title: Intrinsic tryptophan fluorescence as a probe of metal and alpha-ketoglutarate binding to TfdA, a mononuclear non-heme iron dioxygenase.
abstract: 2,4-Dichlorophenoxyacetic acid (2,4-D)/alpha-ketoglutarate (alphaKG) dioxygenase, TfdA, couples the oxidative decarboxylation of alphaKG to the oxidation of the herbicide 2,4-D using a mononuclear non-heme Fe(II) active site. The intrinsic tryptophan fluorescence associated with the four Trp residues in TfdA allows for the use of fluorescence spectroscopy to monitor the binding of iron and alphaKG to the enzyme. The fluorescence spectrum of TfdA is quenched by 50-85% upon addition of Fe(II) or alphaKG, allowing determination of their binding affinities (K(d)=7.45+/-0.61 and 3.35+/-0.35 microM, respectively). Cu, Zn, Mn, Co, Mg, and Ca dictations also quench the TfdA fluorescence with affinities similar to that of Fe(II), whereas monovalent cations such as Na, K, and Li do not. H114A and D116A mutant forms of TfdA, lacking either a histidine or aspartate metallocenter ligand, exhibit weaker affinity for both Fe(II) and alphaKG based on the fluorescence changes. Trp256 is predicted to lie within 5 A of the metal and alphaKG binding sites; however, its substitution by Phe or Leu has negligible effects on the Fe(II)- and alphaKG-dependent fluorescence quenching. Because Trp195 is predicted to be quite distant ( approximately 15 A) from the active site, we conclude that some combination of Trp113 and Trp248 serves as the reporter that senses metal and cofactor binding to TfdA.
authors.affiliation: Department of Microbiology and Molecular Genetics, 160 Giltner Hall, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Julie C
authors.initials: JC
authors.lastname: Dunning Hotopp
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Auchtung
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Hogan
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hausinger
mesh_terms: D001665:Binding Sites; D005453:Fluorescence; D007501:Iron / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D007656:Ketoglutaric Acids; D008670:Metals; D014364:Tryptophan; D007501:Iron; D010105:Oxygenases