Crystal Structures of Zinc-free and -bound Heme Domain of Human Inducible Nitric-oxide Synthase

Abstract

The crystal structures of the heme domain of human inducible nitric-oxide synthase (NOS-2) in zinc-free and -bound states have been solved. In the zinc-free structure, two symmetry-related cysteine residues form a disulfide bond. In the zinc-bound state, these same two cysteine residues form part of a zinc-tetrathiolate (ZnS 4 ) center indistinguishable from that observed in the endothelial isoform (NOS-3). As in NOS-3, ZnS 4 plays a key role in stabilizing intersubunit contacts and in maintaining the integrity of the cofactor (tetrahydrobiopterin) binding site of NOS-2. A comparison of NOS-2 and NOS-3 structures illustrates the conservation of quaternary structure, tertiary topology, and substrate and cofactor binding sites, in addition to providing insights on isoform-specific inhibitor design. The structural comparison also reveals that pterin binding does not preferentially stabilize the dimer interface of NOS-2 over NOS-3. Nitric-oxide synthases (NOS)1 are a family of enzymes that produce nitric oxide (NO) and L-citrulline from L-arginine in the presence of NADPH and O 2 (1). NOS maintains a bidomain structure (2) with the catalytic center residing in the heme domain utilizing electrons derived from the cytochrome P450 reductase-like biflavin domain. The heme domain also contains the binding site for enzyme cofactor, tetrahydrobiopterin (H 4 B).The NOS family currently consists of three mammalian isoforms (3, 4). The endothelial (eNOS or NOS-3) and neuronal (nNOS or NOS-1) isoforms are constitutive and are activated by Ca 2ϩ -dependent calmodulin binding. The inducible isoform (iNOS or NOS-2) entertains a tightly bound calmodulin subunit and is regulated at the level of transcription. Because overproduction of NO by NOS-1 and NOS-2 leads to pathological conditions in stroke (5) and shock (6), respectively, isoformspecific inhibition of NOS has wide therapeutic potential. As a result, there is considerable interest within the pharmaceutical community to develop isoform-specific NOS inhibitors (7). Crystal structures of the catalytic heme domain for the various NOS isoforms are essential if rational drug design is to be applied to NO up/down-regulation.Crane et al. (8) solved the crystal structure of the murine NOS-2 heme domain, which established the overall fold of NOS as well as the location and structure of the pterin and L-Arg binding sites. The structure of the NOS-3 heme domain also has been solved (9), which revealed a novel zinc tetrathiolate (ZnS 4 ) at the bottom of the dimer interface. In the structure of the heme domain of murine NOS-2 (8), a disulfide was identified in the zinc binding site, and no zinc was seen in the structure. To understand the structural consequences of the metal center in other NOS isoforms, we have solved the crystal structure of human NOS-2 in both zinc-free and -bound forms. The availability of a zinc-bound NOS-2 affords direct comparison with NOS-3 and provides a molecular basis for the development of isoform-specific inhibitors. MATERIALS AND METHODS Expression and Pur...

Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes

“…Dimerization of heme-containing iNOS monomers can be enhanced by the presence of BH 4 by hydrophilic interactions (15,16,24,25). In hepatocytes, BH 4 is expressed constitutively at levels sufficient for iNOS activity (10,26).…”

Collisional cooling enhances the ability to observe non-covalent interactions within the inducible nitric oxide synthase oxygenase domain: Dimerization, complexation, and dissociation

“…The dimeric form of iNOS oxygenase domain has been shown through crystallography studies to contain a lone zinc ion coordinated by four cysteine residues along the dimer interface; this structure has been suggested to contribute to the stability of the complex [46]. There was no indication, however, of the presence of zinc in any of the experiments; no adduct of zinc was ever observed, nor was zinc lost as an ion in any CID experiments.…”

Intra-subunit and Inter-subunit Electron Transfer in Neuronal Nitric-oxide Synthase

“…Formation-The crystal structures of the eNOS and iNOS heme domain dimers revealed that L-Arg binds to the active site of the heme domain with hydrogen bonding to Glu-371 of mouse iNOS (Glu-361 in human eNOS, corresponding to Glu-592 in rat nNOS) (23)(24)(25)(26). The E592A mutation resulted in a loss of sensitivity to L-Arg, characterized by a lack of spectroscopic perturbation on its addition (Fig.…”