SPECTROSCOPIC CHARACTERIZATION OF A NOVEL TETRANUCLEAR FE CLUSTER IN AN IRON-SULFUR PROTEIN ISOLATED FROM DESULFOVIBRIO-DESULFURICANS

Mossbauer and EPR spectroscopies were used to characterize the Fe clus ters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATC C 27774). This protein was previously thought to contain hexanuclear F e clusters, but a recent X-ray crystallographic measurement on a simil ar protein isolated from Desulfovibrio vulgaris showed that the protei n contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster a nd a mixed-ligand cluster of novel structure [Lindley et al. (1997) Ab stract, Chemistry of Metals in Biological Systems, European Research C onference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In a ll three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra . The novel-structure cluster was found to be redox active. In the 320 -mV and as-purified samples, the cluster is at a redox equilibrium bet ween its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spect ral components associated with the four Fe sites of cluster 2 in the t hree oxidation states were identified. The spectroscopic parameters ob tained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric a nd antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electr ons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced F e sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin . For the one-electron reduced cluster, the data suggest a half-intege r spin of 9/2 Characteristic fine and hyperfine parameters for all fou r Fe sites were obtained. Structural implications and the nature of th e spin-coupling interactions are discussed.