Nitric Oxide-Generated P420 Nitric Oxide Synthase: Characterization and Roles for Tetrahydrobiopterin and Substrate in Protecting against or Reversing the P420 Conversion

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Abstract
The neuronal NO synthase (nNOS) heme binds self-generated NO, and this negatively regulates NO synthesis. Here we utilized the nNOS oxygenase domain and full-length nNOS along with various spectroscopic methods to (1) study formation of the six-coordinate ferrous NO complex and its conversion to a five-coordinate NO complex and (2) investigate the spectral and catalytic properties of the five-coordinate NO complex following its air oxidation to a ferric enzyme. NO bound quickly to ferrous nNOS oxygenase to form a six-coordinate NO complex (kon and koff values of 1.25 × 10-3 mM-1 s-1 and 128 s-1 at 10 °C, respectively) that was stable in the presence of l-arginine or tetrahydrobiopterin (BH4) but was converted to a five-coordinate NO complex in a biphasic process (k = 0.1 and 0.01 s-1 at 10 °C) in the absence of these molecules. Air oxidation of the ferrous six-coordinate NO complex generated an enzyme with full activity and ferrous−CO Soret absorbance at 444 nm. In contrast, oxidation of the five-coordinate NO complex generated an inactive dimer with ferrous−CO Soret absorbance at 420 nm, indicating nNOS was converted to a ferric P420 form. Incubation of ferric P420 nNOS with BH4 alone or BH4 and l-arginine resulted in time-dependent reactivation of catalysis and associated recovery of P450 character. Thus, nNOS is a heme−thiolate protein that can undergo a reversible P450−P420 conversion. BH4 has important roles in preventing P420 formation during NO synthesis, and in rescuing P420 nNOS.