Distinct catalytic activity of Staphylococcus Aureus nitric oxide synthase compared to other bacterial NOS-like...

To better understand the bacterial nitric oxide synthases (bNOSs), we characterized the properties of proteins from Staphylococcus aureus (saNOS) and Bacillus anthracis (baNOS) relative to Bacillus subtilis (bsNOS) and mammalian NOS using biochemical assays and conventional stopped-flow spectroscopy. Catalysis was examined under single-turnover conditions using ferrous bNOS proteins containing L-arginine (Arg) or N-hydroxy-L-arginine (NOHA) and also in multiple turnover H₂O₂-driven reactions or in a NADPH-driven reconstitution system consisting of bNOS, ferredoxin, and flavodoxin reductase proteins. The optical spectra of their ferric, ferrous, heme-dioxy, ferrous-NO, ferric-NO, and ferrous-CO complexes were highly similar. Imidazole binding affinity had the rank order baNOS > bsNOS > saNOS, while baNOS displayed weaker Arg binding affinity than bsNOS. In peroxide-supported reactions, activities had rank order bsNOS > baNOS > saNOS. Single-turnover reactions revealed saNOS had the fastest Fe^IIO₂ formation rate for both Arg and NOHA oxidation, and the fastest conversion of Fe^IIO₂ to Fe^IIINO during NOHA oxidation. The subsequent Fe^IIINO decay rates for baNOS and saNOS were 10-fold faster than for bsNOS and two-fold slower than mammalian iNOS, whereas their rates of Fe^IINO oxidation were about 41-156 times slower than in mammalian NOSs. In the three component reaction, NO production followed the rank order saNOS > bsNOS > baNOS, with saNOS six-fold higher than baNOS. Together, these findings increase our understanding of bNOS proteins and demonstrate that despite their considerable sequence homology, they display significant differences in their binding and kinetic behaviors that ultimately impact and help explain their divergent catalytic activities.