1. Electron Paramagnetic Resonance Characterization of Tetrahydrobiopterin Radical Formation in Bacterial Nitric Oxide Synthase Compared to Mammalian Nitric Oxide Synthase
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Albane Brunel, Jérôme Santolini, Pierre Dorlet, Stress Oxydants et Détoxication ( LSOD ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Stress Oxydants et Détoxication (LSOD), Département Biochimie, Biophysique et Biologie Structurale (B3S), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)
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MESH: Oxidation-Reduction ,MESH : Escherichia coli ,[SDV]Life Sciences [q-bio] ,Nitric Oxide Synthase Type II ,PROTEIN ,MESH: Biopterin ,Electron donor ,Photochemistry ,01 natural sciences ,chemistry.chemical_compound ,SUBSTRATE ,2ND HALF-REACTION ,LOW-TEMPERATURE ,MESH : Arginine ,Citrulline ,MESH : Bacterial Proteins ,MESH: Animals ,Pterin ,MESH: Bacterial Proteins ,0303 health sciences ,MESH : Free Radicals ,L-ARGININE ,biology ,MESH: Escherichia coli ,Chemistry ,MESH : Rats ,MESH: Arginine ,MESH : Bacillus subtilis ,Tetrahydrobiopterin ,Nitric oxide synthase ,MESH : Electron Spin Resonance Spectroscopy ,MESH: Nitric Oxide Synthase ,MESH: Nitric Oxide Synthase Type II ,Oxidation-Reduction ,Bacillus subtilis ,medicine.drug ,Free Radicals ,MESH: Rats ,Biophysics ,OXYGENASE DOMAIN ,Reaction intermediate ,Arginine ,010402 general chemistry ,Redox ,SUBUNIT DIMERIZATION ,Nitric oxide ,BACILLUS-SUBTILIS ,03 medical and health sciences ,Bacterial Proteins ,Species Specificity ,MESH: Free Radicals ,HEME-DIOXY REDUCTION ,Escherichia coli ,medicine ,Animals ,MESH: Species Specificity ,MESH : Species Specificity ,030304 developmental biology ,SINGLE-TURNOVER ,MESH : Oxidation-Reduction ,[ SDV ] Life Sciences [q-bio] ,Electron Spin Resonance Spectroscopy ,MESH : Nitric Oxide Synthase ,MESH: Bacillus subtilis ,MESH : Nitric Oxide Synthase Type II ,Biopterin ,Rats ,0104 chemical sciences ,biology.protein ,MESH: Electron Spin Resonance Spectroscopy ,MESH : Animals ,Nitric Oxide Synthase ,Proteins and Nucleic Acids ,MESH : Biopterin - Abstract
International audience; 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.
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