Inducible nitric oxide synthase: role of the N-terminal beta-hairpin hook and pterin-binding segment in dimerization and tetrahydrobiopterin interaction

The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residu es 1-498) is a dimer that binds heme, L-arginine and tetrahydrobiopterin (H 4B) and is the site for nitric oxide synthesis. We examined an N-terminal s egment that contains a beta-hairpin hook, a zinc ligation center and part o f the H4B-binding site for its role in dimerization, catalysis, and H4B and substrate interactions. Deletion mutagenesis identified the minimum cataly tic core and indicated that an intact N-terminal beta-hairpin hook is essen tial, Alanine screening mutagenesis of conserved residues in the hook revea led five positions (K82, N83, D92, T93 and H95) where native properties wer e perturbed, Mutants fell into two classes: (i) incorrigible mutants that d isrupt side-chain hydrogen bonds and packing interactions with the iNOSox C -terminus (N83, D92 and H95) and cause permanent defects in homodimer forma tion, H4B binding and activity; and (ii) reformable mutants that destabiliz e interactions of the residue main chain (K82 and T93) with the C-terminus and cause similar defects that were reversible with high concentrations of H4B, Heterodimers comprised of a hook-defective iNOSox mutant subunit and a full-length iNOS subunit were active in almost all cases, This suggests a mechanism whereby N-terminal hooks exchange between subunits in solution to stabilize the dimer.