title: Comparison of oxygen-induced radical intermediates in iNOS oxygenase domain with those from nNOS and eNOS.
abstract: Inducible nitric-oxide synthase (iNOS) produces the reactive oxygen and nitrogen species (ROS/RNS) involved in bacteria killing and is crucial in the host defense mechanism. However, high level ROS/RNS can also be detrimental to normal cells and thus their production has to be tightly controlled. Availability or deficiency of tetrahydrobiopterin (BH4) cofactor and l-arginine substrate controls coupling or uncoupling of NOS catalysis. Fully coupled reaction, with abundant BH4 and l-arginine, produces NO whereas the uncoupled NOS (in the absence of BH4 and/or l-arginine) generates ROS/RNS. In the current work we focus on direct rapid freeze EPR to characterize the structure and kinetics of oxygen-induced radical intermediates produced by ferrous inducible NOS oxygenase domain (iNOSox) in the presence or absence of BH4 and/or l-arginine. Fully reconstituted iNOSox (+BH4, +L-Arg) forms a dimer and yields a typical BH4 radical that indicates coupled reaction. iNOSox (-BH4) remains mainly monomeric and produces exclusively superoxide, that is only marginally affected by the presence of l-arginine. iNOSox (+BH4, -L-Arg) exists as a monomer/dimer mixture and yields both BH4 radical and superoxide. Present study is a natural extension of our previous work on the ferrous endothelial NOSox (eNOSox) [V. Berka, G. Wu, H.C. Yeh, G. Palmer, A.L. Tsai, J. Biol. Chem. 279 (2004) 32243-32251] and ferrous neuronal NOSox (nNOSox) [V. Berka, L.H. Wang, A.L. Tsai, Biochemistry 47 (2008) 405-420]. Overall, our data suggests different regulatory roles of l-arginine and BH4 in the production of oxygen-induced radical intermediates in NOS isoforms which nicely serve individual functional role.
authors.affiliation: Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, United States. Electronic address: Vladimir.Berka@uth.tmc.edu.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Berka
authors.affiliation: Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, United States.
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.affiliation: Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, United States.
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
authors.affiliation: Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030, United States. Electronic address: Ah-Lim.Tsai@uth.tmc.edu.
authors.forename: Ah-Lim
authors.initials: AL
authors.lastname: Tsai
mesh_terms: D020134:Catalytic Domain; D005609:Free Radicals / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry*; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry*; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D013481:Superoxides / Q000737:chemistry
chemical_list: D005609:Free Radicals; D013481:Superoxides; D006418:Heme; D006861:Hydrogen Peroxide; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen
keywords: NOS oxygenase domain; Nitric oxide synthase; Oxygen induced radicals; Rapid-freeze quench EPR; Reactive oxygen species; Superoxide