5.B.2 The Eukaryotic Cytochrome b561 (Cytb561) Family

Cytochromes b561 are a family of transmembrane proteins found in many eukaryotic cells. Three evolutionarily related mammalian cytochromes b561 , are the chromaffin granule cytochrome b, duodenal cytochrome b, and lysosomal cytochrome b (Lane and Lawen, 2009; Oakhill et al., 2008; Su et al., 2006Su and Asard, 2006; Zhang et al., 2006). They contain two haem b prosthetic groups per molecule coordinated with four His residues from four different transmembrane alpha-helices. Cytochromes b561, residing in chromaffin vesicles, are known to have a role for a neuroendocrine-specific transmembrane electron transfer from extravesicular ascorbate to an intravesicular monodehydroascorbate radical to regenerate ascorbate. Some members of the family lack the sequence for putative ascorbate-binding and exhibit a transmembrane ferrireductase activity. Nakanishi et al. (2007) proposed that cytochrome b561 has a specific function facilitating the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the N(delta1) atom of the axial His residue at the extravesicular haem center, as an initial step of transmembrane electron transfer. This mechanism utilizes the well-known electrochemistry of ascorbate for biological transmembrane electron transfer and might be operative for other type of electron transfer reactions from organic reductants (Nakanishi et al., 2007).

Bovine cytochrome b561 is a 6 TMS protein present in secretory vesicles (i.e., adrenal chromaffin granules) which contain catecholamines and amidated peptides. It supplies electrons from cytoplasmic L-ascorbate to intravesicular enzymes such as dopamine β-hydroxylase and α-peptide amidase (Perin et al., 1988). It has about 250 aas, with 6 TMSs and binds two hemes noncovalently, one on the cytoplasmic side of the membrane and one on the external side of the membrane, and each bound to two histidyl residues (Bashtovyy et al., 2003). A homologue of Cytb561 is the duodenyl ferrireductase cytochrome b (Dcytb) which is about 220 aas in length (McKie et al., 2001). This latter enzyme is present in the intestinal mucosa where it takes electrons from cytoplasmic L-ascorbate and transfers them to two Fe3+ yielding two Fe2+. Fe2+ is taken up into the intestinal mucosa cell via an Fe2+ NRAMP2 transporter (TC #2.A.55.2.1). This ferrireductase may also reduce Cu2+ to Cu+ (Kaplan, 2002). Another homologue is the tumor suppressor, the 601F6 protein or the Tsp10 pending protein which is 222 aas in length. All members of the family probably have 6 TMSs. Three-dimensional structural predictions have been published, and a transmembrane electron transport pathway has been proposed (Bashtovyy et al., 2003).

Homologues of cytochrome b561 proteins are found in animals, plants and fungi but not prokaryotes (Verelst and Asard, 2003; Kimball and Saier, 2002). Some of these may be involved in ascorbate regeneration. Mammals (humans and mice) have 4 paralogues, Caenorhabditis elegans, Anopheles gambiae and Drosophila melanogaster each have two, and Arabidopsis thaliana and Orzya sativa each have three. None has been detected in yeast but the fungi, Dugesia japonica and Neurospora crassa each have one (Kimball and Saier, 2002). 

Trans-plasma membrane electron transfer is achieved by b-type cytochromes of different families, and plays a fundamental role in diverse cellular processes involving two interacting redox couples that are physically separated by a phospholipid bilayer, such as iron uptake and redox signaling. Robust electrophysiological evidence demonstrated trans-plasma membrane electron flow mediated by a soybean (Glycine max) cytochrome b561 associated with a dopamine beta-monooxygenase redox domain (CYBDOM) (Picco et al. 2015). In oocytes, two-electrode voltage clamp experiments showed that CYBDOM-mediated currents were activated by extracellular electron acceptors in a concentration- and type-specific manner. Current amplitudes were voltage dependent, strongly potentiated in oocytes preinjected with ascorbate (the canonical electron donor for cytochrome b561), and abolished by mutating a highly conserved His residue (H292L) predicted to coordinate the cytoplasmic heme b group. 

Cyt b561 plays a role in ascorbate recycling and iron absorption. Lu et al. 2014 reported the high-resolution crystal structures of the Arabidopsis thaliana protein in both substrate-free and substrate-bound states. Cyt b561 forms a homodimer, with each protomer consisting of six transmembrane helices and two heme groups. Ascorbate, or monodehydroascorbate, is enclosed in a positively charged pocket on either side of the membrane. Two highly conserved amino acids, Lys81 and His106, play an essential role in substrate recognition and catalysis (Lu et al. 2014).

Transmembrane electron flow reactions catalyzed by Cytb561 family members are:

(1) L-ascorbate (cytosol) + Enzyme (oxidized) (synaptic vesicle) → dehydroascorbate (cytosol) + Enzyme (reduced) (synaptic vesicle)

(2) L-ascorbate (cytosol) + 2Fe3+ (intestinal lumen) → dehydroascorbate (cytosol) + 2Fe2+ (intestinal lumen)

This family belongs to the MFS Superfamily.



Bashtovyy, D., A. Bérczi, H. Asard, and T. Páli. (2003). Structure prediction of the di-heme cytochrome b561 protein family. Protoplasma 221: 31-40.

Bérczi, A. and L. Zimányi. (2014). The trans-membrane cytochrome b561 proteins: structural information and biological function. Curr. Protein. Pept. Sci. 15: 745-760.

Glanfield, A., D.P. McManus, D.J. Smyth, E.M. Lovas, A. Loukas, G.N. Gobert, and M.K. Jones. (2010). A cytochrome b561 with ferric reductase activity from the parasitic blood fluke, Schistosoma japonicum. PLoS Negl Trop Dis 4: e884.

Kaplan, J. (2002). Mechanisms of cellular iron acquisition: another iron in the fire. Cell 111: 603-606.

Kimball, R.A. and M.H. Saier, Jr. (2002). Voltage-gated H+ channels associated with human phagocyte superoxide-generating NADPH oxidases: sequence comparisons, structural predictions, and phylogenetic analyses. Mol. Membr. Biol. 19: 137-147.

Lane, D.J. and A. Lawen. (2009). Ascorbate and plasma membrane electron transport--enzymes vs efflux. Free Radic Biol Med 47: 485-495.

Latunde-Dada, G.O., J. Van der Westhuizen, C.D. Vulpe, G.J. Anderson, R.J. Simpson, and A.T. McKie. (2002). Molecular and functional roles of duodenal cytochrome B (dcytb) in iron metabolism. Blood Cells Mol. Dis. 29: 356-360.

Lu, P., D. Ma, C. Yan, X. Gong, M. Du, and Y. Shi. (2014). Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase. Proc. Natl. Acad. Sci. USA 111: 1813-1818.

Lu, Q., J.P. Lu, X.D. Li, X.H. Liu, H. Min, and F.C. Lin. (2008). Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination. J Zhejiang Univ Sci B 9: 511-519.

McKie, A.T., D. Barrow, G.O. Latunde-Dada, A. Rolfs, G. Sager, E. Mudaly, M. Mudaly, C. Richardson, D. Barlow, A. Bomford, T.J. Peters, K.B. Raja, S. Shirali, M.A. Hediger, F. Farzaneh, and R.J. Simpson. (2001). An iron-regulated ferric reductase associated with the absorption of dietary iron. Science 291: 1755-1759.

McKie, A.T., G.O. Latunde-Dada, S. Miret, J.A. McGregor, G.J. Anderson, C.D. Vulpe, J.M. Wrigglesworth, and R.J. Simpson. (2002). Molecular evidence for the role of a ferric reductase in iron transport. Biochem. Soc. Trans. 30: 722-724.

Nakanishi, N., F. Takeuchi, and M. Tsubaki. (2007). Histidine cycle mechanism for the concerted proton/electron transfer from ascorbate to the cytosolic haem b centre of cytochrome b561: a unique machinery for the biological transmembrane electron transfer. J Biochem 142: 553-560.

Oakhill, J.S., S.J. Marritt, E.G. Gareta, R. Cammack, and A.T. McKie. (2008). Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism. Biochim. Biophys. Acta. 1777: 260-268.

Perin, M.S., V.A. Fried, C.A. Slaughter, and T.C. Sudhof. (1988). The structure of cytochrome b561, a secretory vesicle-specific electron transport protein. EMBO J. 7: 2697-2703.

Picco C., Scholz-Starke J., Naso A., Preger V., Sparla F., Trost P. and Carpaneto A. (2014). How are cytochrome b561 electron currents controlled by membrane voltage and substrate availability? Antioxid Redox Signal. 21(3):384-91.

Picco, C., J. Scholz-Starke, M. Festa, A. Costa, F. Sparla, P. Trost, and A. Carpaneto. (2015). Direct Recording of Trans-Plasma Membrane Electron Currents Mediated by a Member of the Cytochrome b561 Family of Soybean. Plant Physiol. 169: 986-995.

Stewart, M., P. Lau, G. Banks, R.S. Bains, E. Castroflorio, P.L. Oliver, C.L. Dixon, M.C. Kruer, D.M. Kullmann, A. Acevedo-Arozena, S.E. Wells, S. Corrochano, and P.M. Nolan. (2019). Loss of disrupts synaptic AMPA receptor function, and results in neurodevelopmental, motor, cognitive and electrographical abnormalities. Dis Model Mech 12:.

Su, D. and H. Asard. (2006). Three mammalian cytochromes b561 are ascorbate-dependent ferrireductases. FEBS J. 273: 3722-3734.

Su, D., J.M. May, M.J. Koury, and H. Asard. (2006). Human erythrocyte membranes contain a cytochrome b561 that may be involved in extracellular ascorbate recycling. J. Biol. Chem. 281: 39852-39859.

Verelst, W. and H. Asard. (2003). A phylogenetic study of cytochrome b561 proteins. Genome Biology 4: R38.

Zhang, D.L., D. Su, A. Bérczi, A. Vargas, and H. Asard. (2006). An ascorbate-reducible cytochrome b561 is localized in macrophage lysosomes. Biochim. Biophys. Acta. 1760: 1903-1913.


TC#NameOrganismal TypeExample

Cytochrome b561 with an N-terminal cellobiose dehydrogenase (CDH)-like domain and a C-terminal cytb561 - FRRS1-like domain, both containing heme binding sites (Bérczi and Zimányi 2014).

Animals (and plants)

Cytb561 of Bos taurus (P10897)

5.B.2.1.2Tumor suppressor (Tsp10 pending protein)Animals (and plants)Tsp10 of Mus musculus (NP_062694)

Duodenyl cytochrome b ferrireductase, Dcytb. Duodenyl L-Ascorbate (in):Fe3+ iron chelate (out) oxidoreduclase. Present in duodenal enterocyte brush boarder membranes (isoform 1); reduces dietary Fe3+ to Fe2+ to facilitate its transport into the mucosal cell. Binds two hemes non-covalently. Induced by iron deficiency. Also expressed in erythrocytes and respiratory epithelia (Lane and Lawen, 2009).  

Animals (and plants)

Duodenyl cytochrome b reductase of Homo sapiens (Q53TN4)


Secretory vesicle-specific (chromafin granule) cytochrome b561 (isoform 2)


Cytochrome b561 of Homo sapiens (P49447)


The Lysosomal ascorbate:ferric chelate oxidoreductase, Cytochrome b561 (isoform 3).


Lysosomal cytochrome b561 of Homo sapiens (Q6P1H1)


Ascorbate-specific transmembrane electron transporter 1 (Cytochrome b561-1; Zmb561)

PlantsZCYB of Zea mays

Cytochrome b561 ferric reductase (Glanfield et al. 2010).

Parasitic blood flukes

Cyt b561 of Schistosoma japonicum


TC#NameOrganismal TypeExample

Ferric chelate reductase


Ferric chelate reductase of Medicago truncatula


Cytochrome b561 ferric reductase with N-terminal DOMON-related domain (for heme or sugar binding).


Ferric reductase of Arabidopsis thaliana


Ferric-chelate reductase 1. Involved in neurotransmitter synthesis and dietary iron uptake. (Picco et al. 2014).


Ferric-chelate reductase 1 homologue of Drosophila melanogaster


Ferric chelate reductase homologue (N-terminus; 5 TMSs; TC# 5.B.2; may reduce Cu2+ to Cu+) fused to a Ctr copper transporter (C-terminus; 3 TMSs; Ctr family, TC# 1.A.56).


Ctr fusion protein of Galdieria sulphuraria


Uncharacterized protein of 535 aas and 5 C-terminal TMSs with an N-terminal DOMON domain and a C-terminal cytochrome b561 domain.

UP of Chlamydomonas reinhardtii


DOMON domain-containing protein FRRS1L precursor of 293 aas and 2 TMSs, one N-terminal and one central. Loss of Frrs1l disrupts synaptic AMPA receptor function, and results in neurodevelopmental, motor, cognitive and electrographical abnormalities (Stewart et al. 2019).

FRRS1L of Mus musculus


TC#NameOrganismal TypeExample

Uncharacterized protein of 403 aas and 6 TMSs in a 1 (N-termina) plus 5 TMS arrangement.

UP of Setosphaeria turcica (Northern leaf blight fungus) (Exserohilum turcicum)


Uncharacterized protein with an N-terminal TMS followed by a heme binding cytochrome domain of fungal cellubiose dehydrogenases and a C-terminal Cytochrome b-561 / ferric reductase 5 TMS transmembrane domain of 453 aas.

UP of Hypocrea virens (Gliocladium virens) (Trichoderma virens)


Uncharacterized putative cellobiose dehydrogenase of 476 aas and 6 (1 + 5) TMSs. These 5 TMSs may be homologous to those of the CBP family (9.B.306), but homology has not been established.

UP of Ophiocordyceps sinensis


Uncharacterized protein of 563 aas and 6 TMSs in a 1 + 300 residues, largely hydrophilic + 5 TMSs. This protein is homologous to the proteins in the family with TC# 9.B.57.1.1 showing extensive sequence similarity.  The latter protein is the Mtp1 protein, essential for conidiation and conidial germination, of 520 aas and 12 TMSs (Lu et al. 2008). It has an N-terminal Cytb561 domain and a Ytp1 C-terminal domain.

UP of Neurospora crassa


Uncharacterized protein of 272 aas and 6 TMSs

UP of Glycine soja