X-Ray Absorption Spectroscopic Analysis of the High-Spin Ferriheme Site in Substrate-Bound Neuronal Nitric-Oxide Synthase

Abstract

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide through two stepwise oxygenation reactions involving N--hydroxy-L-arginine, an enzyme-bound intermediate. The N^ω-hydroxy-L-arginine-and arginine-bound NOS ferriheme centers show distinct, high-spin electron paramagnetic resonance signals. Iron X-ray absorption spectroscopy (XAS) has been used to examine the structure of the ferriheme site in the N^ω-hydroxy-L-arginine-bound full-length neuronal NOS in the presence of (6R)-5, 6, 7, 8-tetrahydro-L-biopterin. Iron XAS shows that the high-spin ferriheme sites in the N^ω-hydroxy-L-arginine-and arginine-bound forms are strikingly similar, both being coordinated by the heme and an axial thiolate ligand, with an Fe-S distance of ca. 2.29 Å. Cu²⁺ inhibition slightly affects the spin-state equilibrium, but causes no XAS-detectable changes in the immediate ferriheme coordination environment of neuronal NOS. The structure and ligand geometry of the high-spin ferriheme in arginine-bound neuronal NOS are essentially identical to those of the N^ω-hydroxy-L-arginine-bound form and only slightly affected by the divalent cation inhibitor of consitutive NOS.