HEME COORDINATION AND STRUCTURE OF THE CATALYTIC SITE IN NITRIC-OXIDESYNTHASE

Nitric oxide (NO), recently found to play many physiological roles, is generated by the catalysis Of L-arginine and O2 to L-citrulline and N O by nitric oxide synthases (NOSs). Resonance Raman spectra from the h eme of resting, reduced, and CO-bound forms of rat brain NOS firmly es tablish that the enzyme belongs to the P-450 class of enzymes. The ele ctron density marker line (nu4) in the Raman spectrum of ligand-free f errous NOS has a low frequency (1347 cm-1), indicating a thiolate axia l ligand on the heme. The assignment of a thiolate axial ligand is con firmed in the CO-bound form of the enzyme by the frequency of the Fe-C -O bending mode at 562 cm-1. The heme in resting NOS is five-coordinat e high spin and thereby differs from the resting state of most substra te-free P-450s, which are predominantly six-coordinate low spin. The f requency of the Fe-CO stretching mode in the CO-bound enzyme at 491 cm -1, identified by isotope substitution, is higher than that in substra te-free P-450s. Thus, in the ferric and the CO-bound forms of the enzy me, the sixth-ligand binding site on the heme is restricted by steric or hydrophobic interactions. In addition, the Fe-CO stretching mode is broad (30 cm-1) and may be resolved into two overlapping lines of equ al intensity, indicating that the heme domains can adopt two distinct conformations.