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Regulation of Electron Flow in Nitric Oxide Synthase Reductase Domains
Author(s) -
Roman Linda J,
Martasek Pavel,
Masters Bettie Sue S.
Publication year - 2006
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.20.5.a905-c
Subject(s) - nitric oxide synthase , nitric oxide , chemistry , atp synthase , biophysics , microbiology and biotechnology , biochemistry , enzyme , biology , organic chemistry
The nitric oxide synthases (NOSs) catalyze the formation of nitric oxide from L‐arginine. They are modular, cofactor‐containing enzymes, divided into an oxygenase domain, containing heme, and a reductase domain, containing FMN and FAD, as well as a NADPH binding site, connected by a calmodulin (CaM) binding sequence, occupancy of which is required for nitric oxide (NO) production. Two CaM‐modulated regulatory sequences are present in the constitutive isoforms – the autoregulatory insert (AI), which appears to compete with CaM to impede electron flow through the reductase domain, and the C‐terminal tail region, which inhibits electron flow between the flavins. Deletion of the AI reduces CaM stimulation of electron flow from 10‐fold in the wild‐type enzyme to 2‐fold in the mutant. Deletion of the tails from nNOS and eNOS yields enzymes with greatly enhanced electron transfer through the reductase domain, as measured by cytochrome c reduction, in the absence of CaM; activities are 21‐fold and 7‐fold higher, respectively. In the presence of CaM, however, activity is equivalent to that of the wild‐type enzyme. A nNOS mutant in which both the AI and C‐terminus have been deleted completely loses CaM modulation through the reductase domain. Thus, these two CaM‐sensitive elements, the AI and C‐terminus, are completely responsible for transducing the CaM effect through the reductase domains, an example of intramolecular signal regulation in redox proteins. (Grant support‐NIH GM52419 and HL 30050)