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There is still much unknown about how nitric oxide (NO) biosynthesis by NO synthase (NOS) is tightly regulated. This is remarkable because deviated NO production in vivo has been implicated in an increasing number of serious diseases lacking effective treatments, including stroke and cancer.  Unlike inducible NOS, endothelial and neuronal NOS isoforms (eNOS and nNOS) are controlled by calmodulin (CaM) through facilitating catalytically significant interdomain electron transfer (IET) processes. It is proposed that CaM activates NO synthesis in eNOS and nNOS through a conformational change of the flavin mononucleotide (FMN) domain from its shielded electron-accepting (input) state to a new electron-donating (output) state. The FMN-heme IET within the NOS output state is essential for NO synthesis at the catalytic heme. However, the mechanism for formation of the NOS output state remains unclear, and this stands as a critical barrier for development of new therapeutic interventions for treating a wide range of NOS-mediated diseases. We hypothesize that productive FMN/heme interactions and specific binding of CaM to NOS synergistically regulate formation of the NOS output state. This hypothesis will be tested with novel truncated NOS constructs, which are validated models of the output state, through three complementary and synergistic Aims. The experimental design will integrate our state-of-art structural spectroscopy techniques (for determining electronic structure of heme center), and innovative laser flash photolysis methodology (for determining the IET kinetics as a direct measure for formation of the NOS output state), with site-directed mutagenesis (for modulating specific interdomain interactions). This multi-disciplinary study will significantly improve the fundamental understanding of NOS regulation at the molecular level, and will provide new important mechanistic information that has the potential of facilitating the development of effective mechanism-based NOS inhibitors for these clinically important enzymes.