title: A second [2Fe-2S] ferredoxin from Sphingomonas sp. Strain RW1 can function as an electron donor for the dioxin dioxygenase.
abstract: The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954-3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli. This third ferredoxin thus far identified in Sphingomonas sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E'(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp. strain RW1. RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3. In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase.
authors.affiliation: Division of Microbiology, GBF-National Research Center for Biotechnology, D-38124 Braunschweig, Germany. jean.armengaud@ibs.fr
authors.forename: J
authors.initials: J
authors.lastname: Armengaud
authors.forename: J
authors.initials: J
authors.lastname: Gaillard
authors.forename: K N
authors.initials: KN
authors.lastname: Timmis
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D020578:Sphingomonas / Q000201:enzymology*
chemical_list: C081714:2Fe-2S ferredoxin; D005288:Ferredoxins; D011994:Recombinant Proteins; D010105:Oxygenases; C108163:dioxin dioxygenase