Characterization of the Inducible Nitric Oxide Synthase Oxygenase Domain Identifies a 49 Amino Acid Segment Required for Subunit Dimerization and Tetrahydrobiopterin Interaction

Abstract

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-arginine [Ghosh, D. K., & Stuehr, D. J. (1995) Biochemistry 34, 801]. To help 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 bound DTT as a sixth ligand. The iNOSox bound H4biopterin or L-arginine with high affinity, which displaced DTT from the heme and caused spectral changes consistent with a closing up of the heme pocket. The H4biopterin-replete iNOSox could catalyze conversion of Nomega-hydroxyarginine to citrulline and NO in a H2O2-supported reaction. Limited trypsinolysis of the H4biopterin-free iNOSox dimer cut the protein at a single 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 inactive, showed no capacity to bind H4biopterin or l-arginine, and did not dimerize when provided with these molecules, indicating that residues 1-117 were important for iNOSox dimerization and H4biopterin/l-arginine interaction. A deletion mutant missing residues 1-114 was partially dimeric but otherwise identical to the 40 kDa protein regarding its spectral and catalytic properties and inability to respond to l-arginine and H4biopterin, whereas a deletion mutant missing residues 1-65 was equivalent to wild-type iNOSox, narrowing the region of importance to amino acids 66-114. Mutation of a conserved cysteine in this region (C109A) decreased H4biopterin affinity without compromising iNOSox dimeric structure, L-arginine binding, or catalytic function. These results 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 through doing so activates iNOS for NO synthesis.

Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

“…Consistent with this was the observation that radioligand binding with [$H]H % Bip and [$H]NO # Arg revealed a dual allosteric interaction between the H % Bip-binding site and -arginine-binding site of NOS-I [35], whereas for NOS-II this has still remained controversial [37,38]. However, at the crystal level H % Bip was not found to affect substrate binding [34], in direct disagreement with an earlier report [16].…”

“…The reasons for this discrepancy are unclear but might reflect isoform differences or, alternatively, methodological differences, e.g. the use of H % Bip-free NOS and measurements under catalytic [40] compared with non-catalytic conditions used in radioligand binding assays [35][36][37][38][39]. There is therefore uncertainty about the functional significance of allosteric binding site interactions in regulating NOS activity and NO generation.…”

“…During catalysis, NOS can produce significant quantities of reactive oxygen species (O # − d and H # O # ) [21,22,41,50,51] ; H % Bip markedly attenuates this [21,22,37] by a largely unknown mechanism that might involve the haem domain [51]. Importantly, given that NOS-derived reactive oxygen species contribute to enzyme inactivation [41], we considered the possibility that H % Bip stabilizes and protects NOS by interfering with autodamaging O # − d. In agreement with this hypothesis, during enzyme catalysis and in the absence of H % Bip, i.e.…”

“…Allosteric modulation within the catalytic centre of NOS by H % Bip has also been reported [35][36][37][38][39][40]. In a radioligand binding study, Mayer and co-workers demonstrated a 6-fold increase in the affinity of NOS-I for [$H]H % Bip in the presence of -arginine [35].…”

“…In a radioligand binding study, Mayer and co-workers demonstrated a 6-fold increase in the affinity of NOS-I for [$H]H % Bip in the presence of -arginine [35]. For NOS-II, both positive [37] and negative [38] [34] as had previously been demonstrated [16]. Consequently, it remains to be established whether this binding-site co-operativity is a general phenomenon for all NOS isoforms and of relevance during catalysis.…”