Antifungal imidazoles block assembly of inducible NO synthase into an active dimer.

Cytokine-inducible nitric oxide synthase (iNOS) is a homodimeric enzyme that generates nitric oxide (NO) and L-citrulline from L-arginine (L-Arg) and O2. The N-terminal oxygenase domain (amino acids 1-498; iNOSox) in each subunit binds heme, L-Arg, and tetrahydrobiopterin (H4B), is the site of NO synthesis, and is responsible for the dimeric interaction, which must occur to synthesize NO. In both cells and purified systems, iNOS dimer assembly is promoted by H4B, L-Arg, and L-Arg analogs. We examined the ability of imidazole and N-substituted imidazoles to promote or inhibit dimerization of heme-containing iNOSox monomers, or to affect iNOS dimerization in cells. Imidazole, 1-phenylimidazole, clotrimazole, and miconazole all bound to the iNOSox monomer heme iron. Imidazole and 1-phenylimidazole promoted iNOSox dimerization, whereas clotrimazole (30 microM) and miconazole (15 microM) did not, and instead inhibited dimerization normally promoted by L-Arg and H4B. Clotrimazole also bound to iNOSox dimers in the absence of L-Arg and H4B and caused their dissociation. When added to cells expressing iNOS, clotrimazole (50 microM) had no effect on iNOS protein expression but almost completely inhibited its dimerization and consequent NO synthesis over an 8-h culture period, without affecting calmodulin interaction with iNOS. Thus, imidazoles can promote or inhibit dimerization of iNOS both in vitro and in cells, depending on their structure. Bulky imidazoles like clotrimazole block NO synthesis by inhibiting assembly of the iNOS dimer, revealing a new means to control cellular NO synthesis.

Comparative functioning of dihydro- and tetrahydropterins in supporting electron transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase.

The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 can support NO synthesis. Here, we characterize pterin-free inducible NOS (iNOS) and iNOS reconstituted with eight different tetrahydro- or dihydropterins to elucidate how changes in pterin side-chain structure and ring oxidation state regulate iNOS. Seven different enzyme properties that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxidation state (i.e., they required fully reduced tetrahydropterins) and were independent of side chain structure: NO synthesis and the ability to increase heme-dependent NADPH oxidation in response to substrates. In contrast, five properties were exclusively dependent on pterin side-chain structure or stereochemistry and were independent of pterin oxidation state: pterin binding affinity, and its ability to shift the heme iron to its high-spin state, stabilize the ferrous heme iron coordination structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer. These results clarify how structural versus redox properties of the pterin impact on its multifaceted role in iNOS function. In addition, the data reveal that during NO synthesis all pterin-dependent steps up to and including heme iron reduction can take place independent of the pterin ring oxidation state, indicating that the requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.

Analysis of substrate-induced electronic, catalytic, and structural changes in inducible NO synthase.

Inducible nitric oxide synthase (iNOS) catalyzes the NADPH-dependent formation of nitric oxide (NO) and citrulline from L-arginine and O2. In addition to serving as substrate, L-arginine alters the enzyme's heme iron spin equilibrium, increases its NADPH oxidation, and promotes assembly of active dimeric iNOS from inactive monomers. To understand what structural aspects of L-arginine are important for causing these effects, we have studied the interactions of iNOS with several L-arginine and guanidine analogs. Very few analogs supported NO synthesis even when bound to iNOS at saturating or near-saturating levels. In contrast, almost all analogs shifted the heme iron spin equilibrium and either increased or decreased NADPH oxidation by iNOS. The guanidine analogs displayed the same pattern of effects as their amino acid counterparts but exhibited a lower affinity except for analogs containing S-alkylisothiourea or aminoguanidine groups. Most analogs also promoted iNOS dimerization, with hydroxyguanidine and S-ethylisothiourea promoting more dimerization than L-arginine itself. Although the analog concentrations required to promote dimerization of monomers were somewhat higher than those required for binding to dimeric iNOS, they followed the same rank order. The degree of dimerization promoted by each analog did not correlate to its binding affinity, its causing a high- or low-spin shift in heme iron spin state, or to its increasing or decreasing NADPH oxidation. Together, we conclude that the enzyme's high degree of substrate specificity only applies to NO synthesis, in that a number of "inactive" structural analogs still bind to iNOS and affect its heme chemistry and structure in the absence of supporting NO synthesis. These latter affects are mediated through binding of the guanidinium portion of L-arginine and its analogs to a single site within iNOS and are relatively independent of the amino acid portion of the molecule.