Magnetic circular dichroism spectroscopic characterization of the NOS-like protein from Geobacillus stearothermophilus (gsNOS)

Nitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent oxidation of l-arginine (l-Arg) to nitric oxide (NO) and citrulline via an NG-hydroxy-l-arginine (NHA) intermediate. Mammalian NOSs have been studied quite extensively; other eukaryotes and some prokaryotes appear to express NOS-like proteins comparable to the oxygenase domain of mammalian NOSs. In this study, a recombinant NOS-like protein from the thermostable bacterium Geobacillus stearothermophilus (gsNOS) has been characterized using magnetic circular dichroism (MCD) and UV–Vis absorption spectroscopic techniques. Spectral comparisons of ligand complexes (with O2, NO and CO) of substrate-bound (l-Arg or NHA) gsNOS, including the key oxyferrous complex studied at −50 °C in cryogenic mixed solvents, with analogous mammalian NOS complexes indicate overall spectroscopic similarities between gsNOS and mammalian NOSs. However, more detailed spectral comparisons reflect subtle structural differences between gsNOS and mammalian NOSs. This may be due to an incomplete tetrahydrobiopterin (BH4)-binding site and low BH4-binding affinity, which may become even lower in the presence of cryosolvent in gsNOS. Although BH4-binding may be altered, gsNOS appears to require the pterin for NO production since formation of the stable ferric-NO product complex was only observed when excess BH4 (>150 μM) over gsNOS was present upon single turnover reaction in which O2 was bubbled into dithionite-reduced NHA-bound protein solution at −35 °C or −50 °C.

The nitric oxide synthase (NOS)-like protein from Geobacillus stearothermophilus (gsNOS) has been characterized in various states including its O2, NO and CO complexes using magnetic circular dichroism spectroscopy. Despite overall spectral similarities to mammalian NOS heme domain, the ferric-NO product complex formation was only observed when excess tetrahydrobiopterin (BH4) (>150 μM) was present upon single turnover in which O2 was added to dithionite-reduced NG-hydroxy-l-arginine-bound gsNOS in ∼60% glycerol at −50 °C, because of the structurally unfavorable BH4-binding site.Figure optionsDownload as PowerPoint slide