Beschreibung:
<jats:p>4‐Hydroxybutyryl‐CoA dehydratase from <jats:italic>Clostridium aminobutyricum</jats:italic> catalyzes the dehydration of 4‐hydroxybutyryl‐CoA to crotonyl‐CoA. Although dehydration is an overall non‐redox reaction, the enzyme contains FAD and Fe‐S clusters. Previous work has shown that the Fe‐S clusters are difficult to reduce and therefore unlikely to be redox‐active in catalysis. Here, Mössbauer spectroscopy has been used to characterise the Fe‐S clusters in active as well as in air‐inactivated enzyme. In zero magnetic field at 80 K and 4.2 K, the spectra of active dehydratase consisted mainly of one species (95%) with quadrupole splitting, Δ<jats:italic>E</jats:italic><jats:sub>Q</jats:sub>= 1.00 mm s<jats:sup>−1</jats:sup> and isomer shift, δ= 0.43 mm s<jats:sup>−1</jats:sup> Magnetically perturbed Mössbauer spectra indicated a spin of zero. In the presence of 6 mM crotonyl‐CoA, the spectra remained unchanged. Taken together, the data show that there are [4Fe‐4S]<jats:sup>2+</jats:sup> in the enzyme, most probably two clusters/homotetramer, that the four iron atoms in each cluster are coordinated in an identical fashion, and that there is no direct interaction with substrates. We therefore infer that the Fe‐S clusters serve a structural rather than a catalytic role in 4‐hydroxybutyryl‐CoA dehydratase. In air‐inactivated enzyme (10% residual activity), a new doublet appeared (58%) with Δ<jats:italic>E</jats:italic><jats:sup>Q</jats:sup>= 0.72 mm s<jats:sup>−1</jats:sup>, δ= 0.32 mm s<jats:sup>−1</jats:sup> and <jats:italic>S</jats:italic>= 0. The assignment of this subspectrum to [3Fe‐4S]<jats:sup>+</jats:sup> clusters, based on the typical Mössbauer parameters, is contradicted by the finding of spin zero for the species. One possible explanation could be spin‐coupling of two [3Fe‐4S]<jats:sup>+</jats:sup> clusters in close proximity.</jats:p>