48] Purification and characterization of bacterial ferrochelatase

Abstract

Publisher Summary Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme in the heme biosynthetic pathway and catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. Ferrochelatase activity has been detected in a variety of bacteria but has been highly purified from only two. In all organisms examined the enzyme is bound to the cytoplasmic membrane and is only solubilized by detergents or chaotropic agents. This chapter describes the purification and characterization of bacterial ferrochelatase, and summarizes the work on ferrochelatase from the facultative photosynthetic organism R. sphaeroides and from Aquaspirillum itersonii . Two different ferrochelatase assays are used. The most commonly used assay quantitates product formed in a predetermined period of time by measuring hemes formed as their pyridine hemochromogen. The second assay procedure quantitates product radioactivity. The purification scheme described is for a 10 liter culture of R. sphaeroides L or 2.4.1 (ATCC). For the purification of ferrochelatase, cultures were grown at 30° in l-liter flasks containing 500 ml of yeast extract–malate–glutamate (YEMG) media at 140 rpm. A 5 ml, 16 hr culture grown in YEMG broth is used as the starter culture for each 500 ml. The molecular weight of detergent-solubilized purified R. sphaeroides ferrochelatase is 110,000 by gel filtration. The bacterial ferrochelatases seem not to be stimulated by exogeneously added fatty acids or phospholipids. The absorption spectrum of the R. sphaeroides enzyme has a maximum at 278 nm with no absorption in the visible light range. The ferrochelatase of both R. sphaeroides and A. itersonii has similar kinetic parameters and sensitivities to various metal ions. Other properties are also discussed.