TY - JOUR
T1 - The ferrous dioxygen complex of the oxygenase domain of neuronal nitric- oxide synthase
AU - Couture, Manon
AU - Stuehr, Dennis J.
AU - Rousseau, Denis L.
PY - 2000/2/4
Y1 - 2000/2/4
N2 - The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe2O2 nNOSoxy). We detect a line at 1135 cm-1 in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm-1 with 18O2. This line is assigned as the O-O stretching mode (V(o-o)) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or Nω-hydroxy- L-arginine, in the presence or absence of (6R)-5,6,7,8-tetrahydro-L- biopterin, reveal that the V(o-o) line is insensitive to the presence of the substrate and the pterin. The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques.
AB - The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe2O2 nNOSoxy). We detect a line at 1135 cm-1 in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm-1 with 18O2. This line is assigned as the O-O stretching mode (V(o-o)) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or Nω-hydroxy- L-arginine, in the presence or absence of (6R)-5,6,7,8-tetrahydro-L- biopterin, reveal that the V(o-o) line is insensitive to the presence of the substrate and the pterin. The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques.
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U2 - 10.1074/jbc.275.5.3201
DO - 10.1074/jbc.275.5.3201
M3 - Article
C2 - 10652305
AN - SCOPUS:0034603038
SN - 0021-9258
VL - 275
SP - 3201
EP - 3205
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 5
ER -