MUTAGENESIS OF ACIDIC RESIDUES IN THE OXYGENASE DOMAIN OF INDUCIBLE NITRIC-OXIDE SYNTHASE IDENTIFIES A GLUTAMATE INVOLVED IN ARGININE BINDING

The oxygenase domain of the mouse cytokine-inducible nitric-oxide synt hase (iNOSox, amino acids 1-498) binds heme, tetrahydrobiopterin, and the substrate Arg and is the domain responsible for catalyzing nitric oxide synthesis and maintaining the enzyme's active dimeric structure, To further understand iNOSox structure-function, we carried out alani ne point mutagenesis on 15 conserved acidic residues located within a region of iNOSox (amino acids 352-473) that shares sequence homology w ith the pterin-binding module in dihydrofolate reductases and may be i mportant for iNOSox subunit dimerization and/or Arg binding. Five poin t mutants were identical or nearly identical to wild-type, while 10 ex hibited a range of defects that included low heme content (2), heme li gand instability (2), defective dimerization (2), and poor Arg and/or tetrahydrobiopterin binding (4), Mutations that caused defective tetra hydrobiopterin binding were also associated with other defects. In con trast, two mutants (E371A and D376A) exhibited an exclusive defect in Arg binding, These mutants were dimeric, indicating that dimerization of iNOSox in Escherichia coli does not require Arg, In one case (E371A ), the defect in Arg binding was absolute, as assessed by spectral per turbation, radioligand binding, and catalytic studies. We conclude tha t mutagenesis of conserved acidic residues within this region of iNOSo x can lead to exclusive defects in dimerization and in Arg binding, Mo deling considerations predict that the E371 carboxylate may participat e in Arg binding by interacting with its guanidine moiety.