The iron-sulfur clusters in 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans -: Biochemical and spectroscopic investigations

The reversible dehydration of (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA is catalysed by the combined action of two oxygen-sensitive enzymes from Acidaminococcus fermentans, the homodimeric component A (2 x 27 kDa) and the heterodimeric component D (45 and 50 kDa). Component A was purified to homogeneity (specific activity 25-30 s(-1)) using streptavidin-tag affinity chromatography. In the presence of 5 mM MgCl2 and 1 mM ADP or ATP, component A could be stabilized and stored for 4-5 days at 4 degreesC without loss of activity. The purification of component D from A. fermentans was also improved as indicated by the 1.5-fold higher specific activity (15 s(-1)). The content of 1.0 riboflavin 5'-phosphate (FMN) per heterodimer could be confirmed, whereas in contrast to an earlier report only trace amounts of riboflavin (< 0.1) could be detected. Each active component contains an oxygen sensitive diamagnetic [4Fe-4S](2+) cluster as revealed by UV-visible, EPR and Mossbauer spectroscopy. Reduction of the [4Fe-4S](2+) cluster in component A with dithionite yields a paramagnetic [4Fe-4S](1+) cluster with the unusual electron spin ground state S = 3/2 as indicated by strong absorption type EPR signals at high g values, g = 4-6. Spin-Hamiltonian simulations of the EPR spectra and of magnetic Mossbauer spectra were performed to determine the zero field splitting (ZFS) parameters of the cluster and the Fe-57 hyperfine interaction parameters. The electronic properties of the [4Fe-4S](2+,1+) clusters of component A are similar to those of the nitrogenase iron protein in which a [4Fe-4S](2+) cluster bridges the two subunits of the homodimeric protein. Under air component A looses its activity within seconds due to irreversible degradation of its [4Fe-4S](2+) cluster to a [2Fe-2S](2+) cluster. The [4Fe-4S](2+) cluster of component D could not be reduced to a [4Fe-4S](1+) cluster, even with excess of Ti(III)citrate or dithionite. Exposure to oxic conditions slowly converts the diamagnetic [4Fe-4S](2+) cluster of component D to a paramagnetic [3Fe-4S](+) cluster concomitant with loss of activity (30% within 24 h at 4 <degrees>C).