Structures of Tetrahydrobiopterin Binding-Site Mutants of Inducible Nitric Oxide Synthase Oxygenase Dimer and Implicated Roles of Trp457^†,‡

To better understand potential roles of conserved Trp457 of the murine inducible nitric oxide synthase oxygenase domain (iNOS_ox; residues 1−498) in maintaining the structural integrity of the (6R)-5,6,7,8-tetrahydrobiopterin (H₄B) binding site located at the dimer interface and in supporting H₄B redox activity, we determined crystallographic structures of W457F and W457A mutant iNOS_ox dimers (residues 66−498). In W457F iNOS_ox, all the important hydrogen-bonding and aromatic stacking interactions that constitute the H₄B binding site and that bridge the H₄B and heme sites are preserved. In contrast, the W457A mutation results in rearrangement of the Arg193 side chain, orienting its terminal guanidinium group almost perpendicular to the ring plane of H₄B. Although Trp457 is not required for dimerization, both Trp457 mutations led to the increased mobility of the N-terminal H₄B binding segment (Ser112−Met114), which might indicate reduced stability of the Trp457 mutant dimers. The Trp457 mutant structures show decreased π-stacking with bound pterin when the wild-type π-stacking Trp457 position is occupied with the smaller Phe457 in W457F or positive Arg193 in W457A. The reduced pterin π-stacking in these mutant structures, relative to that in the wild-type, implies stabilization of reduced H₄B and destabilization of the pterin radical, consequently slowing electron transfer to the heme ferrous−dioxy (Fe^IIO₂) species during catalysis. These crystal structures therefore aid elucidation of the roles and importance of conserved Trp457 in maintaining the structural integrity of the H₄B binding site and of H₄B-bound dimers, and in influencing the rate of electron transfer between H₄B and heme in NOS catalysis.