title: Electron paramagnetic resonance characterization of tetrahydrobiopterin radical formation in bacterial nitric oxide synthase compared to mammalian nitric oxide synthase.
abstract: H(4)B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation (second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H(4)B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H(4)B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.
authors: CNRS, Laboratoire Stress Oxydant et Détoxication, Gif-sur-Yvette, France.
authors: Albane
authors: A
authors: Brunel
authors: Jérôme
authors: J
authors: Santolini
authors: Pierre
authors: P
authors: Dorlet
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D005609:Free Radicals; D001708:Biopterins; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C003402:sapropterin