HUMAN FERROCHELATASE IS AN IRON-SULFUR PROTEIN

Recombinant human ferrochelatase has been expressed in Escherichia col i 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 (sh oulder, 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 th at varied in intensity for different preparations and is attributed, a t least in part, to a minor component of enzyme-associated high-spin F e(III) heme. The presence of a single [2Fe-2S]2+,+ Cluster as a redox active component of human ferrochelatase was confirmed by variable-tem perature MCD and EPR studies of the dithionite-reduced enzyme which sh owed the presence of a S = 1/2 [2Fe-2S]+ cluster in addition to residu al high spin Fe(II) heme. The reduced enzyme exhibits a S = 1/2 EPR si gnal, 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 obs ervable without significant broadening at temperatures up to 100 K. Th e Fe-S cluster is labile and gradually disappears over period of 24 h, with concomitant loss of enzyme activity, when the enzyme is stored a erobically at 4-degrees-C. Genetically engineered C-terminal truncated human ferrochelatase, as well as the normal yeast and E. coli ferroch elatases, lack the [2Fe-2S] cluster. These data suggest that human fer rochelatase contains a [2Fe-2S]2+,+ cluster that is essential for acti vity and is coordinated by at least one and probably all of the four c ysteines that are conserved in the C-terminal region of mammalian ferr ochelatases.