Inducible Nitric Oxide Synthase (iNOS) catalyzes the formation of nitric oxide (NO) via a two step monooxygenation from Arginine to hydroxy-Arginine (NOHA), then to Citrulline and NO. The importance of NO in normal physiology and disease has made the NOS enzymes an intensely studied group of enzymes. NOS enzymes are unique P450-type heme enzymes with a conserved tryptophan residue hydrogen bonding to the axial cysteinate residue. Several studies were carried out to determine functional properties of the enzyme.

We studied the effect of CO binding to iNOS and its effect on the proximal ligand. CO binds to iNOS producing a 444 nm Soret, which shifts to 420 nm, characteristic for the inactivated “P420 form” of P450 enzymes. The nature of the proximal ligand of inactivated P420 enzymes has been proposed to be a neutral thiol or histidine residue as both produce a 420 nm Soret with CO. We compared the optical absorption and Raman spectra of iNOS P420 to myoglobin and a carbomonoxy-heure thiol model complex and studied the effects of pH on P420 formation. We conclude that the origin of the 420 nm Soret in P420 iNOS is due to protonation of the proximal thiolate ligand to neutral thiol.

Next, we studied the effect of mutating the conserved tryptophan residue to histidine (W188H). During turnover, this mutant was shown to stabilize a previously unobserved intermediate in the reaction, proposed to be compound (Tejero et al. JBC, 283, 33498–33507). We used resonance Raman and optical absorption spectroscopy, to show that the intermediate is a six-coordinate, low-spin, water-bound species. Using EPR we observed that the population of the H4B^·+ radical correlates with the intermediate population.

Finally, we measured the Fe-Cys stretching mode (v _Fe-Cys) of the W188H mutant and two new mutants, W188Y and W188F. The W188H v_Fe-Cys is similar to wild type, while the W188Y and W188F mutants are more similar to the P450 vF6Cys suggesting a broken or weakened hydrogen bond. In each of the mutants and the wild type, NOHA increases the v_Fe-Cys, demonstrating that the substrate (Arg or NORA) determines the mechanism of oxidation.