Human ferrochelatase is an iron-sulfur protein.
Recombinant human ferrochelatase has been expressed in Escherichia coli and purified to homogeneity. Metal analyses revealed approximately 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 approximately 90,000 M-1 cm-1) and bands at 330 nm (epsilon approximately 24,000 M-1 cm-1), 460 nm (shoulder, epsilon approximately 11,000 M-1 cm-1), and 550 nm (shoulder, epsilon approximately 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Dailey
H A
HA
Department of Microbiology, University of Georgia, Athens 30602.
Finnegan
M G
MG
Johnson
M K
MK
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004227:Dithionite; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005294:Ferrochelatase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D004227:Dithionite; D005294:Ferrochelatase