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Calcium binding sites of calmodulin and electron transfer by neuronal nitric oxide synthase

Academic Article
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Overview

authors

  • Stevens-Truss, R.
  • Beckingham, K.
  • Marletta, Michael

publication date

  • 1997

journal

  • Biochemistry  Journal

abstract

  • Although binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) has been demonstrated to act as the trigger necessary for electron transfer and catalytic activity, the exact manner in which this is achieved is unclear. By using a series of single point mutants of Drosophila melanogaster CaM, the role that each Ca2+ binding site plays in the transfer of electrons within nNOS has been examined. In these mutants, the bidentate glutamic acid (E) residue which coordinates Ca2+ at the -Z position in each site has been mutated to a glutamine (Q), preventing Ca2+ binding at that site. The results demonstrate that Ca2+ binding at site I of CaM is critical for all electron transfer reactions. All nNOS activities measured (citrulline formation, NADPH oxidation, and cytochrome c reduction) in the presence of the site I CaM mutant (denoted B1Q) were only 2% of the nNOS activity with wild-type CaM. The B2Q and B4Q mutants activated nNOS to similar levels. These two mutants, however, affected nNOS heme-dependent activities to a greater extent than they affected activities independent of the nNOS heme. The site III CaM mutant (B3Q) activated nNOS to levels similar to activities measured with wild-type CaM. Rates of formation of the ferrous-CO complex were also obtained with each of the mutant CaMs. The relative binding affinities of these mutants do not correlate with the observed differences in electron transfer rates. These results demonstrate that, although binding of CaM to nNOS is necessary for catalysis, specific interactions between the two proteins exist which are required for efficient electron transfer.

subject areas

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Calcium
  • Calmodulin
  • Drosophila
  • Electron Transport
  • Ferrous Compounds
  • Molecular Sequence Data
  • Neurons
  • Nitric Oxide Synthase
  • Point Mutation
  • Protein Binding
  • Spectrophotometry
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Identity

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi970973k

PubMed ID

  • 9315874
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Additional Document Info

start page

  • 12337

end page

  • 12345

volume

  • 36

issue

  • 40

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