A Novel Cryo-Reduction Method to Investigate the Molecular Mechanism of Nitric Oxide Synthases

• Published in: The journal of physical chemistry. B Vol. 116; no. 19; pp. 5595 - 5603
• Main Authors: Bernad, Sophie, Brunel, Albane, Dorlet, Pierre, Sicard-Roselli, Cécile, Santolini, Jérôme
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.05.2012
• Subjects: Activation; Animals; Arginine - chemistry; Bacterial Proteins - chemistry; Biochemistry - methods; Biopterins - analogs & derivatives; Biopterins - chemistry; Catalytic Domain; Chemical Sciences; Cold Temperature; Cryogenic temperature; Dose-Response Relationship, Radiation; Electrons; Gamma Rays; Instability; Isoenzymes - chemistry; Locks; Mammals; Mice; Models, Molecular; Nitric oxide; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase Type II - chemistry; Oxidation-Reduction; Oxygen - chemistry; Radiolysis; Recombinant Proteins - chemistry; Reduction; Spectrum Analysis, Raman
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/jp300749b

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 FeIIO2 complex (extremely fast autoxidation and/or reaction with the cofactor H4B). This imposed a protocol with a stable FeIIO2 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.