Intraprotein electron transfer in inducible nitric oxide synthase holoenzyme.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Feng, C; Dupont, AL; Nahm, NJ; Spratt, DE; Hazzard, JT; Weinberg, JB; Guillemette, JG; Tollin, G; Ghosh, DK

Published Date

  • January 2009

Published In

Volume / Issue

  • 14 / 1

Start / End Page

  • 133 - 142

PubMed ID

  • 18830722

Pubmed Central ID

  • PMC2596912

Electronic International Standard Serial Number (EISSN)

  • 1432-1327

Digital Object Identifier (DOI)

  • 10.1007/s00775-008-0431-2


  • eng

Conference Location

  • Germany