JBIC Journal of Biological Inorganic Chemistry

, Volume 14, Issue 1, pp 133–142 | Cite as

Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme

  • Changjian FengEmail author
  • Andrea L. Dupont
  • Nickolas J. Nahm
  • Donald E. Spratt
  • James T. Hazzard
  • J. Brice Weinberg
  • J. Guy Guillemette
  • Gordon TollinEmail author
  • Dipak K. GhoshEmail author
Original Paper


Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in NO synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN–heme IET in a truncated two-domain construct (oxyFMN) of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present (Feng et al. J. Am. Chem. Soc. 128, 3808–3811, 2006). Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme, and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 ± 5 and 35 ± 3 s−1, respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in neuronal NOS.


Electron transfer Nitric oxide synthase Laser flash photolysis Heme Flavin 




bis-Tris propane







5-Deazariboflavin semiquinone


Endothelial NO synthase


Flavin adenine dinucleotide


Flavin mononucleotide


Flavin mononucleotide semiquinone


Flavin mononucleotide hydroquinone




Intraprotein electron transfer


Inducible NO synthase


Inducible NO synthase oxygenase construct


Neuronal NO synthase


NO synthase


Two-domain NO synthase construct in which only the heme-containing oxygenase and flavin mononucleotide domains along with the calmodulin binding site are present



We thank John C. Salerno for helpful discussions. This work is funded by the NIH (GM081811 and HL091280 to C.F.), the PhRMA Foundation, and UNM HSC RAC grant (to C.F.), and grant 183521 to J.G.G. from the Natural Sciences and Engineering Research Council of Canada.

Supplementary material

775_2008_431_MOESM1_ESM.pdf (2.2 mb)
Supporting information (PDF 2232 kb)


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Copyright information

© SBIC 2008

Authors and Affiliations

  • Changjian Feng
    • 1
    Email author
  • Andrea L. Dupont
    • 2
  • Nickolas J. Nahm
    • 3
  • Donald E. Spratt
    • 2
  • James T. Hazzard
    • 4
  • J. Brice Weinberg
    • 3
  • J. Guy Guillemette
    • 2
  • Gordon Tollin
    • 4
    Email author
  • Dipak K. Ghosh
    • 3
    Email author
  1. 1.College of PharmacyUniversity of New MexicoAlbuquerqueUSA
  2. 2.Department of ChemistryUniversity of WaterlooWaterlooCanada
  3. 3.Department of MedicineDuke University, VA Medical CentersDurhamUSA
  4. 4.Department of Biochemistry and Molecular BiophysicsUniversity of ArizonaTucsonUSA

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