The [2Fe-2S] cluster in mouse liver ferrochelatase was characterized using UV-Visible, EPR, and MOssbauer spectroscopic teclufiques. The as-isolated ferroehdatase duster exhibits a UV/Vis absorption band centered at 330 nm with distinct shoulders at 440 and 525 nm, indicative ofa [2Fe-2S]" cluster, and is EPR-silent, suggesting a diamagnetic ground state. This is confirmed by the Mossbauer spectrum of the S'Fe-enriehed as-isolated protein, which is wall simulated by parameters AEQ = 0.69 + 0.03 mm/s and d/= 0.28 + 0.02 mm/s and assuming diamagnetism. Upon reduction with sodium dithionite, the enzyme shows a near-axial EPR spectrum with g-values at 200, 1.93, and 1.91, consistent with a Fe(lll) site amiferromagnetieally coupled with a Fe(II) site to yield a S = 1/2 ground state. Redox titrations monitored by UVVisible and EPR spectroscopy reveal a midpoint potential of- 400 + 10 mV MOssbauer spectra of the sodium dithionite-redueed Fe-enriehed ferrochelatase collected at 4.2 K in the presence of different magnetic fields were analyzed in the mixed-valent S = 1/2 ground state. From kinetic studies, a positive correlation is observed between the presence of the [2Fe-2S] duster and the enzymatic specific activity.
|Publication status||Published - 1 Dec 1997|