Ferrochelatase
The International Journal of Biochemistry & Cell Biology
1999, 31, 995-1000
Gloria
C. Ferreira
Department of Biochemistry and Molecular Biology, College of Medicine,
University of South Florida, Tampa, FL 33612, USA
Institute for Biomolecular Science, University of South Florida,
Tampa, FL 33612, USA
H. Lee Mofitt Cancer Center and Research Institute, University of
South Florida, Tampa, FL 33612, USA
Abstract
Ferrochelatase, the terminal enzyme of the heme biosynthetic pathway,
catalyzes the insertion of ferrous iron into protoporphyrin IX. It is encoded
by a single gene, and mutations in the human gene are associated with the
inherited disorder, erythropoietic protoporphyria. With the development
of heterologous overexpression systems and the ready availability of recombinant
ferrochelatase, new structural elements have been identified and new aspects
of the ferrochelatase-catalyzed reaction mechanism have been unraveled.
Namely, a [2Fe-2S] cluster is a prosthetic group in mammalian ferrochelatase,
a conserved and essential histidine residue appears to be involved in the
binding of the metal substrate and a conserved glutamate residue has been
proposed to have a catalytic role. The three-dimensional structure for
Bacillus subtilis ferrochelatase, the only known 'water-soluble'
ferrochelatase, revealed that the protein contains two similar domains,
each of which has a four-stranded b-sheet flanked
by a-helices; the active site was modeled to
be in a cleft defined by the two domains. The definition of the structure
and catalytic mechanism of ferrochelatase should help in the interpretation
of the impact caused by erythropoietic porphyria mutations.
1. Introduction
Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal
enzyme of the heme bio-synthetic pathway; it catalyzes the chelation of
ferrous iron into the protoporphyrin IX ring to form protoheme (Scheme
1). This enzyme is ubiquitous in nature, from bacteria to man [1]. Ferrochelatase
activity was first identified in extracts of chicken erythrocytes in 1956,
but it was not until 1974, with the description of a heme auxotrophic mutant
of Aquaspirillum itersonii devoid of ferrochelatase activity, that
the importance of the biological function of ferrochelatase was unequivocally
established [1,2]. It took 7 additional years before ferrochelatase was
purified to homogeneity. Since then the enzyme has been purified from a
wide range of prokaryotic and eukaryotic organisms [1]. With the exception
of the Bacillus subtilis enzyme [1], ferrochelatase has been found
to be a protein as-sociated with the cytoplasmic membrane in prokaryotes
and with the inner mitochondrial membrane in eukaryotes, with its active
site facing the mitochondrial matrix space [1,2]. Since the first
purification studies, research has focused on structure-function relationships
of ferrochelatase, initially on the kinetic characterization and evaluation
of the specificity of enzyme towards its substrates and, more recently,
on the identification of the amino acid residues critical in the binding
of the substrates and catalysis. Further, with the sequencing of
the human ferrochelatase gene and the recent advances in DNA recombinant
technology, erythropoietic protoporphyria, an inherited disorder of heme
biosynthesis characterized by decreased ferrochelatase activity, has been
analyzed at the molecular level. In this review I will concentrate
on the recent advances regarding structural and functional aspects of ferrochelatase
in relation to normal heme biosynthesis and, very briefly, to erythropoietic
protoporphyria.
2. Structure
In the 1990s research was launched on the isolation and sequencing
of genes and cDNAs for ferrochelatase [1]. The cDNA sequences and gene
structures for ferrochelatase of different prokaryotic and eukaryotic organisms
have been determined [1], and presently over twenty nucleotide and amino
acid sequences are available in the GenBank and the Swiss-Prot Databank.
For example, a full-length human ferrochelatase cDNA clone was reported
to encode a protein of 423 amino acids [3], while the human ferrochelatase
gene was demonstrated to contain 11 exons and span approximately 45 kb
[4]. The isolation of ferrochelatase cDNAs and genes was followed
by the rapid development of expression systems, making recombinant ferrochelatase
available in amounts and concentrations not before possible with the conventional
purification methods from natural sources [1]. Thus, structural and
functional questions requiring considerable amounts of protein could start
being addressed. Indeed, new structural features have been discovered in
the past 5 years [5-7] (see below). The initial comparison of the ferrochelatase
amino acid sequences for the different species revealed extensive similarity,
with a catalytic core (approximately 300 amino acids) common to all ferrochelatases
and eukaryotic proteins having an extra 30 to 50 amino acid stretch at
the carboxyl-terminus and an N-terminal extension of 30-80 amino acids
(Fig. 1(A)). In fact, this N-terminal extension, or presequence,
contains the information necessary to target the precursor ferrochelatase
to its final organelle destination.
[1] G.C. Ferreira, R. Franco, S.G. Lloyd, I. Moura, J.J.G. Moura, B.H.
Huynh, Structure and function of ferrochelatase , J. Bioenerg. Biomembr.
27 (1995) 221-229.
[2] H.A. Dailey, Conversion of coproporphyrinogen to protoheme in higher
eukaryotes and bacteria: terminal three enzymes, in: H.A. Dailey (Ed.),
Biosynthesis of Heme and Chlorophylls, McGraw-Hill, New York, 1990,
pp. 123-161.
[3] Y. Nakahashi, S. Taketani, M. Okuda, K. Inoue, R. Tokunaga, Molecular
cloning and sequence analysis of cDNA encoding human ferrochelatase, Biochem.
Biophys. Res. Commun. 173 (1990) 748-755.
[4] S. Taketani, J. Inazawa, Y. Nakahashi, T. Abe, R. Tokunaga, Structure
of the human ferrochelatase gene: exon/intron gene organization and location
of the gene to chromosome 18, Eur. J. Biochem. 205 (1992) 217-222.
[5] G.C. Ferreira, R. Franco, S.G. Lloyd, A.S. Pereira, I. Moura, J.J.G.
Moura, B.H. Huynh, Mammalian ferrochelatase: a new addition to the metalloenzyme
family , J. Biol. Chem. 269 (1994) 7062-7065.
[6] H.A. Dailey, M.G. Finnegan, M.K. Johnson, Human ferrochelatase
is an iron-sulfur protein, Biochemistry 33 (1994) 403-407.
[7] S. Al-Karadaghi, M. Hansson, S. Nikonov, B. Jönsson, L. Hederstedt,
Crystal structure of ferrochelatase: the terminal enzyme in heme biosynthesis,
Structure 5 (1997) 1501-1510.