CHARACTERIZATION OF THE INDUCIBLE NITRIC-OXIDE SYNTHASE OXYGENASE DOMAIN IDENTIFIES A 49-AMINO-ACID SEGMENT REQUIRED FOR SUBUNIT DIMERIZATION AND TETRAHYDROBIOPTERIN INTERACTION

The oxygenase domain of inducible NO synthase (residues 1-498, iNOSox) is the enzyme's catalytic center. Its active form is a homodimer that contains heme and tetrahydrobiopterin (H4biopterin) and binds L-argin ine [Ghosh, D. K., & Stuehr, D. J. (1995) Biochemistry 34, 801], To he lp identify protein residues involved in prosthetic group and dimeric interaction, we expressed H4biopterin-free iNOSox in Escherichia coli. The iNOSox was 80% dimeric but contained a low-spin heme iron that bo und DTT as a sixth ligand. The iNOSox bound H4biopterin or L-arginine with high affinity, which displaced DTT from the heme and caused spect ral changes consistent with a closing up of the heme pocket, The H4bio pterin-replete iNOSox could catalyze conversion of N-omega-hydroxyargi nine to citrulline and NO in a H2O2-supported reaction. Limited trypsi nolysis of the H4biopterin-free iNOSox dimer cut the protein at a sing le site in its N-terminal region (K117). H4biopterin protected against the cleavage whereas L-arginine did not. The resulting 40 kDa protein contained thiol-ligated low-spin heme, was monomeric, catalytically i nactive, showed no capacity to bind H4biopterin or L-arginine, and did not dimerize when provided with these molecules, indicating that resi dues 1-117 were important for iNOSox dimerization and H4biopterin/L-ar ginine interaction. A deletion mutant missing residues 1-114 was parti ally dimeric but otherwise identical to the 40 kDa protein regarding i ts spectral and catalytic properties and inability to respond to L-arg inine and H4biopterin, whereas a deletion mutant missing residues 1-65 was equivalent to wildtype iNOSox, narrowing the region of importance to amino acids 66-114. Mutation of a conserved cysteine in this regio n (C109A) decreased H4biopterin affinity without compromising iNOSox d imeric structure, L-arginine binding, or catalytic function. These res ults suggest that residues 66-114 of iNOSox are involved in productive H4biopterin interaction and subunit dimerization. H4biopterin binding appears to stabilize the protein structure in this region, and throug h doing so activates iNOS for NO synthesis.