Hm. Abusoud et al., THE FERROUS-DIOXY COMPLEX OF NEURONAL NITRIC-OXIDE SYNTHASE - DIVERGENT EFFECTS OF L-ARGININE AND TETRAHYDROBIOPTERIN ON ITS STABILITY, The Journal of biological chemistry, 272(28), 1997, pp. 17349-17353
Nitric oxide synthases (NOS) are hemeproteins that catalyze oxidation
of L-arginine to nitric oxide (NO) and citrulline. The NOS heme iron i
s expected to participate in oxygen activation during catalysis, but i
ts interac tions with O-2 are not characterized. We utilized the heme-
containing oxygenase domain of neuronal NOS (nNOSoxy) and stopped-flow
methods to study forma tion and autooxidative decomposition of the nN
OSoxy oxygenated complex at 10 degrees C. Mixing ferrous nNOSoxy with
air-saturated buffer generated a transient species with absorption max
ima at 427 and similar to 560 nm. This species decayed within 1 s to f
orm ferric nNOSoxy. Its formation was first order with respect to O-2,
monophasic, and gave rate constants for k(on) = 9 x 10(5) M-1 s(-1) a
nd k(off) = 108 s(-1) for an L-arginine- and tetrahydrobiopterin (H4B)
-saturated nNOSoxy. Omission of L-arginine and/or H4B did not greatly
effect O-2 binding and dissociation rates. Decomposition of the oxygen
ated intermediate was independent of O-2 concentration and was either
biphasic or monophasic depending on sample conditions, L-Arginine stab
ilized the oxygenated intermediate (decay rate = 0.14 s(-1)), while H4
B accelerated its decay by a factor of 70 irrespective of L-arginine.
The spectral and kinetic properties of the intermediate identify it as
the (FeO2)-O-II complex of nNOSoxy. Destabilization of a metallo-oxy
species by H4B is unprecedented and may be important regarding the rol
e of this cofactor in NO synthesis.