Harry A. Dailey

The 2.0 Å structure of human ferrochelatase, the terminal enzyme of heme biosynthesis

Human ferrochelatase (E.C. 4.99.1.1) is a homodimeric (86 kDa) mitochondrial membrane-associated enzyme that catalyzes the insertion of ferrous iron into protoporphyrin to form heme. We have determined the 2.0 Å structure from the single wavelength iron anomalous scattering signal. The enzyme contains two NO-sensitive and uniquely coordinated [2Fe-2S] clusters. Its membrane association is mediated in part by a 12-residue hydrophobic lip that also forms the entrance to the active site pocket. The positioning of highly conserved residues in the active site in conjunction with previous biochemical studies support a catalytic model that may have significance in explaining the enzymatic defects that lead to the human inherited disease erythropoietic protoporphyria.

Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters

Abstract. Ferrochelatase (E.C. 4.99.1.1, protoheme ferrolyase) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme (heme). In the past 2 years, the crystal structures of ferrochelatases from the bacterium Bacillus subtilis and human have been determined. These structures along with years of biophysical and kinetic studies have led to a better understanding of the catalytic mechanism of ferrochelatase. At present, the complete DNA sequences of 45 ferrochelatases from procaryotes and eucaryotes are available. These sequences along with direct protein studies reveal that ferrochelatases, while related, vary significantly in amino acid sequence, molecular size, subunit composition, solubility, and the presence or absence of nitric-oxide-sensitive [2Fe-2S] cluster.

The crystal structure of augmenter of liver regeneration: A mammalian FAD-dependent sulfhydryl oxidase

The crystal structure of recombinant rat augmenter of liver regeneration (ALRp) has been determined to 1.8 Å. The protein is a homodimer, stabilized by extensive noncovalent interactions and a network of hydrogen bonds, and possesses a noncovalently bound FAD in a motif previously found only in the related protein ERV2p. ALRp functions in vitro as a disulfide oxidase using dithiothreitol as reductant. Reduction of the flavin by DTT occurs under aerobic conditions resulting in a spectrum characteristic of a neutral semiquinone. This semiquinone is stable and is only fully reduced by addition of dithionite. Mutation of either of two cysteine residues that are located adjacent to the FAD results in inactivation of the oxidase activity. A comparison of ALRp with ERV2p is made that reveals a number of significant structural differences, which are related to the in vivo functions of these two proteins. Possible physiological roles of ALR are examined and a hypothesis that it may serve multiple roles is proposed.

One ring to rule them all: Trafficking of heme and heme synthesis intermediates in the metazoans

The appearance of heme, an organic ring surrounding an iron atom, in evolution forever changed the efficiency with which organisms were able to generate energy, utilize gasses and catalyze numerous reactions. Because of this, heme has become a near ubiquitous compound among living organisms. In this review we have attempted to assess the current state of heme synthesis and trafficking with a goal of identifying crucial missing information, and propose hypotheses related to trafficking that may generate discussion and research. The possibilities of spatially organized supramolecular enzyme complexes and organelle structures that facilitate efficient heme synthesis and subsequent trafficking are discussed and evaluated. Recently identified players in heme transport and trafficking are reviewed and placed in an organismal context. Additionally, older, well established data are reexamined in light of more recent studies on cellular organization and data available from newer model organisms. This article is part of a Special Issue entitled: Cell Biology of Metals.

Ferrochelatase forms an oligomeric complex with mitoferrin-1 and Abcb10 for erythroid heme biosynthesis

In erythroid cells, ferrous iron is imported into the mitochondrion by mitoferrin-1 (Mfrn1). Previously, we showed that Mfrn1 interacts with Abcb10 to enhance mitochondrial iron importation. Herein we have derived stable Friend mouse erythroleukemia (MEL) cell clones expressing either Mfrn1-FLAG or Abcb10-FLAG and by affinity purification and mass spectrometry have identified ferrochelatase (Fech) as an interacting protein for both Mfrn1 and Abcb10. Fech is the terminal heme synthesis enzyme to catalyze the insertion of the imported iron into protoporphyrin IX to produce heme. The Mfrn1-Fech and Abcb10-Fech interactions were confirmed by immunoprecipitation/Western blot analysis with endogenous proteins in MEL cells and heterologous proteins expressed in HEK293 cells. Moreover, Fech protein is induced in parallel with Mfrn1 and Abcb10 during MEL cell erythroid differentiation. Our findings imply that Fech forms an oligomeric complex with Mfrn1 and Abcb10 to synergistically integrate mitochondrial iron importation and use for heme biosynthesis.

Autosomal recessive erythropoietic protoporphyria in the United Kingdom: prevalence and relationship to liver disease

Erythropoietic protoporphyria (EPP; MIM 177000) is an inherited disorder of haem biosynthesis characterised by the onset in early childhood of lifelong acute photosensitivity of sun-exposed skin.1 It results from partial deficiency of ferrochelatase (FECH; E.C. 4.99.1.1.), which leads to accumulation of protoporphyrin IX in erythrocytes, plasma, skin, and liver. Up to 35% of patients have mildly abnormal biochemical tests of liver function, while liver failure caused by the hepatotoxic action of protoporphyrin complicates about 2% of cases.2–5 Over 70 mutations in the FECH gene have been identified in EPP families (Human Gene Mutation Database: http://www.hgmd.org/).6–10 Most individuals who are heterozygous for these mutations are asymptomatic, despite having half-normal FECH activities.11 For protoporphyrin to accumulate sufficiently to cause photosensitivity, reduction of FECH activity to below a critical threshold of about 35% of normal is required.11–14 In most patients, this additional reduction results from inheritance of a low expression FECH allele trans to a severe mutation.10,15–18 The low expression allele is the C variant of a single nucleotide polymorphism (SNP; IVS3-48C/T) in intron 3 of the FECH gene.18 Because the IVS3-48C allele is common in the general population, being present in about 11% of the white inhabitants of France,18 inheritance trans to a severe FECH mutation occurs within EPP families at a frequency that is high enough to produce a pattern of inheritance of overt EPP resembling an autosomal dominant disease with incomplete penetrance. Although co-inheritance of an IVS3-48C allele appears to explain the occurrence of photosensitivity in most EPP families,8,10,17,18 alternative mechanisms may reduce FECH activity to below threshold activity in some patients. These include autosomal recessive inheritance with an FECH mutation on both alleles,15,19–23 deletion of an FECH gene …

Identification of an FAD Superfamily Containing Protoporphyrinogen Oxidases, Monoamine Oxidases, and Phytoene Desaturase EXPRESSION AND CHARACTERIZATION OF PHYTOENE DESATURASE OFMYXOCOCCUS XANTHUS

A large number of FAD-containing proteins have previously been shown to contain a signature sequence that is referred to as the dinucleotide binding motif. Protoporphyrinogen oxidase (PPO), the penultimate enzyme of the heme biosynthetic pathway, is an FAD-containing protein that catalyzes the six electron oxidation of protoporphyrinogen IX. Sequence analysis demonstrates the presence of the dinucleotide binding motif at the amino-terminal end of the protein. Analysis of the current data base reveals that PPO has significant sequence similarities to mammalian monoamine oxidases (MAO) A and B, as well as to bacterial and plant phytoene desaturases (PHD). Previously MAOs have been shown to contain FAD, but there are no publications demonstrating the presence of FAD in purified PHDs. We have carried out the expression and purification of PHD from the bacterium Myxococcus xanthus and demonstrate the presence of noncovalently bound FAD. Sequence analysis demonstrate that PPO is closely related to bacterial PHDs and more distantly to plant PHDs and animal MAOs. Interestingly bacterial MAOs are no more closely related to PPOs, PHDs, and animal MAO’s than they are to the unrelatedPseudomonas phenyl hydroxylase. All of the related sequences contain not only the basic putative dinucleotide binding motif that is found frequently for FAD-binding proteins, but they also have high similarity in an approximately 60-residue long region that extends beyond the dinucleotide motif. This region is not found among any other proteins in the current data base and, therefore, we propose that this region is a signature motif for a superfamily of FAD-containing enzymes that is comprised of PPOs, animal MAOs, and PHDs.

Substrate interactions with human ferrochelatase

Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Human ferrochelatase is a homodimeric, inner mitochondrial membrane-associated enzyme that possesses an essential [2Fe-2S] cluster. In this work, we report the crystal structure of human ferrochelatase with the substrate protoporphyrin IX bound as well as a higher resolution structure of the R115L variant without bound substrate. The data presented reveal that the porphyrin substrate is bound deep within an enclosed pocket. When compared with the location of N-methylmesoporphyrin in the Bacillus subtilis ferrochelatase, the porphyrin is rotated by ≈100° and is buried an additional 4.5 Å deeper within the active site. The propionate groups of the substrate do not protrude into solvent and are bound in a manner similar to what has been observed in uroporphyrinogen decarboxylase. Furthermore, in the substrate-bound form, the jaws of the active site mouth are closed so that the porphyrin substrate is completely engulfed in the pocket. These data provide insights that will aid in the determination of the mechanism for ferrochelatase.

Characterization of a human and mouse tetrapyrrole-binding protein.

The cDNA for p22HBP has been cloned from human and mouse, and the protein expressed, purified, and characterized. Both mouse and human proteins bind heme and porphyrins with micromolar K(d)s, are highly homologous, monomeric, and soluble, and have a cytoplasmic location. The proteins bind metalloporphyrins, free porphyrins, and N-methylprotoporphyrin with similar affinities, and mutations of a selected set of putative metal ligating residues did not have any significant effect on the measured K(d)s. That the presence or absence of metal in the porphyrin has no effect on the binding constants and the observation that the EPR signal for heme does not change upon binding to the protein strongly suggest that p22HBP is a generic tetrapyrrole-binding protein rather than a dedicated heme-binding protein. A role for p22HBP in cellular porphyrin metabolism is discussed.

Human ferrochelatase: crystallization, characterization of the [2Fe-2S] cluster and determination that the enzyme is a homodimer.

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Previously we have demonstrated that the mammalian enzyme is associated with the inner surface of the inner mitochondrial membrane and contains a nitric oxide sensitive [2Fe-2S] cluster that is coordinated by four Cys residues whose spacing in the primary sequence is unique to animal ferrochelatase. We report here the characterization and crystallization of recombinant human ferrochelatase with an intact [2Fe-2S] cluster. Gel filtration chromatography and dynamic light scattering measurements revealed that the purified recombinant human ferrochelatase in detergent solution is a homodimer. EPR redox titrations of the enzyme yield a midpoint potential of -453+/-10 mV for the [2Fe-2S] cluster. The form of the protein that was crystallized has a single Arg to Leu substitution. This mutation has no detectable effect on enzyme activity but is critical for crystallization. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell constants of a=93.5 A, b=87.7 A, and c=110.2 A. There are two molecules in the asymmetric unit and the crystals diffract to better than 2.0 A resolution. The Fe to Fe distance of the [2Fe-2S] cluster is calculated to be 2.7 A based upon the Bijvoet difference Patterson map.