SUMMARY The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized.

It has been generally accepted that biosynthesis of protoheme (heme) uses a common set of core metabolic intermediates that includes protoporphyrin. Herein, we show that the Actinobacteria and Firmicutes (high-GC and low-GC Gram-positive bacteria) are unable to synthesize protoporphyrin. Instead, they oxidize coproporphyrinogen to coproporphyrin, insert ferrous iron to make Fe-coproporphyrin (coproheme), and then decarboxylate coproheme to generate protoheme. This pathway is specified by three genes named hemY, hemH, and hemQ. The analysis of 982 representative prokaryotic genomes is consistent with this pathway being the most ancient heme synthesis pathway in the Eubacteria. Our results identifying a previously unknown branch of tetrapyrrole synthesis support a significant shift from current models for the evolution of bacterial heme and chlorophyll synthesis. Because some organisms that possess this coproporphyrin-dependent branch are major causes of human disease, HemQ is a novel pharmacological target of significant therapeutic relevance, particularly given high rates of antimicrobial resistance among these pathogens.

Variegate porphyria (VP), a low-penetrant autosomal dominant inherited disorder of haem metabolism, is characterised by photosensitivity (Fig. 1) and a propensity to develop acute neuropsychiatric attacks with abdominal pain, vomiting, constipation, tachycardia, hypertension, psychiatric symptoms and, in the worst cases, quadriplegia. Acute attacks, often precipitated by inappropriate drug therapy, are potentially fatal. While earlier workers thought the distal haem biosynthetic enzyme ferrochelatase may be involved in the genesis of VP, it was shown in the early 1980's, and is now accepted, that VP is associated with decreased protoporphyrinogen oxidase activity (PPO) (E.C.1.3.3.4). VP prevalence is much higher in South Africa than elsewhere; probably due to a founder effect with patients descending from a 17th century Dutch immigrant. PPO cDNAs from Bacillus subtilis, Myxococcus xanthus, human placenta and mouse liver have been cloned, sequenced and expressed. Human and mouse cDNAs consist of open reading frames 1431 nucleotides long, encoding a 477 amino acid protein. The human PPO gene contains thirteen exons, spanning approximately 4.5 kb. We have identified a C to T transition in codon 59 (in exon 3) resulting in an arginine to tryptophan substitution (R59W). A protein expressed from an in vitro-mutagenized PPO construct exhibits substantially less activity than the wild type. The R59W mutation was present in 43 of 45 patients with VP from 26 of 27 South African families investigated, but not in 34 unaffected relatives or 9 unrelated British patients with PPO deficiency. Since at least one of these families is descended from the founder of South African VP, this defect may represent the founder gene defect associated causally with VP in South Africa.

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.

SUMMARYFerrochelatase (protoheme ferrolyase, E.C. 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Due to the many critical roles of heme, synthesis of heme is required by the vast majority of organisms. Despite significant investigation of both the microbial and eucaryotic enzyme, details of metal chelation remain unidentified. In this work we present the first structure of the wild-type human enzyme, a lead-inhibited intermediate of the wild-type enzyme with bound metallated porphyrin macrocycle, the product bound form of the enzyme, and a higher resolution model for the substratebound form of the E343K variant. These data paint a picture of an enzyme that undergoes significant changes in secondary structure during the catalytic cycle. The role that these structural alterations play in overall catalysis and potential protein-protein interactions with other proteins, as well as the possible molecular basis for these changes, is discussed. The atomic details and structural rearrangements presented herein significantly advance our understanding of the substrate binding mode of ferrochelatase and reveal new conformational changes in a structurally conserved π helix that is predicted to have a central role in product release.

The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.

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 Ϸ100°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.heme biosynthesis ͉ protoporphyrin IX ͉ x-ray crystallography ͉ metal insertion

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.