Production of nitric oxide (NO) by nitric oxide synthase (NOS) requires
electrons to reduce the heme iron for substrate oxidation. Both FAD and FMN
flavin groups mediate the transfer of NADPH derived electrons to NOS. Unlike
mammalian NOS that contain both FAD and FMN binding domains within a single
polypeptide chain, bacterial NOS is only composed of an oxygenase domain and
must rely on separate redox partners for electron transfer and subsequent
activity. Here, we report on the native redox partners for Bacillus subtilis NOS
(bsNOS) and a novel chimera that promotes bsNOS activity. By identifying and
characterizing native redox partners, we were also able to establish a robust
enzyme assay for measuring bsNOS activity and inhibition. This assay was used to
evaluate a series of established NOS inhibitors. Using the new assay for
screening small molecules led to the identification of several potent inhibitors
for which bsNOS-inhibitor crystal structures were determined. In addition to
characterizing potent bsNOS inhibitors, substrate binding was also analyzed
using isothermal titration calorimetry giving the first detailed thermodynamic
analysis of substrate binding to NOS.
