title: Regulatory role of Glu546 in flavin mononucleotide-heme electron transfer in human inducible nitric oxide synthase.
abstract: Nitric oxide (NO) production by mammalian NO synthase (NOS) is believed to be regulated by the docking of the flavin mononucleotide (FMN) domain in one subunit of the dimer onto the heme domain of the adjacent subunit. Glu546, a conserved charged surface residue of the FMN domain in human inducible NOS (iNOS), is proposed to participate in the interdomain FMN/heme interactions [Sempombe et al. Inorg. Chem.2011, 50, 6869-6861]. In the present work, we further investigated the role of the E546 residue in the FMN-heme interdomain electron transfer (IET), a catalytically essential step in the NOS enzymes. Laser flash photolysis was employed to directly measure the FMN-heme IET kinetics for the E546N mutant of human iNOS oxygenase/FMN (oxyFMN) construct. The temperature dependence of the IET kinetics was also measured over the temperature range of 283-304 K to determine changes in the IET activation parameters. The E546N mutation was found to retard the IET by significantly raising the activation entropic barrier. Moreover, pulsed electron paramagnetic resonance data showed that the geometry of the docked FMN/heme complex in the mutant is basically the same as in the wild type construct, whereas the probability of formation of such a complex is about twice lower. These results indicate that the retarded IET in the E546N mutant is not caused by an altered conformation of the docked FMN/heme complex, but by a lower population of the IET-active conformation. In addition, the negative activation entropy of the mutant is still substantially lower than that of the holoenzyme. This supports a mechanism by which the FMN domain can modify the IET through altering probability of the docked state formation.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico, Albuquerque, New Mexico 87131, United States.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Changyuan
authors.initials: C
authors.lastname: Lu
authors.forename: Bradley O
authors.initials: BO
authors.lastname: Elmore
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Astashkin
authors.forename: Denis L
authors.initials: DL
authors.lastname: Rousseau
authors.forename: Syun-Ru
authors.initials: SR
authors.lastname: Yeh
authors.forename: Changjian
authors.initials: C
authors.lastname: Feng
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman
chemical_list: D006418:Heme; D005486:Flavin Mononucleotide; D052247:Nitric Oxide Synthase Type II