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Oxygen activation in NO synthases: evidence for a direct role of the substrate
Ist Teil von
FEBS open bio, 2016-05, Vol.6 (5), p.386-397
Ort / Verlag
England: John Wiley & Sons, Inc
Erscheinungsjahr
2016
Quelle
Access via Wiley Online Library
Beschreibungen/Notizen
Nitric oxide (NO) and the other reactive nitrogen species (RNOS) play crucial patho‐physiological roles at the interface of oxidative stress and signalling processes. In mammals, the NO synthases (NOSs) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molecular functioning of NOS. Oxygen activation, which is at the core of NOS catalysis, involves a sophisticated sequence of electron and proton transfers. While electron transfer in NOS has received much attention, the proton transfer processes has been scarcely investigated. Here, we report an original approach that combines fast‐kinetic techniques coupled to resonance Raman spectroscopy with the use of synthetic analogues of NOS substrate. We characterise FeII‐O2 reaction intermediates in the presence of L‐arginine (Arg), alkyl‐ and aryl‐guanidines. The presence of new reaction intermediates, such as ferric haem‐peroxide, that was formerly postulated, was tracked by analysing the oxygen activation reaction at different times and with different excitation wavelengths. Our results suggest that Arg is not a proton donor, but indirectly intervenes in oxygen activation mechanism by modulating the distal H‐bond network and, in particular, by tuning the position and the role of the distal water molecule. This report supports a catalytic model with two proton transfers in step 1 (Arg hydroxylation) but only one proton transfer in step 2 (Nω‐hydroxy‐L‐arginine oxidation).
The reactive nitrogen species produced by nitric oxide (NO) synthases play crucial pathophysiological roles at the interface of oxidative stress and signaling processes. Using a combination of fast‐kinetics methods, resonance Raman spectroscopy and synthetic analogs of NOS substrate, we investigated the proton transfer processes involved in NOS catalysis. Our results suggest that arginine is not a proton donor, but indirectly intervenes in oxygen activation mechanism.