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A Novel Cryo-Reduction Method to Investigate the Molecular Mechanism of Nitric Oxide Synthases
journal contribution
posted on 2012-05-17, 00:00 authored by Sophie Bernad, Albane Brunel, Pierre Dorlet, Cécile Sicard-Roselli, Jérôme SantoliniNitric 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.