Handling heme: The mechanisms underlying the movement of heme within and between cells

Article abstract of DOI:10.1016/j.freeradbiomed.2018.08.005

Heme is an essential cofactor and signaling molecule required for virtually all aerobic life. However, excess heme is cytotoxic. Therefore, heme must be safely transported and trafficked from the site of synthesis in the mitochondria or uptake at the cell surface, to hemoproteins in most subcellular compartments. While heme synthesis and degradation are relatively well characterized, little is known about how heme is trafficked and transported throughout the cell. Herein, we review eukaryotic heme transport, trafficking, and mobilization, with a focus on factors that regulate bioavailable heme. We also highlight the role of gasotransmitters and small molecules in heme mobilization and bioavailability, and heme trafficking at the host-pathogen interface.

Full text of DOI:10.1016/j.freeradbiomed.2018.08.005

Author’s Accepted Manuscript
Handling heme: The mechanisms underlying the
movement of heme within and between cells
Rebecca K. Donegan, Courtney M. Moore, David
A. Hanna, Amit R. Reddi
www.elsevier.com
PII:
S0891-5849(18)31327-3
https://doi.org/10.1016/j.freeradbiomed.2018.08.005
FRB13869
DOI:
Reference:
To appear in: Free Radical Biology and Medicine
Received date: 4 July 2018
Revised date: 31 July 2018
Accepted date: 1 August 2018
Cite this article as: Rebecca K. Donegan, Courtney M. Moore, David A. Hanna
and Amit R. Reddi, Handling heme: The mechanisms underlying the movement
of heme within and between cells, Free Radical Biology and Medicine,
https://doi.org/10.1016/j.freeradbiomed.2018.08.005
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Handling heme: The mechanisms underlying the movement of heme within and
between cells.
a
a
a
a,b,c,*
Rebecca K. Donegan , Courtney M. Moore , David A. Hanna , and Amit R. Reddi
a
School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA
3
0332
b
School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332;
Parker Petit Institute for Bioengineering & Biosciences, Georgia Institute of
c
Technology, Atlanta, GA 30332
*
Correspondence should be addressed to: Amit R. Reddi, 315 Ferst Dr, Rm 3313,
Atlanta, GA 30332, amit.reddi@chemistry.gatech.edu

Abstract.
Heme is an essential cofactor and signaling molecule required for virtually all
aerobic life. However, excess heme is cytotoxic. Therefore, heme must be safely
transported and trafficked from the site of synthesis in the mitochondria or uptake at the
cell surface, to hemoproteins in most subcellular compartments. While heme synthesis
and degradation are relatively well characterized, little is known about how heme is
trafficked and transported throughout the cell. Herein, we review eukaryotic heme
transport, trafficking, and mobilization, with a focus on factors that regulate bioavailable
heme. We also highlight the role of gasotransmitters and small molecules in heme
mobilization and bioavailability, and heme trafficking at the host-pathogen interface.
Graphical abstract
Keywords: heme, heme trafficking, heme transport, nitric oxide, hydrogen peroxide,
iron, host-pathogen interactions

Introduction.
Heme (iron protoporphyrin IX, heme b, or protoheme) is an essential but
potentially toxic iron containing cofactor and signaling molecule. In mammals, heme
constitutes the most abundant form of iron due to its requirement as a cofactor for the
2
oxygen (O ) carrier hemoglobin (Hb). Indeed, heme iron constitutes 95% of the total iron
quota in humans,[1] typically 3-4 g of iron, and erythrocytes accumulate as much as ~80
9
mM heme or ~5x10 heme molecules per cell.[2] Beyond the requirement for heme in
2
O transport, virtually every cell and organism on the planet that metabolizes oxygen
requires heme for viability. The Lewis acidity, redox activity, and hydrophobicity of heme
makes it essential for many biological functions, including as a cofactor that enables
electron transfer, catalysis, and gas sensing and transport, and as a signaling molecule
in the regulation of transcription factors, ion channels, kinases, and cell surface
receptors.[3, 4] However, the very same properties that make heme essential also
render it toxic to the cell. Indeed, heme can inappropriately bind to various biomolecules
and alter their structure and function[5, 6] and can catalyze the formation of deleterious
reactive oxygen species (ROS).[7, 8] Therefore, the concentration and bioavailability of
heme must be tightly controlled. Whether heme is synthesized endogenously or
acquired from exogenous sources, it must be safely transported and trafficked to
hemoproteins located in virtually every subcellular compartment, including the nucleus,
ER, golgi, mitochondria, cytosol, and peroxisomes. Given that hemoproteins span a
continuum between being soluble, membrane bound or extracellular, and reside in
different pH and redox environments, heme trafficking mechanisms must be attuned to
and compatible with diverse biological locations. Moreover, since cells can export and
traffic heme between cells in metazoans,[9, 10] there must be mechanisms in place to
safely share and regulate this nutrient on a systemic level. In the context of host-
pathogen interactions and complex microbial communities,[11, 12] cells must even steal
heme to ensure survival in resource-limited settings, necessitating that heme acquisition
factors are constantly interacting with and competing against rival species. Despite the
tremendous importance of heme transport and trafficking for aerobic life, the molecules
and mechanisms underlying intra- and inter-cellular heme mobilization and its dynamics
are poorly understood.

Herein, we review the current state of knowledge on eukaryotic heme transport,
trafficking, and dynamics, including its import and export from cells, as well as highlight
heme homeostatic mechanisms of bacterial and fungal human pathogens that interact
with host heme. We begin by describing conceptual paradigms for heme transport and
trafficking, review the factors that dictate heme concentration and bioavailability, with a
focus on the latter, discuss heme trafficking dynamics and the molecules that may
mobilize heme, and consider heme homeostatic mechanisms in human pathogens in
order to highlight heme trafficking at the inter-species host-pathogen interface.
Altogether, we aim to provide a comprehensive snapshot of the latest advances on the
mechanisms underlying the mobilization of heme and highlight open questions.
Conceptual paradigm for heme trafficking and mobilization.
In order to mitigate heme toxicity, cells carefully control the concentration and
bioavailability of heme. Heme concentration is regulated by its biosynthesis and
degradation, which as discussed below are relatively well-understood processes.
Indeed, the mechanisms and atomic resolution structures of all eight enzymes of the
heme biosynthetic pathway and the heme-degrading enzyme, heme oxygenase, are
known.[3, 4, 13] On the other hand, the molecules and mechanisms that control heme
availability are poorly understood.[3, 4, 13] The speciation of heme in complex biological
matrices, including in cells and tissues, is important for understanding its transport,
trafficking, and mobilization. For reference, “free” or aqueous heme at neutral pH is 5-
th
coordinate, with iron coordinated by the four tetrapyrrolic nitrogens, and a 5 water or
hydroxo axial ligand.[14] Heme can readily form back-to-back π-π dimers, with
Dimer
D
dimerization dissociation constants, K
, on the order of 500 nM over a pH range
between 6.0 and 9.0.[14] The monomer-dimer equilibria of heme is responsible for the
large observed variation in the reduction potential of the “free” heme Fe(III)/Fe(II) redox
couple, determined to be as low as -220 mV vs. the normal hydrogen electrode (NHE)
in concentrated heme solutions or as high as -50 mV vs. NHE in more dilute heme
Cytosol
solutions.[15, 16] Given the reducing environment of the cell, E
m
~ -320 mV vs.
NHE,[17] and assuming heme equilibrates with the primary redox buffer in cells, the
glutathione (GSH)/ glutathione disulfide (GSSG) system, both monomeric and dimeric

“free” heme is expected to be in its reduced state. However, in the cellular milieu, the
speciation of aqueous heme would deviate considerably due to the large compendium
of biomolecules that can bind and buffer heme. As a consequence, a useful formalism
for conceptualizing heme in biology is as the sum of exchange inert and labile
complexes.[3, 4]
Exchange labile and inert heme corresponds to heme-complexes that can or
cannot, respectively, exchange heme with other biomolecules. Exchange inert heme is
bound in a non-dissociable manner and/or is kinetically inaccessible by other factors,
while labile heme is kinetically accessible and/or dissociable. In this context, the
mobilization of heme for intra- or inter-cellular trafficking and/or heme signaling can
occur via two primary mechanisms: a. inducing the release or transfer of heme from a
stable exchange inert heme-bound biomolecule or b. exchanging labile heme between
various buffering factors and downstream clients such as transporters, trafficking
factors, apo-hemoproteins, or other receptors down thermodynamic gradients (Figure
1). In terms of mechanism “a”, an otherwise exchange inert heme-complex may transfer
heme to a specific client protein via protein-protein interactions that drive the release
and transfer of heme. This would be analogous to metallochaperones discovered for
iron, e.g. PCBP1,[18] or copper, e.g. CCS1,[19, 20] which specifically bind and deliver
the metal to the iron storage protein ferritin or the copper enzyme Cu/Zn Superoxide
Dismutase (SOD1), respectively. Alternatively, an exchange inert heme-complex may
release its heme to the labile pool due to any number of molecular events that could
cause heme dissociation, including protonation of heme coordinating His, Cys, or Tyr
residues, oxidation of His or Cys heme binding residues by H
2 2
O and other ROS,
binding of gaseous molecules like NO or H S to the heme iron that could weaken its
2
interactions with coordinating ligands, allosteric interactions that cause changes in
protein structure that weaken the heme affinity, proteolysis, or conditions that promote
protein unfolding. In the context of heme signaling, the intact exchange inert heme-
complex itself could be the substrate for a receptor that controls heme-regulated
processes.
Trafficking or signaling in the context of exchange labile heme complexes would
involve the transfer of heme between various buffering factors to client proteins with

greater heme affinity down thermodynamic gradients. Further complicating matters is
that exchange inert heme complexes can become labile through various covalent and
non-covalent modifications, as previously discussed, or redox events at the heme iron
center that lead to the dissociation of heme if the heme affinity of a given molecule is
oxidation state dependent. Until recently, the concentration and physiological relevance
of labile heme was largely unknown, but often invoked in discussions of heme
homeostasis. Recent studies using enzymatic and fluorescent heme reporters in
Baker’s yeast and various non-erythroid human cell lines have estimated cytosolic labile
heme to be between ~25-300 nM,[21-23] which can be up to ~10% of the total cellular
heme concentration. In the malaria parasite, Plasmodium falciparum, which hyper-
accumulates heme in a food vacuole, cytosolic labile heme was estimated to be about
one order of magnitude higher, ~1.6 μM, using a genetically encoded fluorescent heme
sensor.[24] Moreover, it was recently determined that labile heme may be functionally
significant. In Saccharomyces cerevisiae, sequestration of cytosolic labile heme due to
expression of a high affinity hemoprotein, Cytochrome b562, resulted in the diminution of
the activity of a nuclear heme dependent transcription factor, Hap1.[25] However, the
nature (speciation, oxidation state, and localization) and dynamics of exchange labile
heme pools are not well understood. In cell biological terms, labile heme must be
accessed by buffering factors, transporters, chaperones, or lipid vesicles to promote the
transport and trafficking of heme to hemoproteins throughout the cell. The identification
of heme homeostatic factors have historically relied on biochemical approaches, e.g.
identifying proteins that bind heme to hemin agarose or blue sepharose, or forward
genetic screens to identify genes that contribute to a heme-related phenotype. [3, 4]
More recently, new heme imaging platforms that provide a direct readout for
bioavailable heme have been used to identify heme homeostatic factors in reverse
genetic screens. [3, 4] The continued utilization of these approaches to identify the
molecules that mediate heme transport and trafficking will help inform a better cell
biological and biochemical understanding of heme homeostatic mechanisms.
Heme synthesis and degradation.

The synthesis and degradation of heme are often thought to be the primary
means by which cells control the concentration of heme.[26] With few exceptions, all
eukaryotes synthesize heme via the highly conserved Shemin pathway, which involves
eight enzymatic steps that are partitioned between the mitochondria and cytosol (Figure
2).[27] The first and rate-limiting step of heme synthesis is the condensation of glycine
and succinyl-CoA to form 5-aminolevulinic acid (5-ALA), which is catalyzed by ALA
synthase (ALAS) in the mitochondrial matrix. 5-ALA is then transported from the matrix
into the cytosol where four cytosolic enzymes convert eight molecules of 5-ALA to
coproporphyrinogen III. In the first of the cytosolic biosynthesis steps, two 5-ALA
molecules are condensed by ALA dehydratase (ALAD) to form porphobilinogen (PBG).
Next, four PBG molecules are condensed into hydroxymethybilane via the cytosolic
enzyme PBG deaminase (PBGD). Hydroxymethylbilane is then cyclized by
uroporphyrinogen synthase (UROS) to make uroporphyrinogen III. In the final cytosolic
biosynthesis step, uroporphyrinogen III is decarboxylated to form coproporphyrinogen III
via uroporphyrinogen decarboxylase (UROD). In most eukaryotes, the last three heme
synthesis steps from coproporphyrinogen III to heme occur in the mitochondria.
Coproporphyrinogen III is trafficked to the intermembrane space (IMS) for oxidation to
protoporphyrinogen IX via coproporphyrinogen oxidase (CPOX). CPOX is localized to
the IMS in most eukaryotes requiring protoporphyrinogen IX to be transported across
the inner mitochondrial membrane for the last two heme biosynthesis steps in the
mitochondrial matrix. However, in yeast, CPOX is cytosolic, requiring
protoporphyrinogen IX to be transported across both mitochondrial membranes into the
matrix.[13] In the matrix, protoporphyrinogen IX is oxidized to protoporphyrin IX via
protoporphyrinogen oxidase (PPOX). The final step of heme synthesis occurs on the
matrix-side of the mitochondrial inner-membrane with the ferrochelatase (FECH)
catalyzed insertion of ferrous iron into protoporphyrin IX. Eukaryotic heme synthesis and
its regulation have been previously reviewed comprehensively in the literature[27-31].
In prokaryotes, it has more recently been discovered that heme synthesis occurs
via three different pathways. Variations in the pathways occur during the initial synthesis
of 5-ALA, and the final steps from uroporphyrinogen III to heme. In most metazoans,
fungi and alphaproteobacteria, 5-ALA is synthesized via the Shemin pathway, which

utilizes glycine and succinyl coenzyme A. Most bacteria and archaea, on the other
hand, utilize the C5 pathway, which converts the five carbons of glutamate from
glutamyl-tRNA to 5-ALA in two steps. From 5-ALA to uroporphyrinogen III, eukaryotes
and prokaryotes all share a common pathway utilizing three cytosolic enzymes, with
most prokaryotes synthesizing heme from 5-ALA via the canonical pathway that
eukaryotes utilize. Actinobacteria and firmicutes, however, both phyla of mostly gram-
positive bacteria, synthesize heme from uroporphyrinogen III via a noncanonical
pathway through the intermediates coproporphyrin and iron-coproheme.[32] The
prokaryotic pathways, and their regulation, has been extensively reviewed
elsewhere.[33]
Heme is degraded enzymatically by heme oxygenase. Most heme oxygenases
degrade heme to biliverdin, CO and free iron,[34] though some bacterial heme
oxygenases do not produce CO and biliverdin during heme degradation.[35] In
eukaryotes, two isoforms of heme oxygenase are expressed; inducible HO-1, which is
induced by heme, metal ions, hormones, and stressors like oxidative stress and heat
shock, among others,[36] and the constitutively expressed HO-2, which is most
common in the brain and testis.[37] HO-1 degradation of heme is dependent upon an
electron donor, such as cytochrome P450 (CYP450), and is co-localized with biliverdin
reductase (BVR), which reduces biliverdin to bilirubin preventing inhibition of HO-1. HO-
1
is localized to the ER where it degrades cytosolic heme[38], and has also been
reported to be localized to the nucleus[39], plasma membrane[40] and mitochondria.[41,
2] While the function of HO-1 outside of the ER is debated, co-localization of HO-1
4
with CYP450 and BVR in the plasma membrane and mitochondria suggests HO-1 may
be able to degrade heme in these locations.[34]
Heme synthesis and degradation within the cell is tightly regulated by heme
availability. This regulation is important due to the need to balance the requirement for
heme in cellular processes and the cytotoxicity of excess heme. For instance, ALAS
transcription and mitochondrial translocation is regulated by heme, and heme induces
expression of HO-1.[43] While the synthesis and degradation of heme are well studied,
many questions remain, including those relating to the transport of iron, glycine and
protoporphyrinogen IX into the mitochondria and the transport of ALA into the cytosol

(
Figure 2). Additionally, there is still more to be understood about the role of protein-
protein interactions in regulating heme synthesis. For instance, in differentiating
erythroid cells, three of the mitochondrial resident heme synthesis enzymes, ALAS2,
PPOX, and FECH were found to interact, forming a heme biosynthetic supercomplex, or
metabolon, with Abcb10, which has roles in ALA export and iron homeostasis (Figure
2), and Abcb7, which is involved in Fe-S cluster formation.[44] Identification of additional
proteins in the complex, the role of this association in regulating heme synthesis and
trafficking, and whether the metabolon exists in other cell types remains to be
determined.
Trafficking of endogenously synthesized heme.
Once heme is synthesized on the matrix side of the mitochondrial inner-
membrane (Figure 2), it must then be shuttled for the synthesis of c-type cytochromes
or heme a, which are required for respiration, or trafficked to hemoproteins within the
mitochondrial matrix or IMS, or to extra-mitochondrial hemoproteins that reside in
virtually every subcellular compartment or even outside the cell. The hierarchy of heme
distribution, i.e. the preference of certain organelles to acquire heme before others, or
the factors that regulate it, are not known. In addition, very little is understood about the
transport and trafficking of heme and its spatio-temporal dynamics. This section will
focus on what is known about the trafficking of endogenously synthesized heme both
within and outside the mitochondria.
Trafficking of synthesized heme within the mitochondria
The rate-limiting step of FECH is the dissociation of heme. As a consequence,
there may be chaperones or heme binding proteins that catalyze the dissociation or
transfer of heme from FECH in vivo.[45] Indeed, as discussed below, the putative heme
chaperone, PGRMC1, was found to interact with FECH in the heme metabolon, and
may directly accept heme prior to its trafficking.[46, 47] Second, many matrix or inner-
membrane localized heme binding proteins, such as cholesterol side-chain cleavage
enzyme (also referred to as P450scc[48] or CYP11A1) or cytochrome b of Complex III
(cytochrome bc
1
complex),[49] which are both localized to the mitochondrial inner
membrane may interact directly with FECH to bind newly synthesized heme. Third,

mitochondria, which accumulate as much as ~30 μM heme,[50] have an exceedingly
low amount of labile heme in the matrix, < 1 nM,[22] suggesting that heme is trafficked
from FECH in a manner that limits its dissociation into the labile heme pool. However,
the factors governing the trafficking of heme from FECH are still not well understood
and need to be further elucidated.
For mitochondrial proteins outside the matrix, heme must traverse the inner
membrane to reach soluble hemoproteins within the IMS such as cytochrome c
peroxidase (CCP). In principle, the hydrophobic lipophilic nature of heme could enable
its diffusion across membranes.[4] However, the ability of heme to catalyze the
production of ROS and oxidize lipids,[7, 8] which would lead to the disruption of
membrane structure, implies that heme is either transported (discussed below) or
ferried across the membrane in a chaperoned manner similar to other redox active
metals; albeit the proteins responsible and how this occurs is still largely a mystery.
Recently, the progesterone receptor membrane component 1 (PGRMC1), a heme
binding protein that spans both the inner and outer membrane of the mitochondria and
is found at ER-mitochondrial contact sites, was found to interact with FECH and the
heme synthesis metabolon. [46, 47, 51] The preference for PGRMC1 to interact with
FECH in the “product release” conformation, and the ability of PGRMC1 to transfer
5
heme to cytochrome b , provides evidence for its potential role as a heme chaperone
that acts as a conduit for newly synthesized heme to reach the ER where it can
hemylate a number of ER-localized hemoproteins or be distributed throughout the cell
via the ER-golgi network.[46] However, the exact role and molecular mechanisms of
PGRMC1 in heme trafficking still need to be determined.
Trafficking of synthesized heme via transporters.
After its synthesis in the mitochondria, heme must be trafficked for incorporation
into hemoproteins throughout the cell, and very little is known about the transporters,
chaperones and buffering factors responsible (Figure 3). If heme is trafficked
sequentially from the mitochondria to the cytosol, then into various compartments, heme
transporters likely exist to transport heme from the mitochondria to buffering factors and
hemoproteins within the cytosol. Indeed, over the last 15 years, a number of putative

heme transporters have been identified, including Feline Leukemia Virus Subgroup C
Cellular Receptors (FLVCR), Heme responsive genes (HRG) 1 and 4, and multidrug
resistance protein 5 (MRP-5, also called ABCC5) (Figure 3). The FLVCR proteins,
which are members of the major facilitator superfamily (MFS) have been implicated in
both heme import and export, and though their method of transport is unknown, they do
not appear to utilize the bidirectional gradient-dependent transport used by many
members of the MFS.[52] The heme exporters FLVCR1a and FLVCR1b are splice
variants that are localized to the plasma and mitochondrial membranes, respectively.
FLVCR 1a, a ~60kDa protein of ~560 amino acids, is predicted to have twelve
hydrophobic transmembrane domains (TMDs), as is common for MFS proteins,[53]
while the FLVCR1b isoform is ~28 kDa, with six predicted TMDs.[54] FLVCR1a and b
are ubiquitously expressed with high expression in brain, liver, kidney, intestine and
bone marrow in mammals.[55] FLVCR1b, which is the only putative mitochondrial heme
exporter identified to date, was reported to transport heme from the mitochondria into
the cytosol, and has been shown to be necessary for the differentiation of erythroid
cells.[54] However, there has been limited biochemical characterization of FLVCR1b,
raising many questions about its function and localization. For example, is FLVCR1b
located on the inner or outer mitochondrial membrane, or does it span both
membranes? Does FLVCR1b interact directly with FECH and the heme metabolon?
Interestingly, unlike its plasma membrane isoform FLVCR1a, the mitochondrial
associated FLVCR1b lacks the amino acids associated with binding hemopexin (Hx),
which increases FLVCR1a heme export across the plasma membrane. Does FLVCR1b
also interact with accessory proteins within the mitochondria or cytosol to facilitate heme
transport? Additionally, lower eukaryotes, including yeast, do not encode FLVCR
homologs, which have only been identified in mammals, suggesting the existence of
alternative mechanisms to transport heme out of the mitochondria. Therefore, future
studies are needed to determine if there are additional heme transporters or alternative
mechanisms of heme export.
Once in the cytosol, heme can be trafficked to cytosolic hemoproteins or into
other subcellular compartments. MRP-5 was identified in Caenorhabditis elegans to be
necessary for heme export from the intestines.[56] Studies in yeast and mammalian

cells suggest MRP-5 is necessary for transport of heme from the cytosol into the
secretory pathway.[56] Once in the secretory pathway, heme can be trafficked to
hemoproteins within the pathway or other subcellular locations, the plasma membrane
or trafficked to the extracellular space. Heme may also be trafficked from the cytosol by
binding to heme chaperone proteins that are then translocated into organelles, similar to
the twin-arginine translocation (TAT) system in bacteria.[57] In E. coli, hemoproteins
acquire heme in the cytoplasm and are then translocated outside of the cell via TAT.
The identified hemoprotein, YcdB, can also act as a peroxidase, pointing to a potential
for dual enzymatic and chaperone functions of known hemoproteins.[57] Another
putative cytosolic heme trafficking factor is BVR, which has been implicated in
facilitating the trafficking of heme into the nucleus.[58] The putative heme binding
peptide of BVR was found to have a heme dissociation constant of ~240 nM, which is
similar to the ~25-300 nM span of labile heme concentrations found in mammalian cells
[
21-23], suggesting that BVR may sense and acquire heme from the LH pool. While
BVR induces HO-1 expression,[58] it has not been directly demonstrated that BVR
transports heme into the nucleus and alters nuclear heme concentration. Moreover, the
mechanism of trafficking is unknown.
Heme can also be transported from the cytosol across the plasma membrane
into the extracellular space. FLVCR1a is localized to the plasma membrane and exports
heme from the cell. Heme export via FLVCR1a is necessary to prevent heme toxicity in
hepatocytes,[59] enterocytes,[60] erythroid cells,[61] and endothelial cells.[62]
FLVCR1a has not been found to be bidirectional and only exports heme when heme
binding proteins, such as Hx or albumin are present.[52] Excess heme in the plasma is
mostly (~80%) bound via lipoproteins before being transferred to albumin or to Hx for
CD91 mediated endocytosis of Hx-heme by hepatocytes.[63] It is not known if the
preference of FLVCR1a for Hx is specific, or if are there other unidentified high affinity
hemoproteins that would also enhance FLVCR1a transport. Indeed, many questions
involving heme export via FLVCR1a remain. Is FLVCR1a export necessary in all cells
and is export induced under heme stress? Is the release of heme to Hx a method of
preventing free heme in the plasma or is the transport of heme by Hx to hepatocytes

important for inter-cellular heme trafficking? More work is needed to determine the role
of FLVCR1a in heme homeostasis and trafficking.
Heme buffering factors within the cytosol.
The cytosol contains the greatest repository of labile heme, 20-340 nM [21-23],
even more so than the nucleus or mitochondria, < 1 nM.[22] Heme is likely buffered and
trafficked within the cytosol in a manner that mitigates the toxicity of heme. Over the
years, many heme-binding proteins have been identified based on their affinity for
hemin-agarose or blue sepharose and were proposed to have roles in heme
homeostasis (Figure 3), including heme binding proteins HBP22[64] and HBP23[65]
(
also known as peroxiredoxin-1, PRX-1), so named for their molecular weights of 22
and 23 kDa respectively, glutathione S-transferase (GSH-S transferase),[66] HBP-1
also known as SOUL)[67] and fatty acid binding protein (FABP).[68] These proteins
(
may act to bind and buffer excess heme in the cytosol to prevent toxicity, albeit in vivo
evidence for their roles in heme homeostasis is lacking.
An illustrative but interesting case is that of HBP23/PRX1. PRX1 is a thiol
peroxidase that was recently found to bind heme with a 1:1 stoichiometry and a
dissociation constant of 170 nM [69], which is within the 20-340 nM consensus range of
labile heme in cells [21-23]. PRX1, which binds heme using a catalytic Cys, Cys52,
exhibits a loss in thiol peroxidase activity upon heme binding. [69] Additionally, heme
binding to PRX1 suppresses both the peroxidase activity of heme and the H
mediated degradation of heme. [69] Given the abundance of PRX1 in the cytosol,
20 μM, and ability to bind and potentially detoxify and protect heme from degradation,
2 2
O -
∼
PRX1 may have dual roles as a peroxide scavenging thiol peroxidase and a heme
buffering or storage protein. However, as with many other heme binding proteins
proposed to play a role in heme homeostasis, the in vivo function of PRX1 in heme
homeostasis has yet to be demonstrated [69].
In contrast, the glycolytic enzyme glyceraldehyde phosphate dehydrogenase
(GAPDH) is the only cytosolic protein that has been determined to bind, buffer, and
traffic heme to downstream targets in vivo. In mammalian cells, GAPDH was found to
be required for heme delivery to nitric oxide synthase (NOS).[70] Additionally, in Baker’s

yeast, GAPDH was found to buffer cytosolic heme and regulate its availability to the
nuclear heme-dependent transcription factor Hap1.[22] Moreover, it was recently found
that a highly conserved His residue, His51, is responsible for heme binding and
essential for GAPDH-dependent heme trafficking to NOS and Hap1.[71] The GAPDH
heme binding affinity of 24 nM, which is similar to various estimates of the concentration
of labile heme in the cytosol, 20-340 nM,[21-23] is consistent with the finding that
GAPDH is an important component of the labile heme buffering system. Indeed,
deletion of the major GAPDH isoform in yeast, Tdh3, results in a 2-fold increase in labile
heme as measured by the fluorescent heme sensor, HS1-M7A,[22] consistent with its
role in heme buffering. However, a number of open questions remain. How does
GAPDH interact with client hemoproteins to transfer heme? Binding studies and
structural models predict that heme is bound in a bis-His fashion via coordination from
His51 from two GAPDH monomers at the protomer interface of tetrameric GAPDH.[71]
As such, it is unclear how downstream client hemoproteins can acquire heme if it is
buried at the oligomer interface. It is possible that GAPDH oligomerization may be
dynamic, possibly in a regulated manner via post-translational modifications, to promote
heme accessibility to target hemoproteins. Alternatively, there may be adapter proteins
that facilitate heme exchange between GAPDH and client proteins. Additionally, in
cases where GAPDH traffics heme to other compartments, such as to the nucleus to
activate Hap1, it is not known if GAPDH dynamically shuttles between the cytosol and
nucleus to directly transfer heme to Hap1 or if there are other mediators that shuttle
heme from GAPDH to nuclear Hap1. Apart from GAPDH, the other heme binding
proteins listed above may still yet prove to be important for buffering and/or trafficking
labile heme.
Trafficking of synthesized heme through membranes and vesicles.
Given the hydrophobic nature of heme, once synthesized, heme may be
trafficked via inter-organelle membrane contact points such as mitochondrial-associated
membranes (MAMs) or through vesicular trafficking in mitochondrial derived vesicles
(MDVs). In yeast, the ER-mitochondria encounter structure (ERMES) has been found to
traffic calcium and phospholipids between the mitochondria and the ER (Figure 4).[72]

Similarly in mammals, MAMs are associated with import of calcium and phospholipids
into the mitochondria, and are important for regulating oxidative phosphorylation.[73]
ERMES and MAMs are physical tethers between the ER and mitochondria that could
facilitate the trafficking of heme between these organelles. Once in the ER, which forms
a continuous network of tubules and cisternae that extend throughout the cell and to
many subcellular compartments, as well as communicate with other organelles via
vesicular transport and physical contact,[74] heme can gain access to hemoproteins
residing in virtually any location. For instance, heme could be incorporated into nascent
hemoproteins as they fold and mature prior to their trafficking to other locations via the
trans golgi network and secretory pathway. Alternatively, heme could be shuttled in ER
or golgi-derived vesicles to awaiting hemoproteins in disparate locations such as the
nucleus, where heme regulated transcription factors reside, (e.g. Hap1,[75] p53,[76]
Bach1,[77] Reverb[78]), or peroxisomes, which contain catalase. Indeed, a recent study
has shown that the secretory pathway may be important for heme trafficking after
synthesis in the mitochondria. In HEK293 cells, labile heme was detected by subcellular
reporters to repopulate the ER first, then the Golgi and mitochondria, followed by the
cytoplasm and nucleus, after heme synthesis was restored.[79] Intriguingly, a number of
components of the heme synthesis metabolon, including FECH, Sucla2 and Abcb7[44],
which otherwise reside in the matrix-side of the mitochondrial inner-membrane, as well
as the heme biosynthetic enzyme CPOX, which is localized in the IMS (Figure 2), have
been shown to be localized to MAMs.[80] However, the specific contribution of
ERMES/MAMs to cellular heme distribution has never been directly tested and the
mechanisms underlying how heme is trafficked to and within the pathway is still
unknown.
For MDV-mediated heme trafficking, newly synthesized heme would be trafficked
from the mitochondria to endolysosomes, where it could be exported into the cytosol,
bypassing the secretory pathway. Once in the cytosol, heme could be incorporated into
any number of heme buffering or trafficking factors, including GAPDH. The recent
development of subcellular heme sensors will continue to shed light on the trafficking of
heme throughout the cell.[22, 23, 79]

Import and Trafficking of Exogenous Heme
Exogenous heme can be imported into the cell either directly by heme
transporters or through the endocytosis of extracellular hemoproteins. Once in the
cytosol, exogenous heme may be trafficked to HO-1 for degradation or used intact to
activate any number of hemoproteins.[79] Two putative plasma membrane heme
importers have been identified in mammals (Figure 3), FLVCR2 and heme carrier
protein 1 (HCP-1). FLVCR2, another member of the MFS with 12 predicted TMDs, has
been purported to import heme into the cell across the plasma membrane.[81] Little is
known of the mechanisms of transport for FLVCR2, including whether it is dependent on
heme chaperones or heme binding proteins, and the affinity of FLVCR2 for heme is
unknown. HCP-1 has been shown to import heme into the cell, though the role of HCP-
1
in heme trafficking is debated, as HCP-1 was also identified as the proton-coupled
folate transporter (PCFT).[82] HCP-1/PCFT in mice and humans respectively, is a 459
amino acid protein of ~ 50 kDa with nine predicted transmembrane helices.[83, 84]
Evidence supports HCP-1 may be a low-affinity heme transporter,[85] and may be
involved
in
heme
export
from
the
endosome
in
macrophages.[86]
The paralogous heme responsive genes HRG-1 and HRG-4 were identified in
the heme auxotroph C. elegans, and found to be important for the transport and
utilization of exogenous heme.[87] HRG-1 and HRG-4, are ~19 kDa with four predicted
TMDs. HRG-1 is localized to membranes of endosomal/lysosomal compartments and
transports exogenous heme into the cytosol. HRG-4, which is not conserved in
vertebrates, transports exogenous heme across the plasma membrane into the cytosol
and is involved in intestinal heme uptake in C. elegans.[87] The human homolog of
HRG-1 was found to be necessary for heme recycling by macrophages.[88] Human
HRG-1 transports exogenous heme from the phagolysosome into the cytosol during
erythrophagocytosis (Figure 3).
In the fission yeast, Schizosaccharomyces pombe, heme uptake is induced
under iron starvation, when two iron-regulated plasma membrane proteins designated
Shu1 and Str3 transport heme into the yeast cell.[89, 90] Shu1 is a 25 kDa GPI
anchored transporter with a measured heme affinity of 2.2 μM.[89, 91] S. pombe
utilization of exogenous heme via Shu1 is dependent on the vacuolar transporter

Abc3.[91] Str3, which was discovered to uptake heme in a Shu1 knockout, is predicted
to be a 12 TMD member of the MFS, the same superfamily as mammalian FLVCR
transporters.[90] Str3 has a lower affinity for heme than Shu1 and was unable to rescue
S. pombe growth at low heme concentrations, though Str3 is not dependent on Abc3 for
exogenous heme utilization.[90]
The internalization of extracellular hemoproteins via endocytosis is another
mechanism by which cells acquire heme. Heme and Hb may be released from
erythrocytes into blood plasma, especially during hemolytic stress. Excess heme and
Hb can damage cells, especially the endothelia and vasculature, through a number of
mechanisms, including the lytic action of heme, membrane disruption, heme-catalyzed
ROS production, peroxidation of lipids, or promoting inflammation. As a consequence,
heme and Hb must be scavenged from the blood. Hx is a serum heme scavenger,
exhibiting femtomolar dissociation constants for ferric heme.[63] Heme bound Hx binds
the hepatocyte LDL related receptor CD91, where it is endocytosed. Heme is then
released from Hx due to protonation of coordinating heme residues in acidic
compartments followed by its degradation by HO-1 or possibly its incorporation into the
labile heme pool for use. Similarly, Hb is scavenged by haptoglobin (Hp), which binds
Hb with picomolar affinity.[92] Hb-Hp can then be internalized by binding to the CD163
receptor, which is expressed in macrophages/monocytes and in neurons,[93] followed
by heme dissociation.[92]
Once exogenous heme is imported into the cytosol, either via heme transporters
or through endocytosis, heme may be trafficked to the ER for degradation via HO-1,
incorporated into the secretory pathway via transporters such as MRP-5, or trafficked
throughout the cell and/or incorporated into the labile heme pool. The extent to which
exogenous heme is utilized intact is not entirely clear and the very notion of it being
used whole has been debated.[26] However, a number of lines of evidence suggest that
heme can indeed be used in metabolism without first being degraded. First, heme
auxotrophs, which are either genetically manipulated to lack heme biosynthetic
enzymes, e.g. S. cerevisiae cells lacking the gene for ALAS, hem1∆, or naturally do not
possess the heme biosynthetic machinery, e.g. C. elegans,[94] and the trypanosomatid
parasites Leishmania amazonensis, Trypanosoma cruzi, and Trypanosoma brucei,[95,

96] can be rescued by heme supplementation. Second, in cells that can synthesize
heme, e.g. human embryonic kidney HEK293 cells, exogenous heme is accessible to a
number of subcellular compartments and contributes to the labile heme pool as
measured by a peroxidase-based heme sensor.[79] Third, intravenous heme has been
successfully used to treat porphyrias, which are diseases that arise from defects in
heme synthesis.[97] Finally, heme scavenged by Hx, which is taken up via endocytosis,
can affect BACH1 repression,[98] pointing to the possibility of exogenous heme as an
inter-cellular signal.
Altogether, cells can both transport exogenous heme and incorporate it intact for
use in physiology, albeit the extent to which this happens is still debated.[26] Once
inside the cell, it is unclear how it is transported and trafficked for utilization or to HO-1
for degradation. Interestingly, a recent study using peroxidase-based heme sensors
found that exogenous and endogenous heme is trafficked to various subcellular
compartments differently, suggesting the existence of an alternative set of chaperones
and trafficking factors that can distinguish the source of heme.[79]
Inter-cellular heme trafficking and systemic regulation of heme homeostasis.
The dogma in metazoan heme biology is that every cell lives and dies using its
own heme. That is to say, if heme is imported, it is degraded and the liberated iron is
recycled for new heme synthesis. If the heme concentration exceeds the cellular
requirement for it, excess heme is degraded.[26] However, the discovery of heme
exporters, e.g. FLVCR1a, inter-cellular heme trafficking factors, e.g. HRG-3, and signals
that communicate heme status between cells and organs, e.g. HRG-7, have challenged
old ideas and expanded the cell-centric view of heme homeostasis to a broader
organism-wide model of systemic heme regulation.
As mentioned previously, FLVCR1a is a heme exporter that was proposed to
play an important role in heme detoxification, especially during erythropoiesis when
erythroblasts synthesize large amounts of heme for Hb. However, many non-erythroid
cells that have a much lower requirement for heme express FLVCR1a, including
endothelial cells[62] and enterocytes,[60] raising the interesting question of what the
role of FLCVR1a is when cells are not over-loaded with heme. One intriguing possibility

is that cells can supply each other with heme when demand is not met by heme
synthesis. For instance, nurse macrophages regulate erythroblast development due to
the transmission of signals that promote differentiation and supply of essential
nutrients.[99, 100] It has been proposed that nurse macrophages can supply both iron
and heme to erythroblasts in order to support the sizeable demand for these
metabolites during erythropoiesis.[101]
Using the heme auxotroph, C. elegans, which only acquires heme from its diet,
an inter-cellular heme chaperone, HRG-3, was found to deliver heme from the mother to
embryos. HRG-3 is necessary for embryonic survival but not survival of adult
nematodes.[9] HRG-3 is a soluble 8.1 kDa protein that is secreted by intestinal cells into
the pseudocoelom, the circulatory system of C. elegans, and transports heme to the
extra-intestinal tissues of the worm.[9] The exact mechanisms of heme binding and
trafficking by HRG-3 are still unknown. HRG-3 forms a dimer with a hydrophobic pocket,
and structural and spectroscopic studies of HRG-3 predict heme to be enclosed within
this pocket, instead of direct coordination by heme-binding residues.[102] HRG-3 is one
of only a few known heme chaperones in eukaryotes, however HRG-3 homologs have
not been identified in vertebrates and it remains to be seen if higher organisms have
analogous inter-cellular heme trafficking factors.
If heme can be shuttled between cells, tissues, or organs, there are likely
systemic signals that communicate heme status between distant locations, analogous to
hepcidin regulation of systemic iron homeostasis[103] or the unknown signal that
communicates cardiac copper deficiency to copper storage organs.[104] Indeed, using
C. elegans as a model system, HRG-7, a 37 kDa homolog to A1 aspartic proteases,
was identified as a secreted signaling factor that communicates intestinal heme
deficiency to extra-intestinal tissues.[105] This pathway is regulated through a DBL-1
dependent signal from neurons. Given that many of the components of the HRG-7
dependent pathway for inter-organ signaling of heme are conserved in mammals, it is
tempting to speculate that such a mechanism for the regulation of systemic heme
homeostasis occurs in humans as well.[105] However, HRG-7 itself is not conserved in
vertebrates and it remains to be demonstrated that there are pathways that regulate
systemic heme homeostasis in animals that biosynthesize heme. In total, there is

emerging evidence that cells and tissues in metazoans can share and signal the
availability of heme, challenging the dogma that every cell lives and dies using its own
heme.
Heme Signaling.
In addition to its role as a protein cofactor, heme is a versatile signaling molecule
that dynamically regulates a number of proteins and pathways. Heme can signal directly
through its interactions with transcription factors, kinases, ion channels, and cell surface
receptors, or indirectly via heme-derived metabolites such as CO, bilirubin, and
biliverdin, which are produced from the HO-catalyzed degradation of heme. [3, 4, 106]
Collectively, heme-dependent signaling controls diverse processes that span oxygen
sensing, iron homeostasis, the antioxidant stress response, mitochondrial respiration
and biogenesis, mitophagy, apoptosis, immune function, circadian rhythms, cell cycle
progression, and proliferation.[76, 107-117]
In terms of direct heme signaling, heme must be dynamically mobilized to
activate or repress various physiological processes. In yeast, heme binding to the
transcription factor, Hap1, controls its function as a promoter or repressor of gene
expression in order to facilitate aerobic metabolism, including sterol synthesis and
respiration.[75, 106] In mammalian cells, heme binding to the transcription factors p53
[
76] and Bach1,[77] promote their ubiquitination and proteasomal degradation, and
heme binding to Rev-erb-α, and Rev-erb-β repress the expression of their target
genes.[78] Heme can also regulate ion channels.[118, 119] Heme binding to His and
Cys residues on cardiac KATP channels result in increased currents in a dose dependent
manner, with maximal and half-maximal responses achieved with 500 nM and 100 nM
heme, respectively.[119] Another interesting case of heme signaling is the ability of
heme to regulate micro RNA (miRNA) processing.[120] Ferric heme, but not ferrous
heme, activates DGCR8, an RNA binding protein that cooperates with Drosha to
recognize and cleave long primary transcript pri-miRNA into precursor miRNAs
(premiRNAs) in the nucleus.
Heme can also act on extracellular signaling targets. Heme derived from lysed
erythrocytes, or perhaps even heme exported from FLVRC1a, can bind the cell surface

receptor toll like receptor 4 (TLR4), which results in the inflammatory activation of
macrophages and endothelial cells, [121, 122] and is necessary for the innate immune
response to hemolytic tissue damage.[123-125] Interestingly, heme has the opposite
effect on microglia and astrocytes, suppressing immune signaling and
inflammation.[126] The difference between the immune signaling functions of heme in
macrophages versus resident brain immune cells remains to be determined.
In terms of indirect heme signaling, the heme degradation products, CO,
biliverdin, and bilirubin, the last of which is produced by the action of BVR, can
themselves act on a number of downstream targets to effect cell signaling and
metabolism. For instance, CO regulates vascular tone and has been shown to be both
anti-apoptotic and anti-inflammatory.[114] Biliverdin can regulate the inflammatory
response in macrophages by inhibiting TLR4 expression,[115] and bilirubin binds the
nuclear receptor, peroxisome proliferator-activated receptor-α (PPARα), reducing
accumulation of lipids.[127] Therefore, heme can be regarded as a packaged pro-form
of CO or biliverdin that can be activated by HO to control downstream signaling
pathways.
Dynamics of heme mobilization for trafficking and signaling.
The (on-going) discovery of heme buffering and trafficking factors, including
chaperones and transporters, has greatly transformed our understanding of the
mechanisms that enable heme bioavailability for hemoproteins and signaling. However,
another very important, but much less understood component of heme homeostasis is
the dynamics of heme mobilization and how it can be initiated for the hemylation of
heme enzymes and proteins, regulation of downstream factors, or HO-mediated heme
degradation. One mechanism is that heme synthesis is regulated in a dynamic fashion
to provide transient increases in heme for use in hemoproteins or for signaling. That is
to say, heme is made on demand. Indeed, in yeast, the activity of heme-regulated
2
transcription factor Hap1 is closely tied to the synthesis of heme; exposure to O [75] or
switching from fermentative to respiratory metabolism[128] causes a flood of newly
biosynthesized heme to activate Hap1 so as to support aerobic metabolism. In addition,
the regulation of metabolic cycling in yeast, the periodic oscillation between oxidative,

reductive building, and reductive charging phases, is associated with periodic changes
in the synthesis and degradation of heme.[129, 130] In mammals, heme regulation of
the circadian clock through binding to the nuclear receptors, Rev-erb-α and Rev-erb-β,
is associated with fluctuations in heme synthesis via ALAS expression.[108]
Alternatively, there may be active processes that mobilize pre-existing pools of
heme for use. One mechanism is through oxidation-reduction reactions at the heme iron
center. Given that hemoproteins likely have differential affinities for ferric or ferrous
heme,[120, 131-133] oxidation or reduction at the heme iron center may cause heme
dissociation and release into the labile or bioavailable heme pool. As a consequence,
molecular oxygen, reactive oxygen or nitrogen species, or perturbations in redox poise
of the cell may affect the labilization of heme.
Another possibility is that diffusible signaling molecules can mobilize heme via
the post-translational modification of hemoproteins. Indeed, both H
been demonstrated to labilize heme and affect heme transfer between hemoproteins.
For instance, H was found to induce the transfer of heme between cytochrome c
peroxidase (CCP) and catalase. Respiration-derived H oxidizes a heme-coordinating
histidine in CCP, causing heme dissociation and subsequent binding by catalase
Figure 5A).[134] The H mediated transfer of heme from CCP to catalase is a
remarkable circuit that links peroxide-mediated protein oxidation to activation of a
peroxide scavenging system. While H was not shown to influence cell-wide labile
2 2
O and NO have
2
O
2
2
O
2
(
2 2
O
2
O
2
heme using genetically encoded heme sensors,[22] the finding that a signaling molecule
could labilize otherwise exchange-inert heme for utilization by other proteins could be a
model for thinking about other signaling molecules that can alter the heme binding
properties of hemoproteins, including the gasotransmiters nitric oxide (NO), carbon
2
monoxide (CO) and hydrogen sulfide (H S).
Rather paradoxically, NO has been shown to both inhibit heme binding to a
number of hemoproteins including NOS, Hb and catalase,[70, 135] as well as increase
labile heme.[22] With respect to the former, it was demonstrated that GAPDH regulates
heme insertion into iNOS and that S-nitrosation at Cys152 weakens heme−GAPDH
interactions and prevents the transfer of heme to target hemoproteins. On the other
hand, using genetically encoded heme sensors, NO was found to transiently increase

the labile heme pool in the cytosol and nucleus of yeast by 2-fold and more than 100-
fold, respectively, over 20 minutes, followed by a slower ~90 min restoration back to
initial steady-state levels.[22] Moreover, in endothelial cells, it was proposed that NO
induces the release of heme from a number of hemoproteins due to the NO-mediated
increase in cellular iron pools and enhanced activity of HO.[136] The NO-dependent
release of heme from hemoproteins may be due to nitrosylation of ferrous heme iron
centers, which can lead to dissociation of the trans ligand, weakening of the heme
affinity and release of heme (Figure 5B),[137, 138] or the S-nitrosation of heme-
coordinating cysteine thiols [139] or allosteric Cys sites, which may impact heme
binding[70] and promote heme dissociation (Figure 5C).
Despite the wealth of data demonstrating that CO and H
heme, there is comparatively little known about the ability of these molecules to affect
labile heme and heme trafficking. H S can reduce ferric heme to ferrous heme in
hemoproteins, e.g. cytochrome c oxidase (Figure 5D), or form sulfheme, e.g. Hb and
myoglobin (Figure 5E).[140] The interaction between H S and heme may lead to
2
S can interact with
2
2
dissociation of heme into the labile heme pool, but this remains to be demonstrated.
Similarly, little is known about the effect of CO on heme trafficking. Given the high
affinity of CO for 5- or 6-coordinate ferrous hemoproteins,[141, 142] CO binding may
prevent heme oxidation and subsequent dissociation,[143] which would result in less
labile or bioavailable heme (Figure 5F).
Overall, the discovery that H
2
O
2
and NO can affect the labilization and
S may potentially do the
bioavailability of heme, and that cellular redox state, CO and H
2
same, means that heme mobilization, trafficking, and signaling may be downstream of a
number of key physiological processes, including redox metabolism, NOS-dependent
2 2
NO signaling, NADPH-oxidase mediated H O signaling, sulfur metabolism, and more.
Pathological conditions may also mobilize heme. It was recently demonstrated that
2
+
heavy metal stress, e.g. Pb , increases labile heme, potentially contributing to heme
2
+
toxicity and/or signaling heme to communicate Pb
stress.[25] The continued
development and application of new heme imaging technologies and heme sensors will
help shed light on the dynamics of labile heme and the factors that initiate its
mobilization for trafficking and signaling.[22, 23, 79, 144]

Heme at the host-pathogen axis.
During fungal and bacterial infections, invading microbes must acquire host iron
for virulence. The most abundant source of iron for these pathogens is heme, which is
predominantly bound to hemoproteins such as Hb. As a consequence, host and
pathogen heme homeostatic factors inter-mingle and compete for available heme.
Bacteria and fungi have evolved numerous systems for heme trafficking. Many bacteria
and fungi can both uptake and synthesize heme, while others lack some, or all, of the
heme biosynthetic enzymes and are solely dependent on host heme. The pathogenic
microbes that assimilate host heme face similar challenges; first they must retrieve
heme from the host, transport the heme across the cellular envelope into the cell, and
deliver heme to hemoproteins for utilization or heme oxygenases for degradation, all
while mitigating heme toxicity.
Microbial pathogens have evolved different heme uptake pathways to match the
unique challenges of their environment. During infection, heme within the host may be
available as free heme, Hb, heme-Hx or Hb-Hp. Most microbial pathogens utilize cell-
surface associated heme binding proteins that can transport free or bound heme or
secrete hemophores that sequester free heme or heme binding proteins, such as Hb,
and deliver them back to cell surface receptors for internalization. Once taken up, heme
trafficking within most microbial pathogens is poorly understood. Herein we will briefly
discuss the transport and trafficking of heme within microbial pathogens and how they
interact with host heme.
Heme Uptake in Bacteria
Bacteria have developed varied heme uptake systems to overcome their diverse
cellular envelope compositions and structures, and host microenvironments (Figure 6).
Heme uptake in bacteria is generally divided into direct uptake, hemophore mediated
uptake and, at least in the case of Neisseria meningitides, bipartite surface receptor
mediated uptake for Hb.[145] Herein we present examples of heme uptake in gram-

positive and gram-negative bacteria to point out similarities in uptake and points of
divergence as heme uptake in bacteria has been extensively reviewed in the
literature.[11, 12, 146, 147]
Gram-Positive Bacteria
Arguably the most well characterized heme acquisition system in gram-positive
bacteria is the Isd (Iron-regulated surface determinant) system (Figure 6). Both
Staphylococcus aureus and Bacillus anthracis utilize Isd proteins to transport heme
from the extracellular space, across the cell wall and membrane, and into the
cytoplasm. The hallmark of the Isd system is the use of Near Transporter (NEAT) heme
binding domains to extract host heme. These NEAT domains are found in cell wall
tethered proteins that are attached via sortases in both S. aureus and B. anthracis,
[
148, 149] and in soluble hemophores in B. anthracis. NEAT domains are necessary for
the first step in heme acquisition, which requires retrieving heme from the host. Isd
proteins bind free heme or hemoprotein bound heme via NEAT domains,[150] and
heme is then transferred to other Isd NEAT proteins, such as IsdC, to transport heme
through the cell wall. Finally, heme is transported across the inner membrane into the
cytoplasm via the IsdDEF ATP-binding cassette (ABC) transporter complex. Here
heme can be trafficked to hemoproteins or to an Isd heme oxygenase within the
cytoplasm.[151]
Other gram-positive bacterial heme uptake systems have also been discovered
that do not utilize NEAT heme biding domains. Corynebacterium diptheriae utilizes a
series of proteins termed the hemin uptake system (Hmu). These proteins do not have
NEAT domains and are not attached to the cell surface via sortases.[152] The cell wall
associated heme binding proteins of HtaABC of C. diptheriae have an N-terminal
secretion signal and a C-terminal transmembrane domain allowing them to be tethered
to the cell with an exposed heme binding domain.[153] C. diptheriae have redundant
systems to capture and transport heme to the putative ABC transporter HmuTUV.
Under low iron conditions, the ChtAB and CirAChrC heme binding proteins are

upregulated, and can capture and deliver heme to HmuTUV, to be transported into the
cytosol. Recently in Mycobacterium tuberculosis, proteins have been discovered that
are necessary for M. tuberculosis to grow utilizing heme as a sole iron source (Figure
6).[154, 155] Though the function of these proteins in heme uptake is still not well
understood, heme must be captured from the host, possibly by secreted hemophore
Rv0203,[154] or via cell surface heme binding Proline-Proline-Glutamate (PPE)
proteins.[155] This heme is then transported into the cell, possibly through
Mycobacterial membrane protein large (MMPL) proteins or through PPE
transmembrane proteins, where it can be degraded by the IsdG type Mycobacterial
heme utilization degrader (MhuD) heme oxygenase to release iron.[35]
Gram-Negative Bacteria
The structure of the cellular envelope of gram-negative bacteria requires heme to
be trafficked across the outer membrane, through the periplasm and across the inner
membrane before reaching the cytosol. Most gram-negative bacteria accomplish this
with TonB dependent import across the outer membrane and, similar to gram-positive
bacteria, an ABC transporter at the inner membrane. Shigella dysenteriae utilizes direct
transport of heme into the periplasm the via Shigella heme uptake (Shu) heme binding
protein, ShuA, which is TonB dependent. Heme is then trafficked through the periplasm
via ShuT and delivered to the ABC transporter at the inner membrane, ShuUV. [156-
158] Pseudomonas aeruginosa utilizes a similar uptake system to S. dysenteriae, the
Pseudomonas heme uptake (Phu) system. This utilizes both direct heme import via
PhuRSTUVW[158] and hemophore mediated import via the Heme acquisition system
(Has) proteins HasA and HasR (Figure 6).[159]
Heme Uptake in Fungal Pathogens
Fungal pathogens, such as Candida albicans and Cryptococcus neoformans can
utilize heme as a sole iron source, though their heme uptake mechanisms are not well
understood. The general model is that heme or hemoproteins are bound at the cell
surface and transferred across the cell wall for endocytosis at the inner membrane
(Figure 6). Heme and Hb binding proteins Rbt5, Rbt51[160] and Pga7[161] have been

identified in C. albicans that are anchored within the cell wall and are involved in heme
uptake by binding heme or Hb. Transfer of heme across the plasma membrane is most
likely via endocytosis as proteins involved in the endosomal sorting complex required
for transport (ESCRT) are necessary for heme uptake in both C. albicans[162] and C.
neoformans.[163]
Intracellular heme trafficking in Microbial Pathogens
Exogenous Heme
The fate of exogenous heme once it has entered the cytosol of microbial
pathogens is not well studied. Some pathogens, such as the heme auxotroph
Hemophilus influenzae, must traffic exogenous heme for utilization in all heme
dependent proteins. Though how this occurs is unknown. In P. aeruginosa the cytosolic
protein PhuS delivers heme to the iron regulated heme oxygenase (Figure 6),[164] and
knockout of a homolog in S. dysenteriae, ShuS, increases sensitivity to heme toxicity,
suggesting it plays an important role as a heme chaperone.[165] Some bacteria, such
as S. aureus and B. anthracis, respond to excess heme by expressing efflux proteins
through a heme dependent two-component system, which can alleviate heme toxicity
during hemolytic events.[166]
In the case of pathogenic fungi like C. albicans, it
appears that exogenous heme taken up via endocytosis is mostly, if not entirely,
degraded as growth on exogenous heme is dependent on the heme oxygenase
CaHMX1.[167] It is not yet known whether heme is degraded in the vacuole or
transported to CaHMX1 for degradation.
Endogenous Heme
Endogenously synthesized heme must be trafficked from the site of synthesis to
heme dependent proteins throughout the cell. Specialized heme trafficking systems like
that involved in cytochrome c biogenesis have been identified. In cytochrome c
biogenesis, hemeb is transported to the periplasm of the cell, or in fungi across the
mitochondrial inner membrane, and converted to hemec for incorporation into
cytochrome c. Heme is trafficked to cytochrome c via three known systems. Each
system has unique chaperones and transporters and have been reviewed

extensively.[168] Heme must also be trafficked to other hemoproteins, though few heme
chaperones in pathogens have been identified.
Microbial pathogens are solely dependent on the host for iron and have
developed a number of systems to acquire iron through heme uptake. Fungal and
bacterial pathogens must traffic heme across the cell envelope while avoiding heme
toxicity. The known heme uptake and trafficking systems utilize heme transport via
dedicated transporters, heme trafficking by hemophores and soluble heme chaperones,
and heme trafficking via endocytosis. While much has been studied in relation to heme
uptake in pathogenic microbes, the systems of some pathogens, such as mycobacterial
heme uptake systems, remain to be fully elucidated. Also, as with metazoans, little is
known of the fate of heme once inside the cell. Newly developed methods for sensing
labile heme may provide some insight on heme trafficking within pathogenic
microbes.[22, 23, 79]
Conclusions
The past decade has witnessed a radical transformation of our understanding of
heme homeostatic mechanisms, including heme transport, trafficking, and dynamics. At
one time, heme was viewed as a static cofactor buried in hemoprotein active sites.
However, the development of new heme imaging modalities coupled with integrated
biochemical, cell biological, and genetic approaches have revealed that heme is a
dynamic molecule that can be mobilized in response to a variety of stimuli, including
2 2
signaling molecules like NO and H O . At one time, metazoan heme homeostasis was
viewed from the vantage of individual cells that maintained their own pool of heme,
making it or degrading it as needed. No longer! The discovery of heme importers,
exporters, inter-cellular trafficking factors, and signals that communicate tissue heme
status has created a new paradigm for systemic heme homeostasis. At one time, it was
thought that heme is made on demand and any excess is degraded, obviating the need
for a heme reserve for use in metabolism. The discovery of a labile heme pool that is
highly dynamic has established that a pre-existing supply of bioavailable may be
important for heme utilization and signaling. The rapid emergence of new approaches
and tools promises to reveal the enigmatic rules that govern heme transport, trafficking,

and availability and will no doubt continue to transform and expand our understanding of
heme homeostatic mechanisms in cells and animals.
Acknowledgements:
We acknowledge support from the U.S. National Institutes of Health (ES025661 and
GM118744 to A.R.R.), the U.S. National Science Foundation (MCB-1552791 to A.R.R.),
the Blanchard Professorship (to A.R.R.) and start-up funding from the Georgia Institute
of Technology (to A.R.R.).
Disclosure:
The authors declare no conflict of interest.
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