cells Review From Synthesis to Utilization: The Ins and Outs of Mitochondrial Heme Samantha A. Swenson 1, Courtney M. Moore 2 , Jason R. Marcero 3, Amy E. Medlock 3,4,* , Amit R. Reddi 2,5,* and Oleh Khalimonchuk 1,6,7,* 1 Department of Biochemistry, University of Nebraska, Lincoln, NE 68588, USA; [email protected] 2 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA; [email protected] 3 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA; [email protected] 4 Augusta University/University of Georgia Medical Partnership, Athens, GA 30602, USA 5 Parker Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332, USA 6 Nebraska Redox Biology Center, University of Nebraska, Lincoln, NE 68588, USA 7 Fred and Pamela Buffett Cancer Center, Omaha, NE 68105, USA * Correspondence: [email protected] (A.E.M.); [email protected] (A.R.R.); [email protected] (O.K.); Tel.: +1-402-472-8060 (O.K.) Received: 17 January 2020; Accepted: 23 February 2020; Published: 29 February 2020 Abstract: 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. Keywords: mitochondria; heme; porphyrin; heme biosynthesis; membrane transporters; hemoproteins 1. Introduction Heme b, or iron protoporphyrin IX (Fe-PPIX), is an essential but potentially cytotoxic protein prosthetic group and signaling molecule. The paramount importance of this metallocofactor is highlighted by the plethora of hemoproteins present in virtually every subcellular compartment that fill vital roles within the eukaryotic cell. As a cofactor, heme is essential for mediating gas synthesis, storage and transport; electron transfer; and chemical catalysis [1–6]. As a signaling molecule, heme binding to a number of cellular factors including transcription factors, kinases, ion channels and micro RNA processing proteins [5,7–9] or its catabolism to the signaling molecule, carbon monoxide (CO), collectively regulate diverse physiological processes that include oxygen sensing, iron homeostasis, the oxidative stress response, mitochondrial respiration and biogenesis, mitophagy, apoptosis, circadian rhythms, cell cycle progression and proliferation [1,7,10–19]. Another vital role for heme b is to act as a precursor for the synthesis of other heme types important for eukaryotic physiology, including hemes c, o and a (Figure1). Cells 2020, 9, 579; doi:10.3390/cells9030579 www.mdpi.com/journal/cells Cells 2020, 9, 579 2 of 36 Cells 2020, 9, 579 2 of 38 FigureFigure 1. Chemical 1. Chemical structures structures of of the the various various hemeheme ty typespes in in eukaryotes. eukaryotes. Starting Starting with with the heme the heme b b precursorprecursor (protoheme), (protoheme), heme hemec isc is made made through through covalent attachment attachment to tothe the sulfhydryl sulfhydryl moieties moieties of of either cytochrome c or complex III subunit cytochrome c1; this process is assisted by the cytochrome c either cytochrome c or complex III subunit cytochrome c1; this process is assisted by the cytochrome c lyases.lyases. Heme Hemea is synthesizeda is synthesized from from heme hemeb through b through the the sequential sequential action action of of heme hemeo osynthase synthase and andheme heme a synthase enzymes, going through the intermediate, heme o. a synthase enzymes, going through the intermediate, heme o. All of the aforementioned heme-dependent processes require that heme is dynamically All of the aforementioned heme-dependent processes require that heme is dynamically mobilized mobilized from its site of synthesis on the matrix side of the mitochondrial inner membrane to fromheme-dependent its site of synthesis proteins on the throughout matrix side the of cell the [2 mitochondrial,3]. However, since inner heme membrane is a hydrophobic to heme-dependent and proteinscytotoxic throughout species the[20,21], cell cells [2,3]. are However, challenged since to mi hemetigate is heme a hydrophobic toxicity by andappropriately cytotoxic regulating species [20 ,21], cellscellular are challenged heme concentration to mitigate and heme bioavailability. toxicity by appropriatelyThe concentration regulating of heme is cellular dictated heme by the concentration relative and bioavailability.rates of its synthesis The and concentration degradation of[2,3]. heme Despite is dictated the fact bythat the all relativeof the proteins rates ofinvolved its synthesis in the and degradationsynthesis [ 2and,3]. degradation Despite the of fact heme that have all ofbeen the structurally proteins involved characterized in the to synthesis atomic resolution and degradation and of hemedetailed have kinetic been structurallystudies have revealed characterized their mechan to atomicismsresolution of action [2,3], and our detailed understanding kinetic studies of how have revealedthe relative their mechanisms rates of heme of synthesis action [2 ,are3], ourregula understandingted remains far of from how clear. the relative Furthermore, rates ofthe heme synthesisbioavailability are regulated of heme remains is governed far from by clear. its Furthermore,transport and thetrafficking; bioavailability yet, the of molecules heme is governedand mechanisms that mediate these processes are poorly understood. by its transport and trafficking; yet, the molecules and mechanisms that mediate these processes are Herein, we review the “ins and outs” of heme synthesis, trafficking and utilization, both inside poorlyand understood. outside the mitochondria. In particular, we will first discuss the biosynthesis of heme and how it Herein,may be regulated we review to theaffect “ins heme and bioavailability. outs” of heme Next, synthesis, we will tradiscussfficking the andmechanisms utilization, underlying both inside and outsidehow protoheme the mitochondria. IX, or heme In b particular,, is trafficked we and will converted first discuss to alternative the biosynthesis heme types, of heme including and how it may behemes regulated c, o and to aa andffect how heme it bioavailability.can exit the mitochondria Next, we and will be discuss mobilized the mechanisms for use in hemoproteins underlying how protohemethroughout IX, or the heme cell. Finally,b, is tra wefficked discuss and the converted latest approaches to alternative and technologies heme types, that can including be utilized hemes to c, o and acharacterizeand how it heme can exittransport the mitochondria and trafficking and mechanisms. be mobilized Where for appropriate, use in hemoproteins we will highlight throughout open the cell. Finally,questions we regarding discuss theheme latest homeos approachestatic mechanisms. and technologies The mobilizatio that cann beof utilizedheme for to trafficking characterize and heme signaling, of which little is understood, represents a challenging frontier in heme cell biology. A transport and trafficking mechanisms. Where appropriate, we will highlight open questions regarding better understanding of heme transport is essential for treating numerous diseases associated with heme homeostatic mechanisms. The mobilization of heme for trafficking and signaling, of which little defects in heme homeostasis, including certain cancers, cardiovascular diseases and is understood,neurodegenerative represents disorders a challenging [19,22–25]. frontier in heme cell biology. A better understanding of heme transport is essential for treating numerous diseases associated with defects in heme homeostasis, including2. Heme certain Biosynthesis cancers, cardiovascular diseases and neurodegenerative disorders [19,22–25]. Cells mitigate the toxicity of heme in part by coordinating heme synthesis (see Table 1) with 2. Heme Biosynthesis heme utilization [1]. Animals, fungi and α-proteobacteria make heme via the C4 (Shemin) pathway, Cellswhereas mitigate plants, the archaea toxicity and of other heme bacteria in part utilize by coordinating the C5 (glutamate) heme synthesispathway [26]. (see Here, Table 1we) with focus heme utilizationon the [ 1former,]. Animals, which fungi requires and α eight-proteobacteria enzymes to make construct heme heme via the from C4 (Shemin)glycine, succinyl-CoA, pathway, whereas molecular oxygen (O2) and iron (Fe) [12,27,28]. The first and last three enzymes of the heme plants, archaea and other bacteria utilize the C5 (glutamate) pathway [26]. Here, we focus on the former, synthesis pathway in animal and fungi are localized to the mitochondria and the remaining four which requires eight enzymes to construct heme from glycine, succinyl-CoA, molecular oxygen (O2) enzymes reside in the cytosol. Heme synthesis can therefore be controlled at multiple levels, and iron (Fe) [12,27,28]. The first and last three enzymes of the heme synthesis pathway in animal and including substrate availability, partitioning of heme synthetic intermediates between the fungimitochondria are localized and to thecytosol