[en] Reduction of lactate to propionate by Clostridium propionicum proceeds via acrylate. In cell free extracts, lactate and acrylate are reduced to propionate and acrylate is hydrated to lactate. At no time, however, was acrylate derived from lactate detected, even when acrylate reduction was abolished. Two proteins, E1 and E2, were purified to > 90% homogeneity. Together, they catalyze the hydration of acrylyl CoA to lactyl CoA. There are not detectable intermediates. E1 and E2 form a relatively unstable complex that is catalytically active. Formation of the complex requires hydrolysis of ATP and ADP. E1 is irreversibly inactivated by O₂ with a half-life < 2 seconds. E2 contains 4 cofactors, riboflavine, FMN, a 4Fe-4S cluster and a 3Fe cluster. E1 is a single polypeptide with a molecular mass of 27,000 and E2 consists of polypeptides of molecular mass 41,000 and 48,000. The EPR properties of the 2 Fe-S clusters were characterized. The 4 Fe cluster is unusual because it has axial symmetry but with g/sub perpendicular/ > g/sub parallel/. Addition of lactyl CoA or acrylyl CoA to E2 alters the EPR spectrum of E2, indicating that they alter the environment of the Fe-S clusters. During acrylyl coA hydration, there is a [³H]-H₂O isotope effect of 5,4, indicating that formation of a β carbon-hydrogen bond is at least partially rate determining. Lactyl CoA dehydratase is very specific towards substrates. It does not eliminate chloride from 2-chloropropionyl CoA, and crotonyl CoA is hydrated to α-hydroxybutyryl CoA < 2% as acrylyl CoA is hydrated. Finally, a mechanism for hydration of acrylyl CoA is proposed