Role of an isoform-specific serine residue in FMN–heme electron transfer in inducible nitric oxide synthase

  • Wenbing Li
  • Weihong Fan
  • Li Chen
  • Bradley O. Elmore
  • Mike Piazza
  • J. Guy Guillemette
  • Changjian Feng
Original Paper


In the crystal structure of a calmodulin (CaM)-bound FMN domain of human inducible nitric oxide synthase (NOS), the CaM-binding region together with CaM forms a hinge, and pivots on an R536(NOS)/E47(CaM) pair (Xia et al. J Biol Chem 284:30708–30717, 2009). Notably, isoform-specific human inducible NOS S562 and C563 residues form hydrogen bonds with the R536 residue through their backbone oxygens. In this study, we investigated the roles of the S562 and C563 residues in the NOS FMN–heme interdomain electron transfer (IET), the rates of which can be used to probe the interdomain FMN/heme alignment. Human inducible NOS S562K and C563R mutants of an oxygenase/FMN (oxyFMN) construct were made by introducing charged residues at these sites as found in human neuronal NOS and endothelial NOS isoforms, respectively. The IET rate constant of the S562K mutant is notably decreased by one third, and its flavin fluorescence intensity per micromole per liter is diminished by approximately 24 %. These results suggest that a positive charge at position 562 destabilizes the hydrogen-bond-mediated NOS/CaM alignment, resulting in slower FMN–heme IET in the mutant. On the other hand, the IET rate constant of the C563R mutant is similar to that of the wild-type, indicating that the mutational effect is site-specific. Moreover, the human inducible NOS oxyFMN R536E mutant was constructed to disrupt the bridging CaM/NOS interaction, and its FMN–heme IET rate was decreased by 96 %. These results demonstrated a new role of the isoform-specific serine residue of the key CaM/FMN(NOS) bridging site in regulating the FMN–heme IET (possibly by tuning the alignment of the FMN and heme domains).


Electron transfer Nitric oxide synthase Calmodulin Laser flash photolysis Heme 


Bis–tris propane





Endothelial nitric oxide synthase


FMN hydroquinone




Interdomain electron transfer


Inducible nitric oxide synthase


Neuronal nitric oxide synthase


Nitric oxide synthase


Optical parametric oscillator


Bi-domain nitric oxide synthase construct in which only the heme-containing oxygenase and flavin mononucleotide domains along with the calmodulin binding site are present





This work was supported by grants from the National Institutes of Health (GM081811 and HL091280 to C.F.), an AHA Grant-in-Aid (09GRNT2220310 to C.F.), an NSERC Discovery Grant (183521 to J.G.G.), and grants from the National Center for Research Resources (5P20RR016480-12) and the National Institute of General Medical Sciences (8 P20 GM103451-12). C.F. acknowledges the support of UNM HSC RAC grant.

Supplementary material

775_2012_887_MOESM1_ESM.pdf (861 kb)
Supporting Information Available. Table of primers for constructions of the human iNOS oxyFMN S562K, C563R and R536E mutants; gel electrophoresis of the purified mutants; UV–vis spectra of the proteins; summary of the processes occurring upon CO photolysis; analysis of the FMN content by HPLC; flavin fluorescence spectra of wt iNOS oxyFMN with added EDTA. (PDF 860 kb)


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Copyright information

© SBIC 2012

Authors and Affiliations

  • Wenbing Li
    • 1
  • Weihong Fan
    • 1
  • Li Chen
    • 1
  • Bradley O. Elmore
    • 1
  • Mike Piazza
    • 2
  • J. Guy Guillemette
    • 2
  • Changjian Feng
    • 1
  1. 1.Department of Pharmaceutical Sciences, College of PharmacyUniversity of New MexicoAlbuquerqueUSA
  2. 2.Department of ChemistryUniversity of WaterlooWaterlooCanada

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