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Surface Residue Glu 762 of the FMN Subdomain Impacts Ferrous Heme‐NO reactivity in Neuronal Nitric Oxide Synthase
Author(s) -
HAQUE MOHAMMAD MAHFUZUL,
Wang ZhiQiang,
Fadlalla Mohammed,
Stuehr Dennis J.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.890.2
Subject(s) - heme , chemistry , nitric oxide , ferrous , nitric oxide synthase , hemeprotein , mutant , enzyme , ferric , citrulline , stereochemistry , arginine , biochemistry , inorganic chemistry , organic chemistry , amino acid , gene
Nitric Oxide Synthases (NOSs) are calmodulin (CaM)‐dependent flavoheme enzymes that oxidize L‐arginine to nitric oxide (NO) and L‐citrulline. Their catalytic behaviors are determined by their rates of heme reduction ( k r ), ferric heme‐NO dissociation ( k d ), and ferrous heme‐NO oxidation ( k ox ). We found that point mutation (Glu762N) of a conserved residue on the enzyme's FMN module doubled the NO synthesis activity compared to wild type (WT) nNOS. However, in the absence of Arg, NADPH oxidation rates suggested that electron flux through the heme was slower in E762N nNOS, and this correlated with the mutant having a 60% slower k r . During NO synthesis, little heme‐NO complex accumulated in the mutant, compared to approximately 50‐70% accumulation in WT nNOS. We concluded that the E762N mutation is hyperactive because it minimizes buildup of an inactive ferrous‐NO complex during the steady state. Indeed, we found that k ox was 2 times faster in the E762N mutant than in WT. Computer simulation and experimental measures both indicated that the slower k r coupled with the faster k ox lowers the mutant's apparent K m O 2 for NO synthesis, which in turn enables its hyperactivity in steady‐state NO synthesis assays. This represents a novel and unexpected way for the FMN module to impact the activity of NOS enzymes.This work was supported by NIH Grant CA53914 to Dennis Stuehr.