FUNCTIONAL NECESSITY AND PHYSICOCHEMICAL CHARACTERISTICS OF THE [2FE-2S] CLUSTER IN MAMMALIAN FERROCHELATASE

The recently discovered [2Fe-2S] cluster in mouse liver ferrochelatase has been characterized using UV-vis, EPR, and Mossbauer spectroscopic techniques. Studies are reported here for the recombinant protein pur ified from an overproducing transformed Escherichia coli strain. A pos itive correlation is observed between the presence of the [2Fe-2S] clu ster and the enzymatic specific activity and demonstrates the necessit y of this cofactor. Chemical analysis revealed that the preparations c ontained up to 1.3 Fe/molecule and indicated a 1:1 stoichiometry betwe en Fe and acid-labile sulfide. The [2Fe-2S] cluster in the as-isolated ferrochelatase exhibits a UV-vis spectrum indicative of a [2Fe-2S](2) cluster and is EPR-silent. The 8 T Mossbauer spectrum of the Fe-57-e nriched as-isolated protein is well simulated by parameters Delta E(Q) = 0.69 +/- 0.03 mm/s and delta = 0.28 +/- 0.02 mm/s and confirms the presence of a diamagnetic ground state. Upon reduction with sodium dit hionite, ferrochelatase shows a near-axial EPR spectrum with g-values of 2.00, 1.93, and 1.91, consistent with a S = 1/2 mixed valent Fe3+-F e2+ cluster. The Orbach temperature dependence of the EPR line widths was used to provide an estimate of the exchange coupling J, which was determined to be on the order of 500-650 cm(-1) (+JS(1) . S-2 model). Redox titrations monitored by UV-vis and EPR spectroscopy revealed mid point potentials of -390 +/- 10 and -405 +/- 10 mV, respectively. Moss bauer spectra of the sodium dithionite-reduced Fe-57-enriched ferroche latase collected at 4.2 K in the presence of magnetic fields of 60 mT and 8 T strengths were analyzed in the mixed-valent S = 1/2 ground sta te. Parameters for the ferric site are Delta E(Q) = 1.2 +/- 0.2 mm/s a nd delta = 0.28 +/- 0.03 mm/s, with somewhat anisotropic hyperfine spl ittings; for the ferrous site, Delta E(Q) = 3.3 +/- 0.1 mm/s and delta = 0.67 +/- 0.04 mm/s with anisotropic hyperfine splittings characteri stic of high-spin ferrous ion. The similarities and differences with o ther characterized [2Fe-2S](+) cluster-containing proteins are discuss ed.