title: A structural role for tryptophan 188 of inducible nitric oxide synthase.
abstract: All nitric oxide synthase (NOS) isotypes bear a conserved tryptophan that stacks against the proximal face of the heme cofactor. Recently two hyperactive variants of neuronal NOS were reported in which this residue (W409) was replaced by phenylalanine or tyrosine. We find that mutation of the same residue in the oxygenase domain of inducible NOS (W188) to phenylalanine causes severe destabilization of heme binding. W188F is isolated in a predominantly heme-free state, and axial thiolate ligation to the residual bound heme is unstable. However, W188F is soluble and is expressed at levels comparable to wild type. While circular dichroism spectroscopy demonstrates the loss of some secondary structure, the protein chain is not completely denatured and it retains much of its fold between pH 7.5 and 4. This proximal tryptophan of NOS represents a case where a residue is conserved within an enzyme family but for distinct purposes that are isotype-dependent.
authors.affiliation: Chemistry Department, Trent University, 1600 West Bank Drive, Peterborough, Ontario, Canada K9J 7B8.
authors.forename: D J
authors.initials: DJ
authors.lastname: Wilson
authors.forename: S P
authors.initials: SP
authors.lastname: Rafferty
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics
chemical_list: D006418:Heme; D010649:Phenylalanine; D014364:Tryptophan; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; C496321:Nos1 protein, mouse; C496317:Nos2 protein, mouse