Lys842 in Neuronal Nitric-oxide Synthase Enables the Autoinhibitory Insert to Antagonize Calmodulin Binding, Increase FMN Shielding, and Suppress Interflavin Electron Transfer*

  1. Dennis J. Stuehr‡,2
  1. From the ‡Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195,
  2. the §Department of Chemistry, Southern Illinois University, Edwardsville, Illinois 62026,
  3. the Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250, and
  4. the ‖Department of Molecular Biology, Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037
  1. 2 To whom correspondence should be addressed: Dept. of Pathobiology, NC-22, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195; Tel.: 216-445-6950; Fax: 216-636-0104; E-mail: stuehrd{at}ccf.org.

  1. ↵1 Both of these authors contributed equally to this work.

Abstract

Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOS reductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear. A conserved charged residue (Lys842) lies within a putative CaM binding helix in the middle of the AI. We investigated its role by substituting residues that neutralize (Ala) or reverse (Glu) the charge at Lys842. Compared with wild type nNOS, the mutant enzymes had greater cytochrome c reductase and NADPH oxidase activities in the CaM-free state, were able to bind CaM at lower calcium concentration, and had lower rates of heme reduction and NO synthesis in one case (K842A). Moreover, stopped-flow spectrophotometric experiments with the nNOS reductase domain indicate that the CaM-free mutants had faster flavin reduction kinetics and had less shielding of their FMN subdomains compared with wild type and no longer increased their level of FMN shielding in response to NADPH binding. Thus, Lys842 is critical for the known functions of the AI and also enables two additional functions of the AI as newly identified here: suppression of electron transfer to FMN and control of the conformational equilibrium of the nNOS reductase domain. Its effect on the conformational equilibrium probably explains suppression of catalysis by the AI.

Footnotes

  • ↵* This work was supported, in whole or in part, by National Institutes of Health Grants GM51491, CA53914, and HL76491 (to D. J. S.) and HL58883 (to E. D. G.).

  • ↵Graphic The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S5.

  • ↵4 The electron distributions and extent of electron transfer at each step are also influenced by the relative midpoint potentials of NADPH and the flavin redox couples.

  • ↵5 We used a shorter time frame for the absorbance data collection in the experiments with K842E nNOSred in order to better catch the faster transitions that occur in this mutant.

  • ↵3 The abbreviations used are:

    NOS
    nitric-oxide synthase
    NO
    nitric oxide
    nNOS
    neuronal NOS
    eNOS
    endothelial NOS
    nNOSred
    reductase domain of nNOS
    CaM
    calmodulin
    CT
    C-terminal tail
    AI
    autoinhibitory insert
    FMNsq
    FMN semiquinone
    FADsq
    FAD semiquinone
    EPPS
    4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid
    WT
    wild type.

    • Received March 30, 2009.
    • Revision received November 24, 2009.
View this article with LENS
Table of Contents

This Article

  1. First Published on November 30, 2009 doi: 10.1074/jbc.M109.000810 The Journal of Biological Chemistry 285, 3064-3075.
  1. » Abstract(Free)
  2. Full Text(Free)
  3. PDF(Free)
  4. PDF including Supp Data
  5. Supplemental Data
  6. All Versions of this Article:
    1. M109.000810v1
    2. 285/5/3064 (most recent)

Article Usage Stats

  1. Article Usage Statistics

Submit your work to JBC.

You'll be in good company.