title: Role of an isoform-specific serine residue in FMN-heme electron transfer in inducible nitric oxide synthase.
abstract: In the crystal structure of a calmodulin (CaM)-bound FMN domain of human inducible nitric oxide synthase (NOS), the CaM-binding region together with CaM forms a hinge, and pivots on an R536(NOS)/E47(CaM) pair (Xia et al. J Biol Chem 284:30708-30717, 2009). Notably, isoform-specific human inducible NOS S562 and C563 residues form hydrogen bonds with the R536 residue through their backbone oxygens. In this study, we investigated the roles of the S562 and C563 residues in the NOS FMN-heme interdomain electron transfer (IET), the rates of which can be used to probe the interdomain FMN/heme alignment. Human inducible NOS S562K and C563R mutants of an oxygenase/FMN (oxyFMN) construct were made by introducing charged residues at these sites as found in human neuronal NOS and endothelial NOS isoforms, respectively. The IET rate constant of the S562K mutant is notably decreased by one third, and its flavin fluorescence intensity per micromole per liter is diminished by approximately 24 %. These results suggest that a positive charge at position 562 destabilizes the hydrogen-bond-mediated NOS/CaM alignment, resulting in slower FMN-heme IET in the mutant. On the other hand, the IET rate constant of the C563R mutant is similar to that of the wild-type, indicating that the mutational effect is site-specific. Moreover, the human inducible NOS oxyFMN R536E mutant was constructed to disrupt the bridging CaM/NOS interaction, and its FMN-heme IET rate was decreased by 96 %. These results demonstrated a new role of the isoform-specific serine residue of the key CaM/FMN(NOS) bridging site in regulating the FMN-heme IET (possibly by tuning the alignment of the FMN and heme domains).
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico, Albuquerque, NM 87131, USA.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Li
authors.forename: Weihong
authors.initials: W
authors.lastname: Fan
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Bradley O
authors.initials: BO
authors.lastname: Elmore
authors.forename: Mike
authors.initials: M
authors.lastname: Piazza
authors.forename: J Guy
authors.initials: JG
authors.lastname: Guillemette
authors.forename: Changjian
authors.initials: C
authors.lastname: Feng
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence
chemical_list: D002147:Calmodulin; D020033:Protein Isoforms; D006418:Heme; D005486:Flavin Mononucleotide; D052247:Nitric Oxide Synthase Type II