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

• Published in: Biochemistry (Easton) Vol. 40; no. 43; pp. 12826 - 12832
• Main Authors: Aoyagi, Mika, Arvai, Andrew S, Ghosh, Sanjay, Stuehr, Dennis J, Tainer, John A, Getzoff, Elizabeth D
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 30-10-2001
• Subjects: Animals; Binding Sites; Biopterins - analogs & derivatives; Biopterins - chemistry; Biopterins - genetics; Catalysis; Conserved Sequence; Crystallography, X-Ray; Dimerization; Electron Transport; Escherichia coli - metabolism; Heme - chemistry; Hydrogen Bonding; Mice; Models, Chemical; Models, Molecular; Mutation; Nitric Oxide Synthase - chemistry; Nitric Oxide Synthase - physiology; Nitric Oxide Synthase Type II; Protein Binding; Recombinant Proteins - chemistry; Tryptophan - chemistry
• DOI: 10.1021/bi011183k
• EISSN: 1520-4995
• ISSN: 0006-2960

To better understand potential roles of conserved Trp457 of the murine inducible nitric oxide synthase oxygenase domain (iNOSox; 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 iNOSox dimers (residues 66−498). In W457F iNOSox, 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 (FeIIO2) 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.