10.1021/jp300749b.s001
Sophie Bernad
Sophie
Bernad
Albane Brunel
Albane
Brunel
Pierre Dorlet
Pierre
Dorlet
Cécile Sicard-Roselli
Cécile
Sicard-Roselli
Jérôme Santolini
Jérôme
Santolini
A Novel Cryo-Reduction
Method to Investigate the Molecular
Mechanism of Nitric Oxide Synthases
American Chemical Society
2012
EPR
Nitric Oxide SynthasesNitric oxide synthases
oxygen activation
cofactor H 4B
FeIIO 2
NOS mechanism
electron
resonance Raman spectroscopies
water radiolysis
H 4B
2012-05-17 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/A_Novel_Cryo_Reduction_Method_to_Investigate_the_Molecular_Mechanism_of_Nitric_Oxide_Synthases/2521993
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<sup>II</sup>O<sub>2</sub> complex (extremely fast autoxidation
and/or reaction with the cofactor H<sub>4</sub>B). This imposed a
protocol with a stable Fe<sup>II</sup>O<sub>2</sub> complex (observed
only for one NOS-like protein) and that excludes any redox role for
H<sub>4</sub>B. A relevant approach to the NOS mechanism would use
H<sub>4</sub>B 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.