title: Role of a Conserved Tyrosine Residue in the FMN-Heme Interdomain Electron Transfer in Inducible Nitric Oxide Synthase.
abstract: The interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme domains is essential in the biosynthesis of nitric oxide (NO) by the NO synthase (NOS) enzymes. A conserved tyrosine residue in the FMN domain (Y631 in human inducible NOS) was proposed to be a key part of the electron transfer pathway in the FMN/heme docked complex model. In the present study, the FMN-heme IET kinetics in the Y631F mutant and wild type of a bidomain oxygenase/FMN construct of human inducible NOS were determined by laser flash photolysis. The rate constant of the Y631F mutant is significantly decreased by ∼75% (compared to the wild type), showing that the tyrosine residue indeed facilitates the FMN-heme IET through the protein medium. The IET rate constant of the wild type protein decreases from 345 to 242 s-1on going from H2O to 95% D2O, giving a solvent kinetic isotope effect of 1.4. In contrast, no deuterium isotope effect was observed for the Tyr-to-Phe mutant. Moreover, an appreciable change in the wild type iNOS IET rate constant value was observed upon changing pH. These results indicate that the FMN-heme IET is proton coupled, in which the conserved tyrosine residue may play an important role.
authors.affiliation: College of Pharmacy, University of New Mexico , Albuquerque, New Mexico 87131, United States.
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.affiliation: College of Pharmacy, University of New Mexico , Albuquerque, New Mexico 87131, United States.|Department of Chemistry and Chemical Biology, University of New Mexico , Albuquerque, New Mexico 87131, United States.
authors.forename: Huayu
authors.initials: H
authors.lastname: Zheng
authors.affiliation: College of Pharmacy, University of New Mexico , Albuquerque, New Mexico 87131, United States.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Li
authors.affiliation: College of Pharmacy, University of New Mexico , Albuquerque, New Mexico 87131, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Radiology, University of Colorado Denver , Denver, Colorado 80045, United States.
authors.forename: Yubin
authors.initials: Y
authors.lastname: Miao
authors.affiliation: College of Pharmacy, University of New Mexico , Albuquerque, New Mexico 87131, United States.|Department of Chemistry and Chemical Biology, University of New Mexico , Albuquerque, New Mexico 87131, United States.
authors.forename: Changjian
authors.initials: C
authors.lastname: Feng
mesh_terms: D003903:Deuterium / Q000737:chemistry; D004579:Electron Transport; D006418:Heme / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007834:Lasers; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D012997:Solvents / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: D012997:Solvents; D014443:Tyrosine; D006418:Heme; D003903:Deuterium; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II