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Heme, a near ubiquitous cofactor, is synthesized by most organisms. The essential step of insertion of iron into the porphyrin macrocycle is mediated by the enzyme ferrochelatase. Several ferrochelatases have been characterized, and it has been experimentally shown that a fraction of them contain [2Fe-2S] clusters. It has been suggested that all metazoan ferrochelatases have such clusters, but among bacteria, these clusters have been most commonly identified in Actinobacteria and a few other bacteria. Despite this, the function of the [2Fe-2S] cluster remains undefined. With the large number of sequenced genomes currently available, we comprehensively assessed the distribution of putative [2Fe-2S] clusters throughout the ferrochelatase protein family. We discovered that while rare within the bacterial ferrochelatase family, this cluster is prevalent in a subset of phyla. Of note is that genomic data show that the cluster is not common in Actinobacteria, as is currently thought based on the small number of actinobacterial ferrochelatases experimentally examined. With available physiological data for each genome included, we identified a correlation between the presence of the microbial cluster and aerobic metabolism. Additionally, our analysis suggests that Firmicute ferrochelatases are the most ancient and evolutionarily preceded the Alphaproteobacterial precursor to eukaryotic mitochondria. These findings shed light on distribution and evolution of the [2Fe-2S] cluster in ferrochelatases and will aid in determining the function of the cluster in heme synthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The roundworm Caenorhabditis elegans is a heme auxotroph that requires the coordinated actions of HRG-1 heme permeases to transport environmental heme into the intestine and HRG-3, a secreted protein, to deliver intestinal heme to other tissues including the embryo. Here we show that heme homeostasis in the extraintestinal hypodermal tissue was facilitated by the transmembrane protein HRG-2. Systemic heme deficiency up-regulated hrg-2 mRNA expression over 200-fold in the main body hypodermal syncytium, hyp 7. HRG-2 is a type I membrane protein that binds heme and localizes to the endoplasmic reticulum and apical plasma membrane. Cytochrome heme profiles are aberrant in HRG-2-deficient worms, a phenotype that was partially suppressed by heme supplementation. A heme-deficient yeast strain, ectopically expressing worm HRG-2, revealed significantly improved growth at submicromolar concentrations of exogenous heme. Taken together, our results implicate HRG-2 as a facilitator of heme utilization in the Caenorhabditis elegans hypodermis and provide a mechanism for the regulation of heme homeostasis in an extraintestinal tissue.

Here we identify a previously undescribed protein, HemQ, that is required for heme synthesis in Gram-positive bacteria. We have characterized HemQ from Bacillus subtilis and a number of Actinobacteria. HemQ is a multimeric heme-binding protein. Spectroscopic studies indicate that this heme is high spin ferric iron and is ligated by a conserved histidine with the sixth coordination site available for binding a small molecule. The presence of HemQ along with the terminal two pathway enzymes, protoporphyrinogen oxidase (HemY) and ferrochelatase, is required to synthesize heme in vivo and in vitro. Although the exact role played by HemQ remains to be characterized, to be fully functional in vitro it requires the presence of a bound heme. HemQ possesses minimal peroxidase activity, but as a catalase it has a turnover of over 10(4) min(-1). We propose that this activity may be required to eliminate hydrogen peroxide that is generated by each turnover of HemY. Given the essential nature of heme synthesis and the restricted distribution of HemQ, this protein is a potential antimicrobial target for pathogens such as Mycobacterium tuberculosis.

In a previous study, site-directed mutagenesis experiments identified three of the four ligands to the [2Fe-2S] cluster in animal ferrochelatase as conserved cysteines in the COOH-terminal extension, Cys-403, Cys-406, and Cys-411 in human ferrochelatase (Crouse, B. R., Sellers, V. M., Finnegan, M. G., Dailey, H. A. & Johnson, M. K. (1996) Biochemistry 35, 16222-16229). The nature of the fourth ligand was left unresolved, and spectroscopic studies raised the possibility of one noncysteinyl, oxygenic ligand. In this work, we report two lines of evidence that strongly suggest the fourth ligand is a cysteine residue. Cysteine at position 196 in human recombinant ferrochelatase when changed to a serine results in an inactive enzyme that is lacking the [2Fe-2S] cluster. Furthermore, whole cell EPR studies demonstrate that in the C196S mutant the cluster fails to assemble. Additionally, the cloning and expression of Drosophila melanogaster ferrochelatase has allowed the identification, by EPR and UV-visible spectroscopy, of a [2Fe-2S]2+ cluster with properties analogous to those of animal ferrochelatases. The observation that Drosophila ferrochelatase contains only four conserved cysteines at positions 196, 403, 406, and 411, is in accord with the proposal that these residues function as cluster ligands. In the case of the ferrochelatase iron-sulfur cluster ligands, NH2-Cys-X206-Cys-X2-Cys-X4-Cys-COOH, the position distant from other ligands may lead to a spatial positioning of the cluster near the enzyme active site or at the interface of two domains, thereby explaining the loss of enzyme activity that accompanies cluster degradation and reinforcing the idea that the cluster functions as a regulatory switch.

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 unrelated Pseudomonas 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.

Coproporphyrinogen oxidase (CPO) (EC 1.3.3.3), the antepenultimate enzyme in the heme biosynthetic pathway, catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen IX. Previously, based upon metal analysis and site-directed mutagenesis of purified recombinant enzyme, it has been suggested that CPO contains and requires copper for activity (Kohno, H., Furukawa, T., Tokunaga, R., Taketani, S., and Yoshinaga, T. (1996) Biochim. Biophys. Acta 1292, 156-162). To examine this putative metal site in human CPO, the cDNA encoding human CPO was engineered into an expression vector with a His6 tag at its amino terminus, and the protein was expressed in Escherichia coli and purified to apparent homogeneity using nickel-nitroliotriacetic acid resin. Activity of the purified protein was monitored by a coupled fluorometric assay that employed purified protoporphyrinogen oxidase to convert protoporphyrinogen to protoporphyrin, thereby allowing the direct fluorescent determination of protoporphyrin IX produced. CPO has an apparent Km of 0.6 microM and an apparent Kcat of 16 min-1 with coproporphyrinogen III as substrate. Metal analysis of the enzyme was carried out via ultraviolet and visible spectroscopy, inductively coupled plasma atomic emission spectroscopy metal analysis, and electron paramagnetic resonance spectroscopy. The data presented demonstrate that human CPO contains no metal center, that it is not stimulated in vitro by iron or copper, and that addition of these metals to cultures expressing the protein has no effect.

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.

Ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, is a nuclear encoded protein that is synthesized in the cytoplasm in a precursor form and then is translocated to the matrix side of the inner mitochondrial membrane. Since the product of the enzymatic reaction, protoheme IX, is utilized almost exclusively in the cytoplasmic compartment or on the cytoplasmic side of the inner mitochondrial membrane, it was of interest to determine if the intracellular location of ferrochelatase-deficient strain of the yeast Saccharomyces cerevisiae vectors that coded for full-length ferrochelatase and a truncated form of the enzyme that lacked the mitochondrial targeting sequence were expressed. Both of these transformed cells produce approximately equal total amounts of ferrochelatase, as determined by enzyme assays and Western blot analysis, but only with the full-length construct was ferrochelatase properly localized. In cells containing the truncated construct, ferrochelatase activity was found in all membrane fractions but was not located on the matrix side of the inner mitochondrial membrane. Cells containing either construct produced heme, although the amount of heme synthesized by cells with the truncated construct was significantly less. Interestingly in cells with improperly localized ferrochelatase the amount of b-type cytochrome decreased by 80% as opposed to c- and a-type cytochromes where the decreases were only 60 and 40%, respectively.

The previously cloned hem Y gene of Bacillus subtilis (Hansson, M., and Hederstedt, L. (1992) J. Bacteriol. 174, 8081-8093) has been expressed in Escherichia coli. The expressed protein has been shown to be the penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4) and, thus, the gene designation should be hem G. This represents the first report of the expression of a cloned protoporphyrinogen oxidase from any source. The enzyme is present in the soluble cytoplasmic fraction and is, thus, unlike all previously reported eukaryotic or prokaryotic protoporphyrinogen oxidases, which are membrane-bound. It utilizes molecular oxygen as a terminal electron acceptor, and protoporphyrinogen IX, mesoporphyrinogen IX, and coproporphyrinogen III serve as substrates. The diphenyl ether herbicide acifluorfen, which is a strong inhibitor of the eukaryotic enzyme, is only weakly inhibitory. The enzyme has a predicted molecular weight of 51,200, which corresponds well with molecular weight determination via high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis. In addition the enzyme contains a putative dinucleotide binding region at the amino terminus, which is consistent with the previously demonstrated presence of a flavin moiety in the characterized mammalian enzymes.

Ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Herein we report the expression, purification, and characterization of the mature processed form of human and mouse ferrochelatase in Escherichia coli JM109. Metal analysis of the recombinant normal human ferrochelatase reveals that there are approximately 2 iron atoms/molecule of enzyme. This, along with the presence of spectral absorbance near 320 nm, is strongly suggestive that recombinant mammalian ferrochelatase as expressed in E. coli may contain an iron sulfur cluster. Two human protoporphyric ferrochelatases, F417S and M267I, were also expressed and characterized. The M267I mutant possesses the same Km and Vmax as the normal enzyme but exhibits increased thermolability when compared with normal human ferrochelatase. The F417S mutant has less than 2% of the normal activity. Since the Phe-->Ser substitution in this mutation is both chemically and structurally significant, three single amino acid substitutions (Lys, Tyr, and Trp) were engineered and characterized. None of these resulted in a protein with wild type activity. Additionally the carboxyl-terminal 10-amino acid segment, which contains Phe-417, from the yeast sequence was substituted, but this construct had no activity. Elimination of the carboxyl-terminal 30 amino acid residues (which include Phe-417) results in a protein the same length as the bacterial ferrochelatases, but it is an inactive enzyme.

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes. A cDNA encoding for the mouse erythroid 5-aminolevulinate synthase (Schoenhaut, D. S., and Curtis, P.J. (1986) Gene (Amst.) 48, 55-63) has been expressed in Escherichia coli, using the alkaline phosphatase promoter, to a level of 50-60% of the total bacterial protein. Aminolevulinate synthase was overexpressed in an active form and, therefore, was able to rescue hemA mutants, which are unable to grow in the absence of 5-aminolevulinate. A simple purification from the aminolevulinate synthase-overproducing bacterial strain yielded approximately 50 mg of protein, in a high state of purity, per liter of bacterial culture. Moreover, the expressed aminolevulinate synthase could be easily concentrated up to 6-8 mg/ml. Significantly, recombinant aminolevulinate synthase retained physical and catalytic properties identical to those of natural sources. These include the dimeric structure, subunit molecular mass, and pyridoxal 5'-phosphate as an essential cofactor. Removal of the pyridoxal 5'-phosphate led to complete loss of activity. However, the apoenzyme could be readily reconstituted by incubation with 20 microM 5'-pyridoxal phosphate. The Km values are 51 mM for glycine and 55 microM for succinyl-CoA, in the same range of the Km values determined for the nonrecombinant enzyme. This report describes the overexpression of a mammalian 5-aminolevulinate synthase in E. coli and its purification from an overproducing strain. The ready availability of the pure, cloned, sequenced erythroid 5-aminolevulinate synthase makes it possible now for questions pertinent to the enzyme's structure, mechanism, and regulation to be addressed.

The role of sulfhydryl groups in the activity of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), has been examined by using a variety of sulfhydryl group-specific reagents. The enzyme is rapidly inactivated in a pseudo-first order reaction by N-ethylmaleimide and monobromobimane and more slowly by iodoacetamide and bromotrimethylammoniobimane. Reaction with [3H]N-ethylmaleimide indicates that modification of a single sulfhydryl group is sufficient to inactivate bovine ferrochelatase. The enzyme is protected from inactivation by one substrate, ferrous iron, but not by the porphyrin substrate. Mercury and arsenite are reversible inhibitors. The fluorescence of the bound bimane is blue shifted 8 nm from that obtained in aqueous solutions and is sensitive to quenching by iodide.

The present data show that the carboxyl terminal end of the membrane binding segment (nonpolar peptide) of cytochrome b5 is present on the same side of phospholipid bilayers as the hydrophilic, heme-containing, NH2-terminal segment. This orientation was determined by observing rapid ionization of both tyrosyl residues at positions 5 and 8 from the carboxyl terminus upon addition of sodium hydroxide to the outer aqueous phase of vesicle preparations, and the reaction of one of these residues with a polar, impermeant reagent, diazotized sulfanilic acid. The rate of ionization of both aromatic residues occurred at least 1 order of magnitude faster than ionization of indigo trisulfonate trapped in the inner aqueous compartment of the vesicles. These data and consideration of our earlier characterization of cytochrome b5 structure and binding to membranes support a model for the membrane binding segment that is highly structured, penetrates to the middle of the bilayer, and loops back to the outer surface to place both the NH2 and the carboxyl termini on the same surface of the bilayer.

Ferrochelatase (protohaem ferro-lyase EC 4.99.1.1) has been purified to apparent homogeneity from the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides. The enzyme has been purified 1,640-fold with 43% recovery from isolated membrane fragments. The enzyme has a molecular weight of approximately 115,000 as estimated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography through Sephadex G-150 in the presence of 0.5% sodium deoxycholate. The purification procedure involves solubilization of ferrochelatase with sodium deoxycholate off of salt-washed membranes, followed by ammonium sulfate fraction, ion exchange chromatography on DEAE-Sephacel, followed by chromatography on Amicon dye matrix blue B, and finally Sephadex G-150. The enzyme has an extinction coefficient of 90,000 at 278 nm, and the absorption spectrum reveals no chromophoric cofactors. Purified ferrochelatase is inhibited by iodoacetamide, N-ethylmaleimide, Hg, Pb, Cu, and hemin. The apparent Km values are for mesoporphyrin IX, 20 microM; deuteroporphyrin IX, 95 microM; and iron, 20 microM.

The terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase EC 4.99.1.1), has been purified to apparent homogeneity from bovine liver mitochondria using a scheme similar to that reported by Taketani and Tokunaga (Taketani, S. and Tokunaga, R. (1981) J. Biol. Chem. 256, 12748-12753) for purification of the enzyme from rat liver. The final yield was 49% with a 2000-fold purification. Ferrochelatase has an apparent molecular weight of approximately 40,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and column chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. The purified enzyme was only slightly stimulated by added lipid and was inhibited by Mn2+, Pb2+, and Hg2+. Bovine ferrochelatase utilized proto-, meso-, and deuteroporphyrin, but not disubstituted porphyrins (2,4-disulfonic and 2,4-bisglycol deuteroporphyrin). N-Methylprotoporphyrin, a toxic by-product of the metabolism of some drugs, was found to inhibit ferrochelatase in a competitive fashion with respect to porphyrin with a Ki of 7 nM and uncompetitive with respect to iron. Manganese inhibits ferrochelatase competitively with respect to iron (Ki = 15 microM) and noncompetitively with respect to the porphyrin substrate. Heme, one of the products, is a noncompetitive inhibitor with respect to iron. These findings lead to a sequential Bi Bi kinetic model for ferrochelatase with iron binding occurring prior to porphyrin binding and heme being released prior to the release of two protons.

The COOH-terminal, anionic residues of the membrane binding segment of cytochrome b5 were examined to determine their possible significance in stabilizing the "tight" binding of the cytochrome to phospholipid vesicles. The removal of the 6 COOH-terminal residues, which include the carboxyl groups of Glu 132 and Asn 133, by carboxypeptidase digestion resulted in the loss of the characteristic "tight" binding to either synthetic phospholipid vesicles or isolated microsomes. Chemical modification of the four carboxyl groups of the nonpolar peptide of cytochrome b5 with carbodiimide and methylamine to produce a derivative with no anionic charged residues also resulted in a loss of this type of stable membrane interaction. These results suggest that the short polar COOH-terminal segment, containing two of the four carboxyl groups of the membrane binding domain of cytochrome b5, plays a crucial role in lipid-protein interactions that lead to the normal "tight" binding both in situ and in reconstituted phospholipid bilayer systems.

The role of cationic amino acid residues in the binding of porphyrin substrates by purified bovine ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) have been examined via chemical modification with camphorquinone-10-sulfonic acid, phenylglyoxal, butanedione, and trinitrobenzene sulfonate. The data obtained show that modification of arginyl, but not lysyl, residues results in the rapid inactivation of ferrochelatase. The 2,4-disulfonate deuteroporphyrin, which is a competitive inhibitor of mammalian ferrochelatase, protects the enzyme against inactivation. Ferrous iron has no protective effect. Reaction with radiolabeled phenylglyoxal shows that modification of 1 arginyl residue causes maximum inhibition of enzyme activity. The inactivation does not follow simple pseudo-first order reaction kinetics, but is distinctly biphasic in nature. Comparison of the enzyme kinetics for modified versus unmodified enzyme show that modification with camphorquinone-10-sulfonic acid has no effect on the Km for iron but does alter the Km for porphyrin.

Derivatives of cytochrome b5 that had been selectively shortened at the COOH-terminal, membrane binding segment of this amphipathic protein were employed to examine the minimum structural requirements for binding to phospholipid vesicles and for catalytic interactions in the stearyl-CoA desaturase system. Three derivatives shortened by 6, 18, and 27 amino acid residues were produced by controlled proteolysis with carboxypeptidases. The two largest derivatives bound to synthetic lipid vesicles and interacted with cytochrome b5. The third derivative neither bound to vesicles nor reacted with the desaturase. Whole nonpolar peptide and the nonpllar peptides of the two largest derivates contain only 29 to 34% polar residues, whereas the nonpolar peptide of the third derivative contains 44% polar residues. The secondary structure of the membrane binding segment was studied by circular dichroism of whole nonpolar peptide and the corresponding peptides of the three derivatives. The data for whole nonpolar peptide are consistent with a structure containing approximately 50% helical and 25% beta sheet structure. The CD of the nonpolar peptides of the two largest derivatives are consistent with structures containing 56% helix and 19% beta sheet structure, and 40% helix and 20% beta sheet structure. These data support a predicted model for secondary structure, proposed previously, based upon the primary structure (Fleming, P. J., Dailey, H. A., Corcoran D., and Strittmatter, P. (1978) J. Biol. Chem. 253, 5369-5372).

Purified mouse protoporphyrinogen oxidase (EC 1.3.3.4) and ferrochelatase (EC 4.99.1.1), the two terminal enzymes of the heme biosynthetic pathway, have been reconstituted into phospholipid vesicles, and the kinetics of the enzymes in the reconstituted systems were compared with the values obtained with the free enzymes. The apparent Km for free protoporphyrinogen oxidase in detergent solution is 5.61 +/- 0.62 microM for free protoporphyrinogen. The Km was lower when the enzyme was inserted into phospholipid vesicles (0.78 +/- 0.28 microM) and when both enzyme and substrate were incorporated into phospholipid vesicles (0.61 +/- 0.14 microM). In the presence of cardiolipin, a phospholipid present mainly in the inner mitochondrial membrane, the value of the Km for the substrate decreased 3-fold (0.20 +/- 0.02 microM). For reconstituted ferrochelatase similar kinetic analyses were carried out and it was found that the apparent Km values were only weakly affected by the lipid environment. Studies on the orientation of ferrochelatase demonstrated that approximately 50% of the enzyme in the reconstituted system had the active site located in the inner face of the phospholipid vesicle. This is in contrast to intact mitochondria where the active site is located on the matrix side of the inner mitochondrial membrane. The activation energies for both enzymes were determined for free and reconstituted enzymes. It was found that for both enzymes the activation energies were lower for the reconstituted systems than for the free enzymes.

The primary structure of the membrane bound segment of amphipathic bovine liver microsomal cytochrome b5 has been determined. This 43 residue nonpolar polypeptide is present at the COOH terminus of cytochrome b5. The sequence was obtained by automated sequence analysis and carboxypeptidase digestions. The sequence obtained is: Ile-Thr-Lys-Pro-Ser-Glu-Ser-Ile-Ile-Thr-Ile-Asp-Ser-Asn-Pro-Ser-Trp-Trp-Thr-Asn-Trp-Leu-Ile-Pro-Ala-Ile-Ser-Ala-Leu-Phe-Val-Ala-Leu-Ile-Tyr-His-Leu-Tyr-Thr-Ser-Glu-Asn. Conformational analysis using predictive algorithms is presented along with circular dichroism data on the peptide bound to phospholipid vesicles.

Protoporhyrinogen oxidase (EC 1.3.3.4), the penultimate enzyme of the heme biosynthetic pathway, catalyzes the removal of six hydrogens from protoporphyrinogen IX to form protoporphyrin IX. The enzyme in eukaryotes is associated with the inner mitochondrial membrane. In the present study we have examined requirements for solubilization of this enzyme and find that it behaves as an intrinsic membrane protein that is solubilized only with detergents such as sodium cholate. The in situ orientation of the enzyme with respect to the inner mitochondrial membrane places the active site on the cytosolic face of this membrane rather than the matrix side where the active site of ferrochelatase, the terminal pathway enzyme, is located. Examination of the kinetics of the two terminal enzymes in mitochondrial membranes demonstrates that substrate channeling occurs between these terminal two-pathway enzymes. However, examination of solubilized and membrane-reconstituted enzymes shows no evidence for a stable complex. Based upon these and previous data a model for the terminal three-pathway enzymes is presented.