Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase

M A Noble, A W Munro, S L Rivers, L Robledo, S N Daff, L J Yellowlees, T Shimizu, I Sagami, J G Guillemette, S K Chapman, Simon Daff

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Abstract

Midpoint reduction potentials for the flavin cofactors in the reductase domain of rat neuronal nitric oxide synthase (nNOS) in calmodulin (CaM)-free and -bound forms have been determined by direct anaerobic titration. In the CaM-free form, the FMN potentials are -49 +/- 5 mV (oxidized/semiquinone) -274 +/- 5 mV (semiquinone/reduced:). The corresponding FAD potentials are -232 +/- 7, and -280 +/- 6 mV. The data indicate that each flavin can exist as a blue (neutral) semiquinone. The accumulation of blue semiquinone on the FMN is considerably higher than seen on the FAD due to the much larger separation (225 mV) of its two potentials (cf. 48 mV for FAD). For the CaM-bound form of the protein, the midpoint potentials are essentially identical: there is a small alteration in the FMN oxidized/semiquinone potential (-30 +/- 4 mV); the other three potentials are unaffected. The heme midpoint potentials for nNOS [-239 mV, L-Arg-free; -220 mV, L-Arg-bound; Presta, A., Weber-Main, A. M., Stankovich, M. T., and Stuehr, D. J. (1998) J. Am. Chern. Sec. 120, 9460-9465] are poised such that electron transfer from flavin domain is thermodynamically feasible. Clearly, CaM binding is necessary in eliciting conformational changes that enhance flavin to flavin and flavin to heme electron transfers rather than causing a change in the driving force.

Original languageEnglish
Pages (from-to)16413-16418
Number of pages6
JournalBiochemistry
Volume38
Issue number50
Publication statusPublished - 14 Dec 1999

Keywords

  • HYDROXY-L-ARGININE
  • ELECTRON-TRANSFER
  • ADENINE-DINUCLEOTIDE
  • REDUCTASE DOMAIN
  • REDOX PROPERTIES
  • HEME
  • CALMODULIN
  • MECHANISM
  • BINDING
  • BRAIN

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