Enoate reductase (EC 18.104.22.168) is a protein isolated from Clostridium tyrobutyricum that contains iron, labile sulfide, FAD, and FMN. The enzyme reduces the α,β carbon-carbon double bond of nonactivated 2-enoates and in a reversible way that of 2-enals at the expense of NADH or reduced methyl viologen. UV-visible and EPR potentiometric titrations detect a semiquinone species in redox intermediate states characterized by an isotropic EPR signal at g = 2.0 without contribution at 580 nm. EPR redox titration shows two widely spread mid-point redox potentials (-190 and -350 mV at pH 7.0), and a nearly stoichiometric amount of this species is detected. The data suggest the semiquinone radical has an anionic nature. In the reduced form, the [Fe- S] moiety is characterized by a single rhombic EPR spectrum, observed in a wide range of temperatures (4.2-60 K) with g values at 2.013, 1.943, and 1.860 (-180 mV at pH 7.0). The g(max) value is low when compared with what has been reported for other iron-sulfur clusters. Mossbauer studies reveal the presence of a [4Fe-4S](+2/+1) center. One of the subcomponents of the spectrum shows an unusually large value of quadrupole splitting (ferrous character) in both the oxidized and reduced states. Substrate binding to the reduced enzyme induces subtle changes in the spectroscopic Mossbauer parameters. The Mossbauer data together with known kinetic information suggest the involvement of this iron-sulfur center in the enzyme mechanism.