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

Ghosh, Dipak K.; Crane, Brian R.; Ghosh, Sanjay; Wolan, Dennis; Gachhui, Ratan; Crooks, Carol; Presta, Anthony; Tainer, John A.; Getzoff, Elizabeth D.; Stuehr, Dennis J.

The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1–498) is a dimer that binds heme, L-arginine and tetrahydrobiopterin (H4B) and is the site for nitric oxide synthesis. We examined an N-terminal segment that contains a Β-hairpin hook, a zinc ligation center and part of the H4B-binding site for its role in dimerization, catalysis, and H4B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N-terminal Β-hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side-chain hydrogen bonds and packing inter- actions with the iNOSox C-terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H4B binding and activity; and (ii) reform- able mutants that destabilize 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.

OXYGENASES; AMINO acids; ZINC; NEOVASCULARIZATION; NITRIC oxide; TETRAHYDROBIOPTERIN; OXIDOREDUCTASES

EMBO Journal, 1999, Vol 18, Issue 22, p6260

0261-4189

Academic Journal

10.1093/emboj/18.22.6260