EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or NG-hydroxyarginine

Roman Davydov, Amy Ledbetter-Rogers, Pavel Martásek, Mikhail Larukhin, Masanori Sono, John H. Dawson, Bettie Sue Siler Masters, Brian M. Hoffman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

108 Scopus citations


Reconstitution of the endothelial nitric oxide synthase heme domain (NOS) with the catalytically noncompetent 4-aminotetrahydrobiopterin has allowed us to prepare at -40 °C the oxyferrous-NOS-substrate complexes of both L-arginine (Arg) and NG-hydroxyarginine (NOHA). We have radiolytically cryoreduced these complexes at 77 K and used EPR and ENDOR spectroscopies to characterize the initial products of reduction, as well as intermediates that arise during stepwise annealing to higher temperatures. Peroxo-ferri-NOS is the primary product of 77 K cryoreduction when either Arg or NOHA is the substrate. Proton ENDOR spectra of this state suggest that the peroxo group is H-bonded to a [guanidinium-water] network that forms because the binding of O2 to the ferroheme of NOS recruits H2O. At no stage of reaction/annealing does one observe an EPR signal from a hydroperoxo-ferri state with either substrate. Instead, peroxo-ferri-NOS-substrate complexes convert to a product-state intermediate at the extremely low temperature of 165-170 K. EPR and proton ENDOR spectra of the intermediate formed with Arg as substrate support the suggestion that the reaction involves the formation and attack of Compound I. Within the time/temperature resolution of the present experiments, samples with Arg and NOHA as substrate behave the same in the initial steps of cryoreduction/annealing, despite the different acid/base characteristics of the two substrates. This leads us to discuss the possibility that ambient-temperature catalytic conversion of both substrates is initiated by reduction of the oxy-ferroheme to the hydroperoxo-ferriheme through a coupled proton-electron transfer from a heme-pocket reductant, and that Arg may provide the stoichiometrically second proton of catalysis.

Original languageEnglish (US)
Pages (from-to)10375-10381
Number of pages7
Issue number33
StatePublished - Aug 20 2002

ASJC Scopus subject areas

  • Biochemistry


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