Abstract
Ferrochelatase was purified to homogeneity from yeast mitochondrial membranes and found to be a 40-kDa polypeptide with a pI at 6.3. Fatty acids were absolutely necessary to measure the activity in vitro. The Michaelis constants for protoporphyrin IX (9 x 10(-8) M), ferrous iron (1.6 x 10(-7) M), and zinc (9 x 10(-6) M) were determined on purified enzyme preparations in the presence of dithiothreitol. However, the Km for zinc was lower when measured in the absence of dithiothreitol (K-m(Zn2+) = 2.5 x 10(-7) M, Km(protoporphyrin) unchanged). The maximum velocities of the enzyme were 35,000 nmol of heme/h/mg of protein and 27,000 nmol of zinc-protoporphyrin/h/mg of protein. Antibodies against yeast ferrochelatase were raised in rabbits and used in studies on the biogenesis of the enzyme. Ferrochelatase is synthesized as a higher molecular weight precursor (Mr = 44,000) that is very rapidly matured in vivo to the Mr = 40,000 membrane-bound form. This precursor form of ferrochelatase was immunoprecipitated from in vitro translation (in a rabbit reticulocyte lysate system) of total yeast RNAs. The antibodies were used to characterize two yeast mutant strains deficient in ferrochelatase activity as being devoid of immunodetectable protein in vivo and ferrochelatase mRNA in vitro translation product. The N-terminal amino acid sequence of the purified protein has been established and was found to be frayed.
Highlights
Ferrochelatase was purified to homogeneity from zymes are activated by fatty acids, but thebeef enzyme is not yeast mitochondrial membranes and found to be a 40- [1,2, 4,7]
We have demonstrated that zinc and ferrous iron were competitive substrates for ferrochelatase in yeast [6] and in mammalian mitochondrial membranes [13, 14], with zinc incorporation being strongly inhibited by ferrous iron but not by ferric iron
These results suggested that therewas some modulation of heme synthesis in vivo via the ferrous iron supply and utilization by ferrochelatase
Summary
From the Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod, Centre National dela Recherche Scientifique, Universitk Paris VII, Place Jussieu, F-75251 Paris Cedex 05, France. We have demonstrated that zinc and ferrous iron were competitive substrates for ferrochelatase in yeast [6] and in mammalian mitochondrial membranes (rat or human) [13, 14], with zinc incorporation being strongly inhibited by ferrous iron but not by ferric iron These results suggested that therewas some modulation of heme synthesis in vivo via the ferrous iron supply and utilization by ferrochelatase. All the enzymespurified from vertebrates to date have almost identical molecular masses of -40,000 Da, but they differ in terms of their catalytic properties: the rat and chicken enimportant inthe functioning of the enzyme This is illustrated by the observation that, ayeast mutant which lacks protoporphyrinogen oxidase activity can accumulate and excrete protoporphyrin IX produced by nonenzymatic oxidation of protoporphyrinogen, it cannot synthesize either heme or zinc-protoporphyrin i n vivo, even though ferrochelatase activity measured in vitro is normal with either iron or zinc as substrate [15, 16]. Protein concentrations were determined (i)as described by Lowry et ul. [25] using bovine serum albumin as a standardduring the initial
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