The iron–sulfur clusters in 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans.

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 × 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 °C 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 Mössbauer 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 Mössbauer spectra were performed to determine the zero field splitting (ZFS) parameters of the cluster and the ⁵⁷ Fe 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 °C). [ABSTRACT FROM AUTHOR]