title: Purification and properties of ferredoxinTOL. A component of toluene dioxygenase from Pseudomonas putida F1.
abstract: Toluene dioxygenase oxidizes toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. This reaction is catalyzed by a multienzyme system that is induced in cells of Pseudomonas putida F1 during growth on toluene. One of the components of toluene dioxygenase has been purified to homogeneity and shown to be an iron-sulfur protein that has been designated ferredoxinTOL. The molecular weight of ferredoxinTOL was calculated to be 15,300, and the purified protein was shown to contain 2 g of atoms each of iron- and acid-labile sulfur which appear to be organized as a single [2Fe-2S]cluster. Solutions of ferredoxinTOL were brown in color and showed absorption maxima at 277, 327, and 460 nm. A shoulder in the spectrum of the oxidized protein was discernible at 575 nm. Reduction with sodium dithionite or NADH and ferredoxinTOL reductase resulted in a decrease in visible absorbance at 460 and 575 nm, with a concomitant shift in absorption maxima to 382 and 438 nm. The redox potential of ferredoxinTOL was estimated to be -109 mV. In the oxidized state, the protein is diamagnetic. However, upon reduction it exhibited prominent electron paramagnetic resonance signals with anisotropy in g values (gx = 1.81, gy = 1.86, and gz = 2.01). Anaerobic reductive titrations revealed that ferredoxinTOL is a one-electron carrier that accepts electrons from NADH in a reaction that is mediated by a flavoprotein (ferredoxinTOL reductase). The latter is the first component in the toluene dioxygenase system. Reduced ferredoxinTOL can transfer electrons to cytochrome c or to a terminal iron-sulfur dioxygenase (ISP-TOL) which catalyzes the incorporation of molecular oxygen into toluene and related aromatic substrates.
authors.forename: V
authors.initials: V
authors.lastname: Subramanian
authors.forename: T N
authors.initials: TN
authors.lastname: Liu
authors.forename: W K
authors.initials: WK
authors.lastname: Yeh
authors.forename: C M
authors.initials: CM
authors.lastname: Serdar
authors.forename: L P
authors.initials: LP
authors.lastname: Wackett
authors.forename: D T
authors.initials: DT
authors.lastname: Gibson
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000032:analysis; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000032:analysis*; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D013455:Sulfur / Q000032:analysis
chemical_list: D000596:Amino Acids; D005288:Ferredoxins; D009243:NAD; D013455:Sulfur; D007501:Iron; D010105:Oxygenases; C023172:toluene dioxygenase