ABSTRACT
A novel Cys site is found in the nitric oxide synthase enzymes (Table 4). In endothelial nitric oxide synthase, eNOS or NOS-3, a zinc ion is found tetrahedrally coordinated to pairs of symmetry-related Cys residues near the bottom of the dimer interface [89] (Table 4). The Cys ligands, Cys96 and CyslOl, are part of a small threestranded antiparallel (3 sheet that orientates the Cys ligands in the same direction directly across the antiparallel strands. The zinc site is further stabilized by H bonds between the Cys ligands and the backbone amide NH of Leul02 and Glyl03 as well as an H bond of the amide NH of CyslOl to the carbonyl of Asn468. The zinc is posi-o o tioned equidistant from each heme (21.6 A) and each tetrahydrobiopterin, H4B (12 A). Serl04, two amino acids removed from one of the Cys ligands, H-bonds directly to the pterin side chain hydroxyl group. The metal center is believed to act in a structural capacity to maintain the integrity of the pterin binding site. It is also centered in the most electropositive region of eNOS. It could therefore provide a binding site for the electronegative reductase domain. In addition, if one of the Cys ligands has nucleo philic capacity it could undergo S-nitrosylation [90]. A precedent for this is the DNA repair protein E. coli Ada in which a zinc-bound Cys can be methylated irreversibly in the DNA complex [91]. The nitrosylated Cys might then release zinc which may be controlled by the redox status in situ [92]. The crystal structure of the E. coli expressed human endothethial, eNOS, and the inducible form iNOS or NOS-2 also have the same zinc binding site [93]. An independent study of human iNOS expressed in E. coli found that the zinc site was not present after refolding [94], similar to an earlier study on the murine inducible, iNOS or NOS-2 , where a disulfide was found [95]. However, in the presence of zinc and reducing agents the Zn(Cys)4 site formed readily [94]. These Cys residues are conserved in 20 mammalian e, i, and n NOS enzymes, suggesting that the site may occur in all forms of NOS [89].
