title: A novel cryo-reduction method to investigate the molecular mechanism of nitric oxide synthases.
abstract: Nitric oxide synthases (NOSs) are hemoproteins responsible for the biosynthesis of NO in mammals. They catalyze two successive oxidation reactions. The mechanism of oxygen activation is based on the transfer of two electrons and two protons. Despite structural analogies with cytochromes P450, the molecular mechanism of NOS remains yet to be elucidated. Because of extremely high reaction rates, conventional kinetics methods failed to trap and characterize the major reaction intermediates. Cryo-reduction methods offer a possibility to circumvent this technological lock, by triggering oxygen activation at cryogenic temperatures by using water radiolysis. However, this method is not adapted to the NOS mechanism because of the high instability of the initial Fe(II)O2 complex (extremely fast autoxidation and/or reaction with the cofactor H4B). This imposed a protocol with a stable Fe(II)O2 complex (observed only for one NOS-like protein) and that excludes any redox role for H4B. A relevant approach to the NOS mechanism would use H4B to provide the (second) electron involved in oxygen activation; water radiolysis would thus provide the first electron (heme reduction). In this context, we report here an investigation of the first electron transfer by this alternative approach, i.e., the reduction of native NOS by water radiolysis. We combined EPR and resonance Raman spectroscopies to analyze NOS reduction for a combination of different substrates, cofactor, and oxygen concentrations, and for different NOS isoforms. Our results show that cryo-reduction of native NOS is achieved for all conditions that are relevant to the investigation of the NOS mechanism.
authors.affiliation: Laboratoire de Chimie Physique, CNRS UMR 8000, Univ Paris-Sud, 91405 Orsay Cedex, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Bernad
authors.forename: Albane
authors.initials: A
authors.lastname: Brunel
authors.forename: Pierre
authors.initials: P
authors.lastname: Dorlet
authors.forename: Cécile
authors.initials: C
authors.lastname: Sicard-Roselli
authors.forename: Jérôme
authors.initials: J
authors.lastname: Santolini
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry; D020134:Catalytic Domain; D003080:Cold Temperature; D004307:Dose-Response Relationship, Radiation; D004583:Electrons; D005720:Gamma Rays; D007527:Isoenzymes / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013059:Spectrum Analysis, Raman
chemical_list: D001426:Bacterial Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D001708:Biopterins; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C003402:sapropterin; D010100:Oxygen