HUMAN FERROCHELATASE IS AN IRON-SULFUR PROTEIN

Recombinant human ferrochelatase has been expressed in Escherichia coli and purified to homogeneity. Metal analyses revealed almost-equal-to 2 mol of non-heme Fe per mol of the purified enzyme (M(r) = 40 000). The UV-visible absorption spectrum of the purified enzyme consists of a protein absorption at 278 nm (epsilon almost-equal-to 90 000 M-1 cm-1) and bands at 330 nm (epsilon almost-equal-to 24 000 M-1 cm-1), 460 nm (shoulder, epsilon almost-equal-to 11 000 M-1 cm-1), and 550 nm (shoulder, epsilon almost-equal-to 9000 M-1 cm-1) that are indicative of a [2Fe-2S]2+ cluster. The spectra show an additional band at 415 nm that varied in intensity for different preparations and is attributed, at least in part, to a minor component of enzyme-associated high-spin Fe(III) heme. The presence of a single [2Fe-2S]2+,+ Cluster as a redox active component of human ferrochelatase was confirmed by variable-temperature MCD and EPR studies of the dithionite-reduced enzyme which showed the presence of a S = 1/2 [2Fe-2S]+ cluster in addition to residual high spin Fe(II) heme. The reduced enzyme exhibits a S = 1/2 EPR signal, g = 2.00, 1.94, 1.91 accounting for 0.75 +/- 0.25 spins/molecule, that readily saturates at low microwave powers below 10 K but is observable without significant broadening at temperatures up to 100 K. The Fe-S cluster is labile and gradually disappears over period of 24 h, with concomitant loss of enzyme activity, when the enzyme is stored aerobically at 4-degrees-C. Genetically engineered C-terminal truncated human ferrochelatase, as well as the normal yeast and E. coli ferrochelatases, lack the [2Fe-2S] cluster. These data suggest that human ferrochelatase contains a [2Fe-2S]2+,+ cluster that is essential for activity and is coordinated by at least one and probably all of the four cysteines that are conserved in the C-terminal region of mammalian ferrochelatases.