The importance of ferric reductases for iron uptake in humans

1 Introduction

Human body contains approximately 70 mg of iron per kg of body weight, which serves as the active center for various proteins and enzymes critical to essential physiological processes (Yanatori et al., 2025; Qiu et al., 2025). Each day, one to 2 mg of body iron is lost through unavoidable pathways such as excretion, perspiration, skin desquamation, and hair loss. The total iron content in the body is maintained at a nearly constant level by dietary compensation for these losses. However, only one to 2 mg of iron from an average daily intake of 15–20 mg can be absorbed in the duodenum. This strategy prevents excessive iron accumulation in the body. This poor absorption efficiency makes it difficult to immediately recover the symptoms of iron deficiency. Iron absorption must be delicately balanced to supply sufficient iron for metabolism and to avoid the accumulation of excessive, toxic levels. Body iron levels depend almost exclusively on its duodenal absorption, as there is no regulated excretory pathway for iron. In the absence of sufficient iron, the body is unable to produce red blood cells normally. Consequently, the oxygen supply to the body is compromised, leading to mild hypoxia. This condition is known as iron deficiency anemia. The World Health Organization has identified it as the most severe form of malnutrition, responsible for poor health in approximately 40% of all children aged 6–59 months and 30% of women 15–49 years of age in the global population (World Health Oraganization, 2025; GBD, 2021 Anaemia Collaborators, 2023). Therefore, understanding the mechanism of iron absorption and utilization in humans is an important area of study.

Dietary iron is typically classified as heme iron (iron porphyrin) or non-heme iron (Qiu et al., 2025; Fuqua et al., 2012). Heme iron is abundant in meat and blood as a prosthetic group of hemoproteins, mainly myoglobin and hemoglobin. The low pH of the stomach, coupled with proteolytic enzymes in the stomach and small intestine, helps to release heme from hemoproteins. The released hemes are thought to be taken up into duodenal enterocytes via the heme transporter. Heme carrier protein 1 (HCP1) was first described as a heme transporter in duodenal enterocytes but was subsequently shown to be a proton-coupled folate transporter (Shayeghi et al., 2005). A few years later, heme responsive gene 1 (HRG1, also known as SLC48A1) was identified as a candidate for the heme transporter through a genetic screen in C. elegans (Rajagopal et al., 2008), but not in mammals. To date, no protein has been identified as a definitive heme transporter in mammals, and the molecular mechanism of heme absorption in humans remains unclear. In contrast, non-heme iron is present in diverse forms in plant and animal foods. Dietary iron is typically oxidized to Fe³⁺, which has low water solubility and poor bioavailability. Iron absorption mainly occurs across polarized epithelial cells in the duodenum and has long been known to be enhanced by reducing agents such as ascorbate (Moore et al., 1939). With the discovery of the enteric ferric reductase Dcytb (McKie et al., 2001) and the proton-coupled divalent metal transporter DMT1 (Gunshin et al., 1997), the proteins responsible for the uptake of dietary iron ions have been identified (Figure 1A). In the apical (luminal) membranes of duodenal enterocytes, Fe²⁺ absorption is facilitated by the sequential function of membrane proteins, Dcytb and DMT1. As these membrane proteins control the initial step in iron metabolism, their dysfunction disrupts iron homeostasis in the body. Since DMT1 is selectively permeable to Fe²⁺ (but not Fe³⁺), the reduction of Fe³⁺ by Dcytb serves as the initial step in the uptake of iron nutrients from food sources. Therefore, it is important to investigate the relationship between molecular structure and function of the first key protein, Dcytb, for the understanding of iron absorption in humans. The three-dimensional structure of human Dcytb revealed by the authors and new findings based on the structural information are described in the next section of this review.

Upon internalization within intestinal epithelial cells, Fe²⁺ undergoes a series of intracellular processes, including transport, storage, and membrane transport to capillaries. Subsequently, it binds to transferrin in the blood, adopting the form of Fe³⁺. The Fe³⁺-bound transferrin circulates throughout the body via the bloodstream, reaching peripheral cells such as erythroblasts and hepatocytes. Upon reaching these cells, the Fe³⁺-bound transferrin binds to the transferrin receptor on the cell surface and is internalized by endocytosis (Figure 2A) (Cheng et al., 2004; Hanover et al., 1984). In the acidic environment of endosomes, Fe³⁺ dissociates from transferrin and is reduced to Fe²⁺ by Six-Transmembrane Epithelial Antigen of the Prostate 3 (STEAP3), which is localized on the endosomal membrane (Ohgami et al., 2006; Gomes et al., 2012). STEAP3 is a member of the STEAP family and is composed of a six-transmembrane domain at the C-terminal domain and a cytoplasmic N-terminal oxidoreductase domain, which functions for Fe³⁺ reduction. STEAP3 plays an important role in hematopoiesis, especially in erythroid precursors, by regulating iron metabolism. Its expression, in conjunction with transferrin and the transferrin receptor, is induced during iron deficiency. In contrast to Dcytb in intestinal cells, STEAP3 utilizes NADPH as an electron donor (Ohgami et al., 2006). Fe²⁺ is transported into the cytoplasm by DMT1, which is localized on the endosomal membrane. Subsequently, DMT1 is utilized as the active center of intracellular enzymes and proteins. Subsequent to Fe³⁺ release, the endosome containing the apo-transferrin receptor complex reverts to the cell surface as a recycling endosome. The elevated pH at the cell surface causes the dissociation of apo-transferrin from the receptor, resulting in its release into the bloodstream and subsequent circulation throughout the body (Figure 2A). Recent in vitro studies have indicated that STEAP3 promotes colon cancer cell proliferation and stemness (Gomes et al., 2012; Lv et al., 2024). However, its function in vivo remains to be elucidated and the specific regulatory mechanisms underlying this role warrant further investigation. Therefore, this review in Section 3 focuses on STEAP proteins including their physiological functions and recently elucidated three-dimensional structures (Oosterheert et al., 2018).

2 Dietary iron absorption via Dcytb in human duodenum

Until around 1990, it was understood that iron ions were more efficiently absorbed in the intestine through Fe²⁺ than through Fe³⁺. This was primarily attributed to differences in Fe solubility. However, in the early 1990s, animal experiments demonstrated that Fe³⁺ in food must be reduced to Fe²⁺ for iron uptake into intestinal cells (Wien and Van Campen, 1991; Raja et al., 1992). In 2001, McKie et al. cloned Dcytb as an Fe³⁺ reductase that is expressed in duodenal epithelial cells (McKie et al., 2001). A few years earlier, Gunshin et al. discovered the divalent metal transporter DMT1 as an iron transporter in duodenal epithelial cells (Gunshin et al., 1997), highlighting the role of DMT1 in the uptake of Fe²⁺ reduced by Dcytb. Subsequent reports have indicated that increasing the intracellular ascorbic acid concentration enhances the uptake of iron ions by Dcytb and DMT1, revealing that intracellular ascorbic acid is the electron donor for Dcytb (Raja et al., 1992; Latunde-Dada et al., 2008). Dcytb is a heme-containing enzyme belonging to the ascorbate-dependent oxidoreductase superfamily, which includes the cytochrome b ₅₆₁ protein family (Ludw et al., 2008). This protein family is present in animals including humans, mice (Bérczi et al., 2023), D. melanogaster (Holst et al., 2023), C. elegans (Miura et al., 2025), planarian species (Asada et al., 2002), and plants such as A. thalania (Bérczi et al., 2007) and soybean (Picco et al., 2015). In both animals and plants, cytochrome b ₅₆₁ proteins are membrane proteins and facilitate electron transfer from the cytoplasmic side of the cell membrane to either the extracellular space or intracellular vesicles. These proteins play critical roles in physiological processes such as iron absorption, cellular defense, nitrate reduction, and signal transduction (Zhou et al., 2024). Notably, Dcytb has been extensively studied as a Fe³⁺ reductase involved in dietary iron absorption in humans. In light of this background, the author and coworkers elucidated the three-dimensional structure of human Dcytb using X-ray crystallography to understand the reaction mechanism and reported it in 2018 (Ganasen et al., 2018).

Dcytb forms a dimer, with each monomer consisting of six transmembrane helices. Each monomer binds two heme b molecules. Furthermore, when ascorbic acid and metal ions (Zn²⁺ instead of Fe³⁺) were added during crystal preparation, ascorbic acid, the electron donor, was found to be bound to the cytoplasmic side, while the region (pocket) facing the luminal side contained both metal ions and ascorbic acid (Figure 1B) (Ganasen et al., 2018). This three-dimensional structure enabled the prediction of the electron transfer process across the plasma membrane from ascorbic acid bound to the cytoplasmic side to Fe³⁺ bound to the luminal side during the ferric reduction. Furthermore, it was found that the luminal pocket binds not only ascorbic acid but also sugars and organic acids contained in food, along with Fe³⁺, promoting the reduction of Fe³⁺ (Ganasen et al., 2018). The C-terminus of Dcytb is exposed to the cytoplasm and contains an extramembrane region of approximately 60 amino acids that is not found in other proteins of the cytochrome b ₅₆₁ family members. This region is believed to comprise a highly flexible loop devoid of secondary structure, a feature that has not been observed using X-ray crystallography. One significant difference in the Dcytb relative to other members of cytochrome b ₅₆₁ family is the presence of this C-terminal region (Ganasen et al., 2018). Other proteins belonging to the cytochrome b ₅₆₁ family, which are Lcytb in late endosomal-lysosomal membrane and CGcytb in chromaffin granule, differ in the length of this C-terminal region and in their cellular locations (Zhang et al., 2006). Given that Dcytb and Lcytb function as Fe³⁺ reductases, while CGcytb acts as a monodehydroascorbate reductase, the C-terminal region may be linked to where these enzymes are found within cells or to their specific biological roles. For instance, it has been shown that the C-terminal region of human Lcytb contains a signal sequence directing it to the lysosomal membrane (Su and Asard, 2006). A similar example of a physiological role for such regions is found in another transmembrane protein involved in iron metabolism: one of the cytoplasmic loops of DMT-1 helps regulate its interaction with an iron chaperone protein (Yanatori et al., 2025). Likewise, a cytoplasmic lysyl residue on the iron exporter ferroportin-1 serves as a site for ubiquitination (Yanatori et al., 2025). These examples suggest that the disordered C-terminal region of Dcytb could play a part in controlling duodenal iron absorption. Further research is needed to clarify the functional significance of the C-terminal region of Dcytb.

To elucidate the structural basis for ascorbate and metal ion binding to Dcytb, co-crystallization trials were conducted with Dcytb in the presence and absence of metal ions (Fe³⁺, Cu²⁺, and Zn²⁺) and ascorbate. Successful X-ray diffraction was achieved only with crystals soaked in Zn²⁺ and ascorbate, allowing the determination of the crystal structure (Figure 1B) (Ganasen et al., 2018). Electron density corresponding to substrate binding was detected at both the cytoplasmic and apical surfaces of the protein. On the apical side, near the apical heme group, both Zn²⁺ and an ascorbate molecule were observed (Figure 1B). Another ascorbate molecule, acting as an electron donor, was found on the cytoplasmic surface of Dcytb in proximity to the heme located near the cytoplasmic side (Figure 1B). Each monomer contains two heme b groups which are sandwiched between four transmembrane helices, and each heme is hexacoordinated with His86 and His159 to the iron of the cytoplasmic heme and His50 and His120 to the apical heme iron (Figure 1B). Computational analysis identified three possible electron transfer pathways from cytoplasmic ascorbate to apical Fe³⁺: one via cytoplasmic heme → Tyr131 → Phe58 → apical heme → Fe³⁺, and two others through covalent and hydrogen bonds along helix α2 or α4 (Ganasen et al., 2018).

To date, the crystal structures of only two cytochrome b ₅₆₁ proteins have been determined: human Dcytb and Arabidopsis AtCytb ₅₆₁ (Lu et al., 2014). The sequence of the human Dcytb core structure (residues 6–230) shares 36% identity and 52% similarity with that of AtCytb ₅₆₁ (residues 9–220). Comparison of these two structures reveals that the six α helices are superimposable and the relative orientations of monomers within these two dimers differ by only 5°. The structural environments of the two heme groups of Dcytb are identical to those of the heme groups occurring in AtCytb ₅₆₁ in terms of bis-histidyl coordination and interactions with surrounding residues. On the other hand, the structure of the protein intervening between the two heme groups that is expected to participate in intramolecular electron transfer differs significantly between Dcytb and AtCytb ₅₆₁. Lu and colleagues examined the AtCytb ₅₆₁ structure and proposed that a water molecule and Phe129 play a role in electron transfer. They suggested that the arrangement of this water and the phenyl ring of Phe129 is well-suited for electron tunneling from the cytoplasmic heme to the non-cytoplasmic heme (Lu et al., 2014). For Dcytb, possible electron-transfer routes are identified via main chain NH–OC interactions extending from the cytoplasmic heme ligand through six residues along helix α2 or α4, as well as the apical heme ligand (Figure 1B, right panel) (Ganasen et al., 2018). A key difference between Dcytb and AtCytb ₅₆₁ is that Phe129 in AtCytb ₅₆₁ aligns with Tyr131 in Dcytb; no water molecule is part of the pathway in Dcytb, and the phenyl group of Dcytb residue Phe58 sits between Tyr131. As a result, Dcytb residues Phe58 and Tyr131 may facilitate electron tunneling across a suitable distance (3.6–4.9 Å). This hypothesis aligned with in vivo Fe³⁺ reductase assays investigating intramolecular electron transfer in Dcytb variants with substitutions at these positions (Ganasen et al., 2018).

The cooperative binding of ascorbate and Zn²⁺ at the apical substrate binding site in the Dcytb structure (Figure 1B, upper left panel) provided significant mechanistic insights into the function of Dcytb. Members of the Cytb ₅₆₁ family are known to reduce monodehydroascorbate to ascorbate, utilizing ascorbate bound at the cytoplasmic substrate binding site to supply electrons to monodehydroascorbate on the apical side. Like other Cytb ₅₆₁ proteins, Dcytb features an ascorbate-binding site at the cytoplasmic surface; however, it uniquely possesses an apical ascorbate-binding site that coincides with an Fe³⁺ substrate binding location, potentially facilitating the formation of monodehydroascorbate during Fe³⁺ reduction to Fe²⁺. The generated monodehydroascorbate can subsequently be reduced back to ascorbate by Dcytb. This proposed mechanism indicated that Dcytb not only reduces Fe³⁺, but also regenerates ascorbate from monodehydroascorbate at the apical binding site, employing electrons provided by ascorbate at the cytoplasmic binding site (Ganasen et al., 2018).

3 Iron uptake to peripheral cells via STEAP proteins

STEAP family comprises a group of highly conserved metalloreductases that play critical roles in various cellular processes, including iron homeostasis, oxidative stress regulation, and cell proliferation. The first identification of STEAP was reported by Hubert and coworkers in 1999 as a prostate cell surface-specific antigen. Its elevated expression in cancer-specific cells has led to its investigation as a potential cancer marker (Hubert et al., 1999; Gomes et al., 2012). Subsequently, four isoforms were identified, and their expression was analyzed in various organs. STEAP1 and STEAP2 are highly expressed in the prostate, while STEAP3 and STEAP4 show high levels of expression in the bone marrow (Ohgami et al., 2006). All STEAP proteins are transmembrane metalloreductases, that facilitate the reduction of iron (Ohgami et al., 2006; Hubert et al., 1999; Gomes et al., 2012). Excluding STEAP1, STEAP2-4 are composed of a six-transmembrane domain with conserved heme-binding sites at the C-terminal domain, and a cytoplasmic N-terminal domain homologous to the F₄₂₀H₂:NADP⁺ oxidoreductase (Fno)-binding domain, which catalyzed the reversible reduction of NADP⁺ to NADPH using a reduced flavin cofactor (F₄₂₀ ⁻) as the hydride donor (Warkentin et al., 2001; Howard et al., 2021). These three STEAP proteins utilize intracellular NADPH and the flavin cofactor as electron donors for iron reduction (Ohgami et al., 2006). In contrast, STEAP1 requires its heme group and interaction with STEAP2 or STEAP4 to participate in metal metabolism (Kim et al., 2016; Oosterheert and Gros, 2020).

STEAP proteins are predominantly located on the plasma membrane, Golgi apparatus, trans-Golgi network, and endosomal-vesicular compartments (Hubert et al., 1999; Korkmaz et al., 2005; Kor et al., 2002). Their colocalization with endosomal-vesicular compartments and transferrin suggests a role in molecular trafficking through exocytosis and endocytosis, and transferrin cycle (Ohgami et al., 2006). Since the seminal report by Ohgami et al. (2005), which demonstrated the involvement of STEAP3 in serum iron uptake in erythroid cells, STEAP3 has studied as a ferric reductase involved in iron uptake (Ohgami et al., 2006; Ohgami et al., 2005). Humans utilize approximately 80% of the iron present in their bodies to produce 200 billion red blood cells per day (Hentze et al., 2004). STEAP3 has been thoroughly studied as an enzyme crucial for the differentiation of erythroblasts into red blood cells (Ohgami et al., 2005). STEAP3 expression is tightly regulated by iron responsive protein (IRP), and its expression is induced along with transferrin and its receptor during iron deficiency. In 2018, the three-dimensional structure of STEAP4 was reported by single-particle analysis using cryo-electron microscopy. His316 and His409 in the transmembrane domain are ligands for heme b, suggesting that electrons are transferred from NADPH in the cytoplasm to Fe³⁺ via FAD and heme b (Figure 2B) (Oosterheert et al., 2018). In addition, the three-dimensional structure of STEAP4 with Fe³⁺-nitrilotriacetic acid (NTA) was analyzed. Although the results were unclear, they indicated that a Fe³⁺-binding site exists near the propionic acid of the heme b. Furthermore, Ser138, located at the interface between the N- and C-terminal domains, is thought to be present in the cavity leading from NADPH to FAD and contributes to electron transfer. The three-dimensional structure of STEAP4 forms a trimer with the N- and C-terminal transmembrane domains swapped (Figure 2B). However, as STEAP3 has been reported to function as a dimer (Oosterheert et al., 2018), it remains unclear whether the reaction mechanisms of STEAP3 and STEAP4 are the same. Recent studies reported the cryo-electron microscopic structure and potential enzymatic activity of human STEAP1 (Oosterheert and Gros, 2020). Although STEAP1 lacks an intracellular NADPH-binding domain and was previously considered inactive in Fe³⁺ reduction, structural analysis demonstrated that STEAP1 adopts a reductase-like conformation. Additionally, STEAP1 facilitates Fe³⁺ reduction when fused to the intracellular NADPH-binding domain from its family member STEAP4, indicating that STEAP1 may function as a Fe³⁺ reductase within STEAP heterotrimers (Oosterheert and Gros, 2020). These new findings regarding STEAP proteins could inform the development of novel therapeutic strategies for targeting STEAP proteins in cancer, broadening applications beyond their established role in iron uptake.

STEAP proteins serve as key regulators of iron homeostasis and are associated with both acute and chronic inflammation-related diseases, as well as the development and progression of malignant tumors. Recent studies have demonstrated that STEAP proteins exhibit abnormal expression across multiple cancer types, contributing to enhanced tumor cell proliferation, migration, and malignant transformation (Yuan et al., 2025; Sun and Yang, 2024; Wang et al., 2021; Grunewald et al., 2012; Zhao et al., 2021). Therefore, STEAP proteins have been recognized as a promising biomarker and therapeutic target for tumor immunotherapy, particularly in prostate cancer (Singh and Kyte, 2025; Cai et al., 2025). Understanding the functional mechanisms of the STEAP proteins in cancer and inflammation is important. However, the details remain unclear due to their dependence on risk factors such as metabolic disorders, hormonal imbalances, and chronic inflammation.

4 Discussion and conclusion

This review summarized iron uptake in humans and the heme-containing ferric reductases that function in these processes. Given the presence of ferric reductases in the metal nutrient uptake systems of higher plants (Yi and Guerinot, 1996; Waters et al., 2002; Li et al., 2004), yeast (Shatwell et al., 1996; Dancis et al., 1990), and bacteria (Schröder I et al., 2003; Cain and Smith, 2021), it can be hypothesized that these enzymes have evolved to implement a shared strategy across biological species for metal ion uptake. This strategy involves the conversion of poorly soluble Fe³⁺ to Fe²⁺, thereby enhancing its water solubility and facilitating its uptake by metal transporters located in the plasma membrane. This common strategy involves transferring electrons received from intracellular electron donors across the plasma membrane to extracellular metal ions via a prosthetic molecule, such as heme, which is bound to the transmembrane domain. These structural and functional characteristics are commonly observed in the transmembrane cytochrome b family. Notably, this mechanism may have evolved to facilitate the uptake of metal ions from the external environment. However, the absence of detailed information regarding the electron transfer mechanism and three-dimensional structures, such as the selective binding modes of metal ions and electron donors, makes it challenging to compare and discuss these strategies from a molecular perspective.

Iron is an essential metal for sustaining life. However, its high reactivity poses a considerable risk of generating reactive oxygen species if present in excess in cells. The strategy that utilizes ferric reductases, as introduced in this review, suggests the existence of a system for the safe and efficient uptake of Fe²⁺ produced by the reduction of Fe³⁺ in cells. Further elucidation of the mechanisms of intracellular utilization of highly reactive Fe²⁺ by iron delivery chaperones (drawn as PCBP in Figures 1A, 2A) and intracellular accumulation of less water-soluble Fe³⁺ are needed. A better understanding of metal utilization strategies will likely contribute to the development of safe and efficient methods for the intake of metal nutrients in humans and the prevention and treatment of diseases caused by metal metabolism disorders in humans.

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Footnotes

Edited by: Craig C. McLauchlan, Illinois State University, United States

Reviewed by: Urszula Katarzyna Komarnicka, University of Wrocław, Poland

Hanna Kwon, University of Leicester, United Kingdom