Heme is a cofactor essential for a multitude of biological reactions. The terminal step of heme synthesis occurs in the mitochondrial matrix which means that heme must be trafficked from there to other locales in the cell. Thus, identifying intracellular heme chaperones is crucial to understanding regulation of global cellular metabolism. The heme-binding protein progesterone receptor membrane component 1 (PGRMC1) has been proposed to function as a chaperone for several biologically active molecules including heme, but its cellular role is not fully understood. Here, we investigate the function of PGRMC1 in heme metabolism. By monitoring intracellular heme location and concentrations in Saccharomyces cerevisiae, we show that mutants lacking damage associated protein 1 (Dap1), the yeast ortholog of PGRMC1, have altered nuclear heme trafficking which can be corrected by complementation with DAP1 or PGRMC1. Biochemical analyses reveal that PGRMC1 co-localizes with known mitochondrial-associated membrane (MAM) proteins and proteomic comparison of interaction partners shows enrichment of MAM-associated proteins and pathways. Metabolomics profiling of wild-type and PGRMC1 knockout cells identifies significant changes of several metabolites, including heme, several amino acids, long chain acyl-carnitine, ethanolamine phosphate, and mevalonic acid. Together, these results provide evidence that PGRMC1 is involved in heme trafficking and homeostasis through MAMs.

Ferrochelatase (FECH) is the terminal enzyme in human heme biosynthesis, catalyzing the insertion of ferrous iron into protoporphyrin IX (PPIX) to form protoheme IX (Heme). Phosphorylation increases the activity of FECH, and it has been confirmed that the activity of FECH phosphorylated at T116 increases. However, it remains unclear whether the T116 site and other potential phosphorylation modification sites collaboratively regulate the activity of FECH. In this study, we identified a new phosphorylation site, T218, and explored the allosteric effects of unphosphorylated (UP), PT116, PT218, and PT116 + PT218 states on FECH in the presence and absence of substrates (PPIX and Heme) using molecular dynamics (MD) simulations. Binding free energies were evaluated with the MM/PBSA method. Our findings indicate that the PT116 + PT218 state exhibits the lowest binding free energy with PPIX, suggesting the strongest binding affinity. Additionally, this state showed a higher binding free energy with Heme compared to UP, which facilitates Heme release. Moreover, employing multiple analysis methods, including free energy landscape (FEL), principal component analysis (PCA), dynamic cross-correlation matrix (DCCM), and hydrogen bond interaction analysis, we demonstrated that phosphorylation significantly affects the dynamic behavior and binding patterns of substrates to FECH. Insights from this study provide valuable theoretical guidance for treating conditions related to disrupted heme metabolism, such as various porphyrias and iron-related disorders.

Ferrochelatases (E.C. 4.99.1.1) catalyze the insertion of ferrous iron into either protoporphyrin IX to make protoheme IX or coproporphyrin III to make coproheme III. Ferrochelatase activity in extracts or purified protein can be measured via several assays. Here, we describe a rapid real-time direct spectroscopic ferrochelatase assay for both protoporphyrin and coproporphyrin ferrochelatases.

Gender gaps in academic performance have been reported at a variety of educational levels including several national standardized exams for medical education, with men scoring higher than women. These gaps potentially impact medical school acceptance and residency matching and may be influenced by curricular design. Performance data for our 4-year integrated hybrid curriculum, which features a large proportion of active learning, revealed a gender gap with men performing better early in the curriculum and on the first national standardized exam. This gap in performance almost entirely disappeared for years 2–4 of the curriculum and the second national standardized exam.

Heme is an essential cofactor for multiple cellular processes in most organisms. In developing erythroid cells, the demand for heme synthesis is high, but is significantly lower in non-erythroid cells. While the biosynthesis of heme in metazoans is well understood, the tissue-specific regulation of the pathway is less explored. To better understand this, we analyzed the mitochondrial heme metabolon in erythroid and non-erythroid cell lines from the perspective of ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Affinity purification of FLAG-tagged-FECH, together with mass spectrometric analysis, was carried out to identify putative protein partners in human and murine cell lines. Proteins involved in the heme biosynthetic process and mitochondrial organization were identified as the core components of the FECH interactome. Interestingly, in non-erythroid cell lines, the FECH interactome is highly enriched with proteins associated with the tricarboxylic acid (TCA) cycle. Overall, our study shows that the mitochondrial heme metabolon in erythroid and non-erythroid cells has similarities and differences, and suggests new roles for the mitochondrial heme metabolon and heme in regulating metabolic flux and key cellular processes.

Metals are key elements for the survival and normal development of humans but can also be toxic to cells when mishandled. In fact, even mild disruption of metal homeostasis causes a wide array of disorders. Many of the metals essential to normal physiology are required in mitochondria for enzymatic activities and for the formation of essential cofactors. Copper is required as a cofactor in the terminal electron transport chain complex cytochrome c oxidase, iron is required for the for the formation of iron-sulfur (Fe-S) clusters and heme, manganese is required for the prevention of oxidative stress production, and these are only a few examples of the critical roles that mitochondrial metals play. Even though the targets of these metals are known, we are still identifying transporters, investigating the roles of known transporters, and defining regulators of the transport process. Mitochondria are dynamic organelles whose content, structure and localization within the cell vary in different tissues and organisms. Our knowledge of the impact that alterations in mitochondrial physiology have on metal content and utilization in these organelles is very limited. The rates of fission and fusion, the ultrastructure of the organelle, and rates of mitophagy can all affect metal homeostasis and cofactor assembly. This review will focus of the emerging areas of overlap between metal homeostasis, cofactor assembly and the mitochondrial contact site and cristae organizing system (MICOS) that mediates multiple aspects of mitochondrial physiology. Importantly the MICOS complexes may allow for localization and organization of complexes not only involved in cristae formation and contact between the inner and outer mitochondrial membranes but also acts as hub for metal-related proteins to work in concert in cofactor assembly and homeostasis.

Porphyrin and iron are ubiquitous and essential for sustaining life in virtually all living organisms. Unlike iron, which exists in many forms, porphyrin macrocycles are mostly functional as metal complexes. The iron-containing porphyrin, heme, serves as a prosthetic group in a wide array of metabolic pathways; including respiratory cytochromes, hemoglobin, cytochrome P450s, catalases, and other hemoproteins. Despite playing crucial roles in many biological processes, heme, iron, and porphyrin intermediates are potentially cytotoxic. Thus, the intersection of porphyrin and iron metabolism at heme synthesis, and intracellular trafficking of heme and its porphyrin precursors are tightly regulated processes. In this review, we discuss recent advances in understanding the physiological dynamics of eukaryotic ferrochelatase, a mitochondrially localized metalloenzyme. Ferrochelatase catalyzes the terminal step of heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to produce heme. In most eukaryotes, except plants, ferrochelatase is localized to the mitochondrial matrix, where substrates are delivered and heme is synthesized for trafficking to multiple cellular locales. Herein, we delve into the structural and functional features of ferrochelatase, as well as its metabolic regulation in the mitochondria. We discuss the regulation of ferrochelatase via post-translational modifications, transportation of substrates and product across the mitochondrial membrane, protein-protein interactions, inhibition by small-molecule inhibitors, and ferrochelatase in protozoal parasites. Overall, this review presents insight on mitochondrial heme homeostasis from the perspective of ferrochelatase.

During erythropoiesis, there is an enormous demand for the synthesis of the essential cofactor of hemoglobin, heme. Heme is synthesized de novo via an eight enzyme-catalyzed pathway within each developing erythroid cell. A large body of data exists to explain the transcriptional regulation of the heme biosynthesis enzymes, but until recently much less was known about alternate forms of regulation that would allow the massive production of heme without depleting cellular metabolites. Herein, we review new studies focused on the regulation of heme synthesis via carbon flux for porphyrin synthesis to post-translations modifications (PTMs) that regulate individual enzymes. These PTMs include cofactor regulation, phosphorylation, succinylation, and glutathionylation. Additionally discussed is the role of the immunometabolite itaconate and its connection to heme synthesis and the anemia of chronic disease. These recent studies provide new avenues to regulate heme synthesis for the treatment of diseases including anemias and porphyrias.

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium exchange, resonance Raman, molecular dynamics, and high level quantum mechanic studies have added to our understanding of the catalytic cycle of the enzyme. However, an understanding of how the metal ion is delivered and the specific role that active site residues play in catalysis remain open questions. Data are consistent with metal binding and insertion occurring from the side opposite from where pyrrole proton abstraction takes place. To better understand iron delivery and binding as well as the role of conserved residues in the active site, we have constructed and characterized a series of enzyme variants. Crystallographic studies as well as rescue and kinetic analysis of variants were performed. Data from these studies are consistent with the M76 residue playing a role in active site metal binding and formation of a weak iron protein ligand being necessary for product release. Additionally, structural data support a role for E343 in proton abstraction and product release in coordination with a peptide loop composed of Q302, S303 and K304 that act a metal sensor.

As part of the inflammatory response by macrophages, Irg1 is induced, resulting in millimolar quantities of itaconate being produced. This immunometabolite remodels the macrophage metabolome and acts as an antimicrobial agent when excreted. Itaconate is not synthesized within the erythron but instead may be acquired from central macrophages within the erythroid island. Previously, we reported that itaconate inhibits hemoglobinization of developing erythroid cells. Herein we show that this action is accomplished by inhibition of tetrapyrrole synthesis. In differentiating erythroid precursors, cellular heme and protoporphyrin IX synthesis are reduced by itaconate at an early step in the pathway. In addition, itaconate causes global alterations in cellular metabolite pools, resulting in elevated levels of succinate, 2-hydroxyglutarate, pyruvate, glyoxylate, and intermediates of glycolytic shunts. Itaconate taken up by the developing erythron can be converted to itaconyl-coenzyme A (CoA) by the enzyme succinyl-CoA:-glutarate-CoA transferase. Propionyl-CoA, propionyl-carnitine, methylmalonic acid, heptadecanoic acid, and nonanoic acid, as well as the aliphatic amino acids threonine, valine, methionine, and isoleucine, are increased, likely due to the impact of endogenous itaconyl-CoA synthesis. We further show that itaconyl-CoA is a competitive inhibitor of the erythroid-specific 5-aminolevulinate synthase (ALAS2), the first and rate-limiting step in heme synthesis. These findings strongly support our hypothesis that the inhibition of heme synthesis observed in chronic inflammation is mediated not only by iron limitation but also by limitation of tetrapyrrole synthesis at the point of ALAS2 catalysis by itaconate. Thus, we propose that macrophage-derived itaconate promotes anemia during an inflammatory response in the erythroid compartment.

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 [2Fe2S] 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.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synthases. We employ apocytochrome c and peptide analogs containing CXXCH as substrates, examining recognition determinants, thioether attachment, and subsequent release and folding of cytochrome c. Peptide analogs reveal very different recognition requirements between HCCS and CcsBA. For HCCS, a minimal 16-mer peptide is required, comprised of CXXCH and adjacent alpha helix 1, yet neither thiol is critical for recognition. For bacterial CcsBA, both thiols and histidine are required, but not alpha helix 1. Heme attached peptide analogs are not released from the HCCS active site; thus, folding is important in the release mechanism. Peptide analogs behave as inhibitors of cytochrome c biogenesis, paving the way for targeted control. eLife digest From tiny bacteria to the tallest trees, most life on Earth carries a protein called cytochrome c, which helps to create the energy that powers up cells. Cytochrome c does so thanks to its heme, a molecule that enables the chemical reactions required for the energy-creating process. Despite both relying on cytochrome c, animals and bacteria differ in the enzyme they use to attach the heme to the cytochrome. Spotting variations in how this 'cytochrome c synthase' works would help to find compounds that deactivate the enzyme in bacteria, but not in humans. However, studying cytochrome c synthase in living cells is challenging. To bypass this issue, Sutherland, Mendez, Babbitt et al. successfully reconstituted cytochrome c synthases from humans and bacteria in test tubes. This allowed them to examine in detail which structures the enzymes recognize to spot where to attach the heme onto their target. The experiments revealed that human and bacterial synthases actually rely on different parts of the cytochrome c to orient themselves. Different short compounds could also block either the human or bacterial enzyme. Variations between human and bacterial cytochrome c synthase could lead to new antibiotics which deactivate the cytochrome and kill bacteria while sparing patients. The next step is to identify molecules that specifically interfere with cytochrome c synthase in bacteria, and could be tested in clinical trials. Introduction The structure of cytochrome c (cyt c), as well as its key function in electron transport for aerobic respiration, have been known for over half a century (Dickerson et al., 1971; Ernster and Schatz, 1981). Scores of newly discovered and extraordinary electron transport chains with unique cyt c proteins in bacteria are now known, such as extracellular multiheme nanowires comprised of many c-type hemes (e.g. Deane, 2019; Wang et al., 2019). In addition to its role in respiration, cyt c is known to play other important functions, such as activation of programmed cell death in eukaryotes (apoptosis) (Ow et al., 2008; Tait and Green, 2010). Regardless of its function, each c-type heme contains two thioether attachments to a conserved CysXxxXxxCysHis (CXXCH) motif, where the histidine acts as an axial ligand to the heme iron in the native cyt c (Figure 1—figure supplement 1a,b; Dickerson et al., 1971). It is generally agreed that the covalently attached heme makes these energy conversion proteins particularly stable (e.g. Allen et al., 2005). In fact, recent engineering of novel and stable heme-based catalysts has used c-heme polypeptides produced in vivo (Kan et al., 2017; Kan et al., 2016; Watkins et al., 2017). To form c-heme, heme is attached stereochemically to each CXXCH motif and it appears that in the case of cyt c, folding into its native structure occurs after attachment (Kranz et al., 2009). Cyt c biogenesis requires accessory proteins that are needed to attach the heme group and complete maturation. Three pathways have been discovered and characterized genetically, called Systems I, II, III (Figure 1—figure supplement 1b,c) (reviewed in Kranz et al., 2009; Ferguson et al., 2008; Kranz et al., 1998; Bowman and Bren, 2008; Simon and Hederstedt, 2011; Verissimo and Daldal, 2014; Gabilly and Hamel, 2017). Systems I and II have evolved in bacteria, while System III is in most mitochondria. Each system possesses a cyt c synthase (Figure 1—figure supplement 1c, orange), which attaches the two vinyl groups of heme to cysteines of CXXCH. However, the cyt c biogenesis process, starting with CXXCH recognition, to heme attachment, to release and final folding, remains largely unknown. While in vivo studies have suggested some requirements (Babbitt et al., 2017; Babbitt et al., 2016; Corvest et al., 2010; San Francisco et al., 2013), such cyt c genetic studies do not examine problems of instability, recognition, release, or folding of the cyt c variants. Direct testing of substrates without these limitations awaited the development of in vitro reconstitution. The mitochondrial System III is composed of a cyt c synthase called HCCS (holocyt c synthase) in the intermembrane space (Figure 1a and Figure 1—figure supplement 1c, Pollock et al., 1998; Dumont et al., 1987; Babbitt et al., 2015). Bacterial systems are unrelated to HCCS and more complicated, heme attachment occurs 'outside' the cells; thus, these pathways export the heme and attach it to secreted, unfolded cyt c. System II is composed of a large integral membrane protein complex called CcsBA (Beckett et al., 2000; Dreyfuss et al., 2003; Xie and Merchant, 1996) (sometimes called ResBC [Ahuja et al., 2009; Le Brun et al., 2000]), which is proposed to both export heme and then attach it to cyt c CXXCH motifs (Feissner et al., 2006; Frawley and Kranz, 2009). Specific factors for thiol reduction of the CXXCH motifs have also been proposed (Bonnard et al., 2010; Kranz et al., 2009). Figure 1 with 7 supplements see all Download asset Open asset Cyt c is biosynthesized in vitro by mitochondrial HCCS. (a) Schematic of the in vitro heme attachment reaction of HCCS with apocytochrome c (apocyt c). Mitochondrial cyt c synthase, HCCS, positions heme (orange) and attaches it to apocyt c. Cyt c is released and folds into its native structure. Insets show the UV–vis spectra of heme. (b) UV–vis spectra of heme signal from the anaerobic reaction of WT and H154A HCCS (±heme loading [HL]) with apocyt c as outlined in a black line – initial, red line – 1 hr post-addition of DTT. Inset shows magnification of the β / α region. (c) In vitro biosynthesis of cyt c was monitored by heme stain. WT HCCS biosynthesized 12 kDa cyt c product (lanes 3 and 8). HCCS H154A, a mutant defective for heme binding, did not (lanes 5 and 10). Total protein for in vitro reaction shown by Coomassie. For (b) and (c), representative data is shown from three biological replications (independent purifications of HCCS). (d) Time course of HCCS in vitro activity. A single trial showed heme-stained cyt c product is first observed after 10 min (red, lane 5). Sypro stain shows total protein levels, α -cyt c shows total cyt c in reaction. The apocyt c dimer observed upon SDS–PAGE is due to aggregation and does not impact the results or conclusions. (e) HPLC profiles of the indicated reaction products representative of two trials. Large gaps in the cyt c biogenesis field remain such as CXXCH recognition requirements by each cyt c synthase and whether other general factors in the cell are needed for recognition, heme attachment, and folding. While specific proteins have been identified and functions hypothesized for each system (reviewed in Babbitt et al., 2015; Ferguson et al., 2008; Gabilly and Hamel, 2017; Kranz et al., 2009; Verissimo and Daldal, 2014), there has been no in vitro reconstitution studies with purified cyt c synthases, which will be needed to address these gaps. Only recently was our group able to purify the cyt c synthases, after recombinant expression in Escherichia coli (Frawley and Kranz, 2009; Merchant, 2009; Richard-Fogal et al., 2009; San Francisco et al., 2013; Sutherland et al., 2018b). Here we develop and characterize the first in vitro reconstitutions of cyt c synthases, using purified human HCCS and the bacterial CcsBA. No protein factors other than the cyt c synthases are needed in vitro for attachment and folding into a native cyt c structure. In vitro reactions with a variety of peptides containing CXXCH show that the CXXCH substrates for each cyt c synthase are quite different and that post-attachment folding of cyt c is important in release from the synthase active sites. Key differences between HCCS and CcsBA include thiol (cysteine) requirements and the alpha helix sequence adjacent to CXXCH. Peptide analogs behave as inhibitors. Because bacteria and humans (mitochondria) use very different cyt c synthases, shown here to recognize distinct features of the CXXCH substrate, specific inhibitors could constitute targeted antimicrobials, facilitating chemical control of cyt c levels in selected organisms. Results In vitro reconstitution of HCCS using apocyt c as substrate Using purified human HCCS, we reconstituted cyt c synthase activity with equine apocyt c as substrate, initially assaying formation of a peak at 550 nm, diagnostic of cyt c's typical UV–vis spectra (Figure 1a). Recombinant human HCCS (GST-tagged) is functional in vivo, attaching heme to co-expressed apocyt c (San Francisco et al., 2013) in E. coli. We have previously shown that HCCS co-purifies with heme, which is liganded to His154 (San Francisco et al., 2013). UV–vis spectra of purified HCCS shows a 423 nm and broad 560 nm absorption, typical of heme proteins, while HCCS H154A variant does not bind heme (Figure 1b, −HL). We developed a 'heme-loading (HL)' protocol to increase the levels of heme bound in HCCS (+HL, ~30% occupied) above the co-purified levels of endogenous heme (−HL, ~10% occupied). HL was also advantageous since the HL protocol removes excess heme, thus minimizing spectral interference from free heme in reactions. HL was shown to depend on the natural His154 ligand (Figure 1b, +HL black line), and loading was saturated at 2–5 µM heme (Figure 1—figure supplement 2). Initial reconstitutions were performed with wild type (wt) HCCS (±HL) and the HCCS His154Ala variant that does not bind heme (Figure 1b). Upon incubation for 1 hr in the presence of apocyt c and dithiothreitol (DTT), a sharp 550 nm peak emerged, indicative of a c-type cytochrome (Figure 1b, red with wt). This occurred with wt HCCS containing endogenous heme (−HL) and in vitro loaded heme (+HL), while HCCS H154A did not produce the 550 nm peak. A second method to determine if heme has been covalently attached to the apocyt c is to separate reactions with denaturing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by heme staining, whereby covalently attached heme electrophoreses with the polypeptide (Figure 1c). Reactions with wt HCCS (−HL and +HL) and apocyt c confirmed that heme is covalently attached to cyt c (12 kDa) in the 1 hr reaction (Figure 1c, lanes 3, 8). As expected, no cyt c was formed with the HCCS H154A variant (Figure 1c, lanes 5, 10). Pyridine hemochrome spectra is often used to determine if two, one, or no covalent bonds to heme are present, with two thioether bonds showing a 550 nm peak (c-heme) and 560 nm for none (b-heme). The in vitro synthesized product has two thioether bonds, indicated by a 550 nm peak in pyridine hemochrome spectra (Figure 1—figure supplement 3a). In vitro reconstitutions were studied for optimal conditions and requirements. Synthesis is optimal at 37°C (Figure 1—figure supplement 4), required DTT (Figure 1—figure supplement 5), with the cyt c product observed in 10 min (e.g. Figure 1d, lane 5). While cyt c is formed in both aerobic (Figure 1—figure supplement 6) and anaerobic conditions (Figure 1b,c), we decided to use anaerobic conditions for all studies since peptide substrates (below) under aerobic conditions required varying DTT concentrations, likely due to distinct thiol reducing requirements of individual peptides in air. To further characterize HCCS, substrates and products, we employed analytical HPLC size exclusion chromatography (SEC), whereby UV–vis spectra of each separated species was recorded (Figure 1e). HCCS (brown profile) elutes earlier than cyt c (green profile), and because these are 424 nm (heme) profiles, it is observed in the reaction (blue profile) that heme in HCCS decreases while cyt c product increases. These results also demonstrate that the cyt c product is released from the HCCS active site since it elutes at the same time as purified cyt c (holocyt c). We conclude that we have recapitulated in vitro the four-step process proposed previously (Figure 1—figure supplement 7) for HCCS-mediated cyt c biogenesis: heme binding (step 1), apocyt c binding (step 2), thioether formation (step 3), and release (step 4) (Babbitt et al., 2015; San Francisco et al., 2013). Next, we further characterize the released cyt c product to establish whether proper folding to the native state resulted from in vitro biogenesis. We developed a HCCS-tethered (to glutathione beads) release assay to isolate HCCS reaction product(s), confirm that cyt c is released, and obtain high yields for product characterization (Figure 2a). Spectra of the released product (Figure 2b) is identical to holocyt c. SDS–PAGE of stages in the bead release protocol (Figure 2c) showed a released product of 12 kD that heme stained and reacted with cyt c antisera (Figure 2c, lane 2). We determined spectrally that the released cyt c has folded properly, forming the Met81 ligand as well as His19 (Figure 1—figure supplement 3b). Redox titrations (Figure 2d) showed that the redox potential of the cyt c in vitro product is the same as cyt c produced in vivo, +253 mV (Figure 2d). Analyses of supernatants (released), washes, and bead-retained material allowed for an estimate that at least 62% of cyt c is released from HCCS (Figure 2c). Since heme in all cyt c's is attached stereochemically (Figure 2e), we performed circular dichroism (CD) spectra to compare the released (in vitro) product to cyt c made in vivo (Figure 2f). CD absorption of heme (~420 nm) is reduced in globins when heme binds in multiple orientations compared to a single orientation (Aojula et al., 1986; Nagai et al., 2014). Cyt c synthesized in vitro by HCCS shows an identical CD spectral profile as in vivo synthesized (Figure 2f). We conclude that in vitro reconstitution with purified HCCS results in stereochemical heme attachment, release, and proper folding of cyt c. Figure 2 Download asset Open asset Cyt c biosynthesized in vitro is released by mitochondrial HCCS. (a) Schematic of HCCS released product assay. In vitro reaction is carried out with bead tethered GST-HCCS. Centrifugation separates the beads (GST-HCCS) and supernatant (e.g. released products). (b) UV–vis spectra of supernatant from the released product assay shows characteristic 550 nm cyt c peak, indicating cyt c is matured and released from GST-HCCS beads. (c) SDS–PAGE analysis of released product assay fractions. Lane two shows released cyt c as compared to purified holocyt c (lane 8). (b) and (c) are representative of three biological replicates. The standard deviation is provided. (d) The redox potential of the released cyt c was determined by a modified Massey method (Efimov et al., 2007) and determined to be +253 mV, similar to the published value for cyt c. This is data from one of three biological replicates. (e) Schematic of heme attached to cyt c from PDB: 3ZCF with heme rotated 180o (from Babbitt et al., 2015). (f) Circular dichroism (CD) spectra of in vivo (orange, gray, yellow) vs in vitro (blue) biosynthesized cyt c. In vivo cyts c represent three independent preparations. © 2015, Elsevier permissions. Panel e is reproduced with permission from Figure 1, Babbitt et al., 2015, with permission from Elsevier. It is not covered by the CC-BY 4.0 licence and further reproduction of this figure would need permission from the copyright holder. Peptide analogs of apocyt c are recognized by HCCS and heme is covalently attached In vitro reconstitution of the cyt c synthases provides an opportunity to investigate chemically synthesized apocyt c peptides and analogs as substrates. For example, there are in vivo genetic results suggesting that alpha helix 1, adjacent to the CXXCH motif (Figure 3a), of native cyt c is necessary for maturation by HCCS (San Francisco et al., 2013; Zhang et al., 2014; Kleingardner and Bren, 2011). In fact, the bacterial cyt c has a natural deletion of Met13 in alpha helix 1, recently shown in vivo to be the basis for the inability of HCCS to mature bacterial cyt c (Babbitt et al., 2016; Verissimo et al., 2012). We wanted to determine if cyt c peptides are recognized in vitro and if so the minimal sequence for recognition and heme attachment. Initially, we examined three peptides, an 11mer, 16mer, and 20mer with the 11mer lacking the sequence of alpha helix 1 (Figure 3a). Heme stains of tricine SDS–PAGE were used to detect whether heme was covalently attached to peptides (Figure 3b). After 1 hr, reactions showed that the 16mer (Figure 3b, lane 6) and 20mer (Figure 3b, lane 8) possessed an intense heme-stained peptide of 2.8 kDa, whereas the 11mer did not (Figure 3b, lane 4). Spectral analyses showed that the 11mer reaction looked like HCCS alone (no peptide added), whereas the reactions with the 16mer and 20mer showed a 552–553 nm peak (Figure 3c). We have previously shown that some recombinant HCCS is co-purified with cyt c remaining bound (and heme attached) (San Francisco et al., 2013). UV/vis absorption of these HCCS/cyt c complexes exhibits a peak in the reduced state of 553–555 nm (Figure 1—figure supplement 7), whereas a purified heme attached peptide shows a 550 nm peak (Figure 1—figure supplement 7b). Spectral results of HCCS reactions with 16mer and 20mer peptides (i.e. 552–553 nm peaks, see Figure 3c) suggest heme is covalently attached to the peptides, but that they remain in complex with HCCS, unlike full-length cyt c produced in vitro. To further test CXXCH peptide recognition, we tested a 56mer (with alpha helix 1 and 2 of cyt c) and a 9mer (Figure 3—figure supplement 1). While the 56mer was recognized and heme attached, the 9mer was not, consistent with the in vivo results that alpha helix 1 is required for heme attachment (Babbitt et al., 2016). Because HCCS reaction with the 56mer yields a 555 nm absorption (Figure 3—figure supplement 1a), it is likely not released. Figure 3 with 3 supplements see all Download asset Open asset HCCS requires alpha helix 1 of cyt c for heme attachment to peptides containing CXXCH. (a) Sequence of three CXXCH containing peptides with alpha helix 1 and CXXCH designated. Three-dimensional structures of peptides with heme were generated from the cyt c 3D crystal structure PDB: 3ZCF, alpha helical structure is predicted, but not experimentally confirmed. In vitro reaction (as in Figure 1a) of HCCS and the peptides in 3 (a) was performed and analyzed by (b) SDS–PAGE followed by heme stain and (c) UV–vis spectra to assess heme signal. Black – initial, red – 1 hr post-addition of DTT. Inset shows magnification of the β / α region. Data is representative of three biological replicates. To confirm that heme-attached peptides remain bound to HCCS, we used both HPLC SEC and the bead release assay described above (Figure 2a). HPLC separation (Figure 4a) showed that HCCS with the 20mer reaction (blue profile) eluted at the same time as HCCS alone (brown profile), not unexpected since a small 2.8 kD unreleased product would not significantly alter size exclusion properties. However, the spectra of the 20mer reaction from the HPLC SEC shows the signature of a HCCS-bound cyt c product, with a peak at 553 nm. This supports the conclusion that the heme attached 20mer remains bound to HCCS upon HPLC SEC, explaining why no heme-peptide product elutes separately (Figure 4a, compare blue and green profiles). Results of the bead release assay also show there is very little release of the heme-attached peptides from HCCS. Spectra of the reaction supernatant (red) exhibits very little heme (Figure 4b), unlike with full cyt c (Figure 2b, red). However, eluted HCCS from the beads show a spectra consistent with heme-attached peptide still bound, with a 555 nm peak (Figure 4b, purple). Quantitation of the level of heme-attached 20mer released from HCCS was carried out using the bead release assay (Figure 4c), with 14 ± 3% of the heme-attached peptide released from HCCS. Figure 4 Download asset Open asset Peptides not released by HCCS can inhibit HCCS in vitro biosynthesis of cyt c. (a) The 20mer reaction was analyzed by SEC-HPLC (blue) and compared to HCCS alone (brown). The 'heme attached peptide' serves as a positive control for a released peptide (green). It is commercially available MP-11 (Sigma), an 11mer with heme attached that is purified from trypsinized cyt c. Insets show the spectra of the respective peaks. (b) The 'released product assay' (see Figure 2a) was performed with HCCS and the 20mer peptide. Glutathione eluted beads had a heme signal of 555 nm (purple) indicating a complex of HCCS with 20mer. The supernatant has little heme signal (red). (c) Tris–Tricine SDS–PAGE of the reaction supernatant (lane 1) and the elution from the beads (lane 4) shows that 14 ± 3% of the 20mer is released from HCCS. (b and c) are representative of seven trials and the estimated release is based on all trials. The standard deviation is provided. (d) Schematic of peptide inhibition assay with HCCS. T0 – The in vitro reaction components HCCS and peptide are combined under anaerobic conditions, T1a – Addition of DTT initiates the reaction. Reaction incubates for 1 hr at 37 C, then the reaction is measured. T1b– Apocyt c is added to the reaction to determine whether the peptide inhibits HCCS heme attachment to apocyt c. DTT is added to the reaction after T1b and incubated at 37 C for 1 hr. T2– The final reaction products were analyzed by SDS–PAGE to determine if holocyt c was matured. (e) Reactions were separated by Tris–tricine SDS–PAGE and heme- and protein-stained. The 16 and 20mers inhibit HCCS maturation of apocyt c (lanes 11, 12, 15, 16). The 11mer or no peptide do not inhibit maturation of apocyt c (lanes 3, 4, 7, 8) (see boxed bands with holocyt c). The data is representative of three biological replicates. Synthetic peptides as inhibitors of cyt c synthase activity We evaluated whether peptides recognized by HCCS would act as inhibitors of heme attachment to subsequent addition of apocyt c. We carried out reactions with the three peptides for 1 hr, then added apocyt c, taking samples throughout (Figure 4d). The 11mer behaved as expected, as if no other substrate was present, with synthesis of cyt c occurring (in Figure 4e, compare lanes 1–4 and 5–8 boxed bands). This also suggests that the 11mer is not recognized by HCCS, in that it does not prevent apocyt c from binding. However, both the 16mer (Figure 4e, lanes 9–12) and 20mer (lanes 13–16) showed heme attached to the peptides, but not to the apocyt c. We consider this inhibition of cyt c biogenesis (see Discussion). We conclude that alpha helix 1 is necessary and sufficient for recognition and attachment to the adjacent CXXCH motif. Our findings suggest that folding of cyt c is required for optimal release from the HCCS active site (see Discussion). In vitro reconstitution of CcsBA using apocyt C as substrate Our previous studies with CcsBA have used recombinant GST-tagged CcsBA (from Helicobacter), shown to be functional in vivo and co-purify with endogenous heme (Feissner et al., 2006; Frawley and Kranz, 2009; Sutherland et al., 2018b). We concluded that CcsBA is both a heme exporter and a cyt c synthase with two heme binding sites (Figure 6a). To increase CcsBA yields for in vitro and future structural studies, we explored various tagging and expression strategies, ultimately selecting a C-terminal hexahistidine tagged CcsBA which gave high yields (Figure 5a). For unknown reasons, yields were higher when the GST ORF (with stop codon), as well as a new ribosome binding site upstream of ccsBA were used (threefold higher than GST-tagged or without the GST gene: Figure 5a,b). The purified hexahistidine tagged CcsBA still possessed the natural proteolysis site we have previously characterized (Frawley and Kranz, 2009; Sutherland et al., 2018b), resulting in two polypeptides (Figure 5c, lane 4, boxed). The GST*CcsBA:His construct is hereafter referred to as CcsBA:His. Using the anaerobic in vitro reconstitution conditions described above for HCCS, both the purified GST-CcsBA and metal-affinity purified CcsBA:His, both with endogenous heme, were active for heme attachment to apocyt c in vitro (Figure 6a–d). For further studies here, we used the CcsBA:His due to its higher yields. We have previously shown that while wt CcsBA has heme in both the P-His/WWD and TM-His sites (Figure 6a), the P-His variants possess heme only in the TM-His site (Sutherland et al., 2018b). GST:CcsBA P-His mutants are unable to attach heme in vivo to cyt c4, yet co-purified with heme (Sutherland et al., 2018b). Since heme is proposed to attach to apocyt c from the P-His/WWD site (Figure 6a), we tested whether the P-His variant functions in vitro, representing ideal negative controls for genuine in vitro attachments. Importantly, the GST:CcsBA P-His variant did not attach heme to apocyt c in vitro (Figure 6b). In vitro reactions with the wt CcsBA:His shows initial spectral signatures of b-heme (Figure 6c, black spectra). Within 1–3 hr, the wt CcsBA shows two peaks of reduced heme, one at 560 nm and a 550 nm peak that is characteristic of covalent heme attached in c-type cytochromes (Figure 6c, red spectra). It is likely that the b-heme (in the TM-His site) is responsible for the absorption remaining at 560 nm. These results were confirmed by SDS–PAGE and heme stains at the different time points (Figure 6d), confirming that the wt CcsBA formed cyt c. We conclude that purified wt CcsBA acts as a cyt c synthase in vitro and that heme is attached from the P-His/WWD domain, as hypothesized from in vivo results. Figure 5 Download asset Open asset Construction of CcsBA with a C-terminal 6XHis affinity tag. (a) Schematics of CcsBA constructs used for overexpression and affinity purification. Gray, GST ORF; red, ccsB; blue ccsA; purple, C-terminal 6XHis tag. Site of natural proteolysis is shown with expected molecular weights of polypeptides. *Insertion of a stop/RBS/start cassette. UV–vis spectra Soret (~412 nm) is used to determine relative heme levels of 50 µg of purified CcsBA protein from the indicated construct. Spectra are representative of three independent purifications. (b) Affinity purifications of constructs in a. Affinity tag used for purification and relevant polypeptides are labeled. Boxed lane four is the His-tagged CcsBA used for these studies (except in Figure 6b). Data is representative of three biological replicates. Figure 6 Download asset Open asset In vitro biosynthesis of cyt c by the bacterial synthase, CcsBA. (a) Schematic of the heme attachment reaction of CcsBA with apocyt c. Note, CcsBA has two heme binding sites, one in the periplasmic WWD domain (P-heme site, orange heme) and one in the transmembrane region (TM-heme site, green heme). CcsBA is proposed to traffick heme from the TM-heme site to the P-heme site for attachment to apocyt c. CcsBA model was generated by docking the TM-region (Sutherland et al., 2018b), with a cartoon of the periplasmic region. CcsBA is combined with apocyt c under anaerobic conditions and the reaction is initiated with DTT. UV–vis spectra (insets) show the initial reaction before (black) and after addition of DTT (red). After 3 hr, reaction products are holocyt c and monoheme CcsBA. (b) In vitro reaction with WT and P-His1/2Gly GST:CcsBA (5 µM) and apocyt c (20 µM). Samples were analyzed at 0, 1, 3 hr post-addition of DTT, separated by SDS–PAGE and maturation of holocyt c monitored by heme stain. CcsBA P-His1/2Gly is defective for heme binding in the P-heme site. (c) In vitro reaction with CcsBA:His (5 or 10 µM) and apocyt c (20 µM). Black, initial spectra; blue, 1 hr; red, 3 hr; 550 nm peak indicative of holocyt c; inset shows magnification of the β / α region. (d) Samples from (c) were analyzed at 0, 1, 3 hr post-DTT addition and analyzed as in (b, e), time course of in vitro reaction with CcsBA:His (5 µM) and apocytc (20 µM). Samples were taken at indicated timepoints and analyzed as in (b, f), UV–vis spectra of selected timepoints from (e), 550 nm peak indicative of holocyt c. Magnification of the β / α region is shown. (g), HPLC SEC separation of CcsBA (orange) and an in vitro reaction (blue). Monitored at 412 nm to detect heme. Insets show full spectra of indicated fractions. A time course of in vitro reactions with wt CcsBA shows that the covalent attachment to apocyt c is measurable at 20 min, reaching a maximum at approximately 3 hr (Figure 6e). Spectra at selected time points confirm these results (Figure 6f, see 550 nm formation). To determine whether cyt c is released from CcsBA and folds into its native state, we performed HPLC SEC on CcsBA alone and from a 3

Heme is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans the heme biosynthetic pathway is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. The pathway has been extensively studied and its biosynthetic enzymes structurally characterized to varying extents. Nevertheless, understanding of the regulation of heme synthesis and factors that influence this process in metazoans remains incomplete. Therefore, we investigated the molecular organization as well as the physical and genetic interactions of the terminal pathway enzyme, ferrochelatase (Hem15), in the yeast Saccharomyces cerevisiae. Biochemical and genetic analyses revealed dynamic association of Hem15 with Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS). Loss of MICOS negatively impacts Hem15 activity, affects the size of the Hem15 high-mass complex, and results in accumulation of reactive and potentially toxic tetrapyrrole precursors that may cause oxidative damage. Restoring intermembrane connectivity in MICOS-deficient cells mitigates these cytotoxic effects. These data provide new insights into how heme biosynthetic machinery is organized and regulated, linking mitochondrial architecture-organizing factors to heme homeostasis.

Heme is a ubiquitous and essential iron containing metallo-organic cofactor required for virtually all aerobic life. Heme synthesis is initiated and completed in mitochondria, followed by certain covalent modifications and/or its delivery to apo-hemoproteins residing throughout the cell. While the biochemical aspects of heme biosynthetic reactions are well understood, the trafficking of newly synthesized heme-a highly reactive and inherently toxic compound-and its subsequent delivery to target proteins remain far from clear. In this review, we summarize current knowledge about heme biosynthesis and trafficking within and outside of the mitochondria.

Heme is an essential cofactor and signaling molecule. All heme-dependent processes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins in virtually every subcellular compartment. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. To address this, we developed a pulse-chase assay in which, upon the initiation of heme synthesis, heme mobilization into the mitochondrial matrix, cytosol and nucleus is monitored using fluorescent heme sensors. Surprisingly, we found that heme trafficking to the nucleus occurs at a faster rate than to the matrix or cytosol. Further, we demonstrate that GTPases in control of mitochondrial fusion, Mgm1, and fission, Dnm1, are positive and negative regulators of mitochondrial-nuclear heme trafficking, respectively. We also find that heme controls mitochondrial network morphology. Altogether, our results indicate that mitochondrial dynamics and heme trafficking are integrally coupled.

ADVERTISEMENT RETURN TO BOOKPREVChapterNEXTThe Development and Use of Case StudiesSarah Baas RobinsonSarah Baas Robinson Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 20602, United StatesMore by Sarah Baas Robinson, Erin DolanErin Dolan Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 20602, United StatesMore by Erin Dolan, Kathleen CornelyKathleen Cornely Department of Chemistry and Biochemistry, Providence College, Providence, Rhode Island 02918, United StatesMore by Kathleen Cornely, Amy E. MedlockAmy E. Medlock Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 20602, United StatesMore by Amy E. Medlock, Jin Kyu LeeJin Kyu Lee Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 20602, United StatesMore by Jin Kyu Lee, and Paula P. Lemons*Paula P. Lemons Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 20602, United States*E-mail: [email protected]More by Paula P. LemonsDOI: 10.1021/bk-2019-1337.ch006Publication Date (Web):December 5, 2019Publication History Published online5 December 2019Published inprint 1 January 2019RIGHTS & PERMISSIONSBiochemistry Education: From Theory to PracticeChapter 6pp 127-141ACS Symposium SeriesVol. 1337ISBN13: 9780841236332eISBN: 9780841236325 Copyright © 2019 American Chemical SocietyChapter Views87Citations2LEARN ABOUT THESE METRICSChapter Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (2 MB) Get e-Alerts

Heme is an iron-containing cofactor and signaling molecule that is essential for much of aerobic life. All heme-dependent processes in eukaryotes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins located throughout the cell. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. Herein, using genetically encoded fluorescent heme sensors, we developed a live cell assay to monitor heme distribution dynamics between the mitochondrial inner-membrane, where heme is synthesized, and the mitochondrial matrix, cytosol, and nucleus. We found that heme distribution occurs simultaneously via parallel pathways. In fact, surprisingly, we find that trafficking to the nucleus is ∼25% faster than to the cytosol or mitochondrial matrix. Moreover, we discovered that the heme biosynthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of the mitochondrial dynamics machinery, Mgm1 and Dnm1, and ER contact sites, Gem1, regulate the flow of heme between the mitochondria and nucleus. Altogether, our results indicate that the nucleus acquires heme faster than the cytosol or mitochondrial matrix, presumably for mitochondrial-nuclear retrograde signaling, and that GTPases that regulate mitochondrial dynamics and ER contact sites are hard-wired to cellular heme distribution systems. Summary Statement The factors that govern the trafficking of heme, an essential but potentially cytotoxic cofactor and signaling molecule, are poorly understood. Herein, we developed a live-cell assay to monitor heme distribution kinetics and identified the first enzyme in the heme synthesis pathway and GTPases in control of mitochondrial-ER contact sites and dynamics as being critical modulators of heme trafficking.

Heme is an essential cofactor in metazoans that is also toxic in its free state. Heme is synthesized by most metazoans and must be delivered to all cellular compartments for incorporation into a variety of hemoproteins. The heme biosynthesis enzymes have been proposed to exist in a metabolon, a protein complex consisting of interacting enzymes in a metabolic pathway. Metabolons enhance the function of enzymatic pathways by creating favorable microenvironments for pathway enzymes and intermediates, facilitating substrate transport, and providing a scaffold for interactions with other pathways, signaling molecules, or organelles. Herein we detail growing evidence for a mitochondrial heme metabolon and discuss its implications for the study of heme biosynthesis and cellular heme homeostasis.