Purification and characterization of membrane-bound ferrochelatase from Rhodopseudomonas sphaeroides.

Ferrochelatase (protohaem ferro-lyase EC 4.99.1.1) has been purified to apparent homogeneity from the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides. The enzyme has been purified 1,640-fold with 43% recovery from isolated membrane fragments. The enzyme has a molecular weight of approximately 115,000 as estimated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography through Sephadex G-150 in the presence of 0.5% sodium deoxycholate. The purification procedure involves solubilization of ferrochelatase with sodium deoxycholate off of salt-washed membranes, followed by ammonium sulfate fraction, ion exchange chromatography on DEAE-Sephacel, followed by chromatography on Amicon dye matrix blue B, and finally Sephadex G-150. The enzyme has an extinction coefficient of 90,000 at 278 nm, and the absorption spectrum reveals no chromophoric cofactors. Purified ferrochelatase is inhibited by iodoacetamide, N-ethylmaleimide, Hg, Pb, Cu, and hemin. The apparent Km values are for mesoporphyrin IX, 20 microM; deuteroporphyrin IX, 95 microM; and iron, 20 microM.