INOS dimerization

An unusual and unexpected feature of NOS is the presence of a Zn ion tetrahedrally coordinated to pairs of symmetry-related Cys residues along the dimer interface 81, 82) (Fig. 4). The original structure of the mouse iNOS dimer did not have the Zn but instead two symmetry-related Cys residues formed a disulfide bond 80). The structure of the human iNOS heme domain also had a disulfide, but the disulfide could readily be broken and Zn reconstituted to give a ZnS4 center indistinguishable from that found in eNOS (S3). An independent structure... 

BBS-2 Inhibits iNOS dimerization Also weakly inhibits nNOS and eNOS... 

Figure 10. Proposed model for the iNOS dimer indicating domain swapping and electron transfer pathway. (Adapted from Ref. [98].)...

Although the first purification of bNOS was a monomer, it is now clear that the enzyme in all cases is effective as a dimer. A purified macrophage iNOS was used by Baek and coworkers98 to separate the holoenzyme from the monomers. The subunits do not have NOS activity but do have the ability to oxidize reduced triphosphopyridine nucleotide with either ferricyanide, cytochrome c or dichlorophenolindophenol. When all of the missing factors are present, but not when any is missing, the authors find recombination, as shown in Figure 13. 

Crane et al. first established the three-dimensional fold of NOS by solving the structure of a monomeric form of the mouse iNOS heme domain (78). This version of iNOS was missing the first 114 residues, which are known to be critical for dimer formation and activity (79). The monomer structure was soon followed by the dimeric heme domain structures of mouse iNOS (80), bovine eNOS (81), and the human isoforms of iNOS (82, 83) and eNOS (82). A comparison of eNOS and iNOS reveals that the structures are essentially the same with an overall root-mean-square deviation in backbone atoms of 1.1 A (S3). The sequence identity between human iNOS and bovine eNOS is 60% for 420 residues compared in the crystal structures (83). 

A comparative analysis of the dimer interface between NOS isoforms is important since there are questions on significant variation in dimer stability between isoforms (84, 85). The dimer interface is extensive with approximately 2700 A of surface area buried per monomer. The interface contacts involve a mix of nonpolar and polar interactions, including hydrogen bonding. Approximately 60% of the interface in both iNOS and eNOS is hydrophobic, although the higher resolution eNOS... 

The infrared spectra of pyridinyls are measured conveniently with the thin film spectroscopy apparatus and the changes which ensue on dimerization have been examined. A complete inO red spectrum for 4 at 77 K has been reported External sapphire windows on the thin film apparatus allow spectra to be measured... 

Figure 11. Schematic ribbon drawing of the iNOS oxygenase dimer generated from the X-ray coordinates [107] illustrating the locations of heme, L-arginine, tetrahydrobiopterin, and the zinc ion. The zinc ion is tetrahedrally coordinated to its protein ligands.

The recent advances in NOS structure analysis have shed light on the roles of the pterin, the substrate, and the newly discovered zinc ion in dimer formation and stabilization as well as catalytic activity. Work by numerous groups on iNOS (murine and human) [100, 101, 105-107], and eNOS (human and bovine) [101, 102] provides an intimate view of the various interactions between cofactors and protein-protein interplay in dimer formation. Structural work on the neuronal isoform of NOS is presently ongoing, with preliminary results indicating high structural similarity to published structures of the other NOS isoforms (T.L. Poulos, personal communication). 

Prior to the elucidation of these structures, it was known that NOS must dimerize through the oxygenase domain for catalytic function [99]. Structures of the oxygenase dimer of both iNOS and eNOS reveal that dimer formation reinforces the substrate binding channel and sequesters two pterin molecules within two symmetry-related lariats (see Figure 11) [100-102]. The dimer interface is extensive with between 1200 and 2800 of buried surface [100-102]. Additionally, conformational changes upon dimerization of iNOS expose the heme edge opposite of the eenter channel, and provide a possible interaction surface for the complementarily shaped reduetase domain [100]. 

Fig. (5). Schematic functioning of NOS [56]. NOS is represented as an homodimer (dimerization occurs through the heme domain of each subnit). Calmodulin (CaM) binding may tether the oxygenase and the reductase domain close together, thus promoting the electron transfer from NADPH to the heme center (H) via FAD and FMN. nNOS and eNOS depend upon Ca2+-calmodulin for activation, whereas the activity of iNOS is Ca2+ independent.

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