Mossbauer study of 4-hydroxybutyryl-CoA dehydratase - Probing the role of an iron-sulfur cluster in an overall non-redox reaction

4-Hydroxbutyryl-CoA dehydratase from Clostridium aminobutyricum 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, Mossbauer 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 speetra of active dehydratase consisted mainly of one species (95%) with quadrupole splitting, Delta E-Q = 1.00 mm s(-1) and isomer shift, delta = 0.43 mm s(-1). Magnetically perturbed Mossbauer 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](2+) in the enzyme, most probably two clusters/homotetramer, that the four iron atoms in each cluster Lire 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-CDA dehydratase. In air-inactivated enzyme (10% residual activity), a new doublet appeared (58%) with Delta E-Q = 0.72 min s(-1), delta = 0.32 mm s(-1) and S = 0. The assignment of this subspectrum to [3Fe-4S](+) clusters, based on the typical Mossbauer parameters. is contradicted by the finding of spin zero for the species. One possible explanation could be spin-coupling of two [3Fe-4S](+) clusters in close proximity.