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Calmodulin Induced Changes in the Active Site of Nitric Oxide Synthase
Author(s) -
Sanchez Austin Johnny,
Esquerra Raymond,
Refuerzo Ruszel
Publication year - 2019
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.2019.33.1_supplement.779.32
Subject(s) - nitric oxide , heme , active site , nitric oxide synthase , calmodulin , chemistry , tetrahydrobiopterin , nos1 , biochemistry , cofactor , biophysics , enzyme , biology , organic chemistry
Nitric oxide is a transient signaling molecule in mammalian physiology, known as a vasodilating neurotransmitter and effector during innate immune responses. As such, studies about the family of nitric oxide synthases (NOS) responsible for the rapid conversion of amino acid L‐arginine into nitric oxide have been of great interest. The heme domain within the NOS active site critically binds molecular oxygen which combines with L‐arginine in a series of redox reactions. NOS activity is regulated by a number of cofactors, most notably the ubiquitous regulatory protein calmodulin (CaM) which, in the presence of calcium, binds NOS and promotes nitric oxide production. Though this relationship is well‐documented, the molecular mechanism is not understood. Previously, time resolved spectroscopy has been used to probe the active sites of heme proteins and observe differences in absorption on a nanosecond time frame during changes in the catalytic pockets. Our investigation studies the kinetic effects of CaM binding NOS during the photolysis of carbon monoxide from its heme domain. CaM has been shown to directly influence the active site, promoting availability to substrates and rebinding of ligands to the NOS heme, thus enhancing enzymatic activity. A major facet of our project involves further investigation of the various domains of NOS to determine their significance in CaM‐regulated binding. To this end, we prepared a number of knockout NOS variants which, through assaying via time‐resolved spectroscopy, further elucidate the molecular mechanism of active site regulation. Support or Funding Information NIH MARC: T34‐GM008574 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .