Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase

Michael A Noble, Andrew W Munro, Stuart L Rivers, Laura Robledo, Simon N Daff, Lesley J Yellowlees, Toru Shimizu, Ikuko Sagami, J Guy Guillemette, Stephen K Chapman

    Research output: Contribution to journalArticlepeer-review

    117 Citations (Scopus)

    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
    DOIs
    Publication statusPublished - 14 Dec 1999

    Keywords

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

    Fingerprint

    Dive into the research topics of 'Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase'. Together they form a unique fingerprint.

    Cite this