PMID:12610725

Citation	Thamer, W, Cirpus, I, Hans, M, Pierik, AJ, Selmer, T, Bill, E, Linder, D and Buckel, W (2003) A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. Arch. Microbiol. 179:197-204
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.
Links	PubMed Online version:10.1007/s00203-003-0517-8
Keywords	Amino Acid Sequence; Bacteria, Anaerobic/genetics; Bacteria, Anaerobic/growth & development; Bacteria, Anaerobic/metabolism; Clostridium/genetics; Clostridium/metabolism; Culture Media; Electron Spin Resonance Spectroscopy; Electron Transport; Ferredoxins/chemistry; Ferredoxins/genetics; Ferredoxins/metabolism; Flavodoxin/biosynthesis; Hydro-Lyases/metabolism; Iron/metabolism; Molecular Sequence Data; Oxidation-Reduction; Sequence Homology, Amino Acid
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