title: A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans.
abstract: The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from ( R)-2-hydroxyglutaryl-CoA to ( E)-glutaconyl-CoA catalyzed by 2-hydroxyglutaryl-CoA dehydratase, a two-component enzyme system. The actual dehydration is mediated by component D, which contains 1.0 [4Fe-4S](2+) cluster, 1.0 reduced riboflavin-5'-phosphate and about 0.1 molybdenum (VI) per heterodimer. The enzyme has to be activated by the extremely oxygen-sensitive [4Fe-4S](1+/2+)-cluster-containing homodimeric component A, which generates Mo(V) by an ATP/Mg(2+)-induced one-electron transfer. Previous experiments established that the hydroquinone state of a flavodoxin (m=14.6 kDa) isolated from A. fermentans served as one-electron donor of component A, whereby the blue semiquinone is formed. Here we describe the isolation and characterization of an alternative electron donor from the same organism, a two [4Fe-4S](1+/2+)-cluster-containing ferredoxin (m=5.6 kDa) closely related to that from Clostridium acidiurici. The protein was purified to homogeneity and almost completely sequenced; the magnetically interacting [4Fe-4S] clusters were characterized by EPR and Mössbauer spectroscopy. The redox potentials of the ferredoxin were determined as -405 mV and -340 mV. Growth experiments with A. fermentans in the presence of different iron concentrations in the medium (7-45 microM) showed that flavodoxin is the dominant electron donor protein under iron-limiting conditions. Its concentration continuously decreased from 3.5 micromol/g protein at 7 microM Fe to 0.02 micromol/g at 45 microM Fe. In contrast, the concentration of ferredoxin increased stepwise from about 0.2 micromol/g at 7-13 microM Fe to 1.1+/-0.1 micromol/g at 17-45 microM Fe.
authors.affiliation: Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, 35032 Marburg, Germany.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Thamer
authors.forename: Irina
authors.initials: I
authors.lastname: Cirpus
authors.forename: Marcus
authors.initials: M
authors.lastname: Hans
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Pierik
authors.forename: Thorsten
authors.initials: T
authors.lastname: Selmer
authors.forename: Eckhard
authors.initials: E
authors.lastname: Bill
authors.forename: Dietmar
authors.initials: D
authors.lastname: Linder
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
mesh_terms: D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D003013:Clostridium / Q000235:genetics / Q000378:metabolism; D003470:Culture Media; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005418:Flavodoxin / Q000096:biosynthesis; D006836:Hydro-Lyases / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid
chemical_list: D003470:Culture Media; D005288:Ferredoxins; D005418:Flavodoxin; D007501:Iron; C054464:2-hydroxyglutaryl-CoA dehydratase; D006836:Hydro-Lyases