed that mesohaem substitution triggered higher rates of Fe(II)O₂ conversion and altered other key kinetic parameters. We elucidated the first crystal structure of a NOS substituted with mesohaem and found essentially identical features compared with the structure of iNOS carrying native haem. This facilitated the dissection of structural and electronic effects. Mesohaem substitution substantially reduced the build-up of species P in W188H iNOS during catalysis, thus increasing its proficiency towards NO synthesis. The marked structural similarities of iNOSoxy containing native haem or mesohaem indicate that the kinetic behaviour observed in mesohaem-substituted iNOS is most heavily influenced by electronic effects rather than structural alterations.
authors.affiliation: *Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, U.S.A.
authors.forename: Luciana
authors.initials: L
authors.lastname: Hannibal
authors.affiliation: ‡Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, U.S.A.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Page
authors.affiliation: *Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, U.S.A.
authors.forename: Mohammad Mahfuzul
authors.initials: MM
authors.lastname: Haque
authors.affiliation: *Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, U.S.A.
authors.forename: Karthik
authors.initials: K
authors.lastname: Bolisetty
authors.affiliation: *Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, U.S.A.
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Yu
authors.affiliation: ‡Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, U.S.A.
authors.forename: Saurav
authors.initials: S
authors.lastname: Misra
authors.affiliation: *Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, U.S.A.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D055162:Biocatalysis; D019281:Dimerization; D004795:Enzyme Stability; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D006900:Hydroxylation; D007700:Kinetics; D008652:Mesoporphyrins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D008652:Mesoporphyrins; D050505:Mutant Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; C031468:mesoporphyrin IX; D001120:Arginine; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse