4-Hydroxybutyryl-CoA Dehydratase from Clostridium aminobutyricum: Characterization of FAD and Iron−Sulfur Clusters Involved in an Overall Non-Redox Reaction

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Abstract
4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which involves cleavage of an unactivated β-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 °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 °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 β-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.