title: 4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum: characterization of FAD and iron-sulfur clusters involved in an overall non-redox reaction.
abstract: 4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which involves cleavage of an unactivated beta-C-H bond. The enzyme also catalyzes the apparently irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. Addition of crotonyl-CoA to the dehydratase, which contains FAD as well as non-heme iron and acid labile sulfur, led to a decrease of the flavin absorbance at 438 nm and an increase in the region from 500 to 800 nm. The protein-bound FAD was easily reduced to the semiquinone (redox equilibration within seconds) and only slowly to the hydroquinone (redox equilibration minutes to hours): the redox potentials were not unusual for flavoproteins (Eox/sq = -140 +/- 15 mV and Esq/red = -240 +/- 15 mV; pH 7.0, 25 degrees C). There was no equilibration of electrons between the flavin and the Fe-S cluster, which was difficult to reduce. After extensive photoreduction, an EPR signal indicative of a [4Fe-4S]+ cluster was detected (g-values: 2.037, 1.895, 1.844). Upon exposure to air at 0 degrees C, the enzyme lost dehydration activity completely within 40 min, but isomerase activity dropped to about 40% of the initial value and persisted for more than a day. The properties of the protein-bound FAD are consistent with a mechanism involving transient one-electron oxidation of the substrate to activate the the beta-C-H bond. The putative [4Fe-4S]2+ cluster could serve a structural role and/or as Lewis acid facilitating the leaving of the hydroxyl group.
authors.affiliation: Laboratorium für Mikrobiologie am Fachbereich Biologie der Philipps Universität Marburg, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Müh
authors.forename: I
authors.initials: I
authors.lastname: Cinkaya
authors.forename: S P
authors.initials: SP
authors.lastname: Albracht
authors.forename: W
authors.initials: W
authors.lastname: Buckel
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D003013:Clostridium / Q000201:enzymology*; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000378:metabolism*; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D007536:Isomerism; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010777:Photochemistry; D012765:Shikimic Acid / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: C101347:4-hydroxybutyryl-coenzyme A; D000214:Acyl Coenzyme A; D007506:Iron-Sulfur Proteins; C101348:vinylacetyl-coenzyme A; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D012765:Shikimic Acid; C029276:flavin semiquinone; D000105:Acetyl Coenzyme A; C010701:crotonyl-coenzyme A; C066370:4-hydroxybutyryl-CoA dehydratase; D006836:Hydro-Lyases; D010100:Oxygen