Fluorescence detection of obligatory conformations in catalysis and control of NOS

Abstract

Nitric Oxide synthases produce NO as a signal and as a cytotoxin in immune response. In the tethered shuttle model for NOS reductase kinetics, the FMN domain moves between NADPH dehydrogenase and oxygenase catalytic sites. FMN fluorescence is sensitive to NOS conformation; eNOS and nNOS holoenzymes have states with lifetimes of ~4.3ns, 1 ns, and 90 ps. Two domain oxyFMN constructs (truncated before the FAD binding domain) and holoenzyme share open conformations with lifetimes of ~4.3 ns. The holoenzyme majority state has a lifetime of ~90 ps due to FAD/FMN proximity as in crystal structures (Wang et al, Garcin et al). In oxyFMN ~25% of FMN has a lifetime of 0.9 ns, which we attribute to quenching by heme. Calmodulin activation of NOS releases the FMN binding domain from the ‘dehydrogenase’ complex, increasing the populations of the long lifetime states. Calmodulin binding shortens the average lifetime of oxyFMN constructs, because CaM promotes association of the FMN binding domain with the oxygenase domain, and favors the 1 ns state over the 4.3 ns state. Similar effects help explain modulation of NOS activity by covalent modification and protein‐protein interactions, e.g., in the HSP90‐eNOS complex.