FORMATION OF NITRIC-OXIDE SYNTHASE-IRON(II) NITROSOALKANE COMPLEXES -SEVERE RESTRICTION OF ACCESS TO THE IRON(II) SITE IN THE PRESENCE OF TETRAHYDROBIOPTERIN

Nitric oxide synthases (NOS) are heme proteins, closely related to cyt ochromes P450, that catalyze oxidation of L-arginine (L-Arg) to nitric oxide (NO) and citrulline. To get further insight into their active s ite, we have studied the ability of recombinant mouse inducible NOS (i NOS) and rat brain neuronal NOS (nNOS), and of their oxygenase domains (iNOS(oxy) and nNOS(oxy)), to form Fe(II)-nitrosoalkane complexes. In the absence of BH4, iNOS(oxy), nNOS(oxy), and full-length iNOS readil y form complexes characterized by Soret peaks around 448 nm, after rea ction with various nitroalkanes and sodium dithionite. These complexes displayed physicochemical characteristics very similar to those of pr eviously reported microsomal cytochrome P450-Fe(II)-nitrosoalkane comp lexes: (i) a Soret peak around 450 nm, (ii) a clear stability in the p resence of CO, and (iii) a fast destruction upon oxidation of the iron by ferricyanide. Thus, in the absence of L-Arg and BH4, NOSs Fe(II) a ppear to be largely opened to even large R-NO ligands with R = cyclohe xyl or p-Cl-C6H4-CH2CH(CH3) for instance, in a manner similar to micro somal P450s Fe(II). As expected, the presence of L-Arg inhibits the fo rmation of NOSs Fe(II)-RNO complexes. More surprisingly, the presence of BH4 also strongly inhibits the formation of the NOSs Fe(II) complex es even with the smallest nitrosoalkane ligand, CH3NO (IC50 values of 0.5 and 4 mu M for nNOS(oxy) and iNOS(oxy), respectively). Accordingly , recombinant full-length nNOS containing BH4 and L-Arg is completely unable to form Fe(II)-nitrosoalkane complexes, even with CH3NO. These results suggest that, in the absence of L-Arg and BH4, the distal pock et of NOSs Fe(II) is largely opened even to bulky ligands, in a manner similar to that of microsomal cytochromes P450. On the contrary, the distal heme pocket of iNOS and nNOS seems to be closed after binding o f L-Arg and BH4, particularly in the Fe(II) state. This results in a h ighly restricted access for Fe(II) ligands, except very small ones suc h as CO, NO, and O-2. Such effects of BH4 in controlling the size of t he distal heme pocket of NOS Fe(II) correspond to a new role of biopte rins in biological systems.