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

Cytochromes b561 are a family of transmembrane proteins found in many eukaryotic cells (animals and plants). 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, each on opposite sides of the membrane. 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). Mammalian cytochrome b5 (cyt b5) and cytochrome b5 reductase (b5R) are electron carrier proteins for membrane-embedded oxidoreductases. Both b5R and cyt b5 have a cytosolic domain and a single TMS. The cytosolic domains of b5R and cyt b5 contain cofactors required for electron transfer. b5R and cyt b5 form a stable binary complex, and so do cyt b5 and stearoyl-CoA desaturase-1 (SCD1). b5R, cyt b5 and SCD1 form a stable ternary complex, and the TMSs are required for the assembly of stable binary and ternary complexes where electron transfer rates are greatly enhanced (Shen et al. 2022).

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: Major Facilitator (MFS) Superfamily.

References associated with 5.B.2 family:

Bashtovyy, D., A. Bérczi, H. Asard, and T. Páli. (2003). Structure prediction of the di-heme cytochrome b561 protein family. Protoplasma 221: 31-40. 12768339
Bérczi, A. and L. Zimányi. (2014). The trans-membrane cytochrome b561 proteins: structural information and biological function. Curr. Protein. Pept. Sci. 15: 745-760. 25163754
Bérczi, A., Z. Márton, K. Laskay, A. Tóth, G. Rákhely, &.#.1.9.3.;. Duzs, K. Sebők-Nagy, T. Páli, and L. Zimányi. (2023). Spectral and Redox Properties of a Recombinant Mouse Cytochrome 561 Protein Suggest Transmembrane Electron Transfer Function. Molecules 28:. 36903505
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. 21103361
Gradogna, A., L. Lagostena, S. Beltrami, E. Tosato, C. Picco, J. Scholz-Starke, F. Sparla, P. Trost, and A. Carpaneto. (2023). Tonoplast cytochrome b561 is a transmembrane ascorbate-dependent monodehydroascorbate reductase: functional characterisation of electron currents in plant vacuoles. New Phytol. [Epub: Ahead of Print] 36806214
Kaplan, J. (2002). Mechanisms of cellular iron acquisition: another iron in the fire. Cell 111: 603-606. 12464171
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. 12126231
Lane, D.J. and A. Lawen. (2009). Ascorbate and plasma membrane electron transport--enzymes vs efflux. Free Radic Biol Med 47: 485-495. 19501649
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. 12547225
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. 24449903
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. 18600780
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. 11230685
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. 12196176
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. 17905810
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. 18194661
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. 2460342
Perozzo, A.M., P.M.G.E. Brown, and D. Bowie. (2023). Alternative splicing of the flip/flop cassette and TARP auxiliary subunits engage in a privileged relationship that fine-tunes AMPA receptor gating. J. Neurosci. [Epub: Ahead of Print] 36931708
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. 24410448
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. 26282237
Shen, J., G. Wu, A.L. Tsai, and M. Zhou. (2022). Transmembrane helices mediate the formation of a stable ternary complex of bR, cyt b, and SCD1. Commun Biol 5: 956. 36097052
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:. 30692144
Su, D. and H. Asard. (2006). Three mammalian cytochromes b561 are ascorbate-dependent ferrireductases. FEBS J. 273: 3722-3734. 16911521
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. 17068337
Verelst, W. and H. Asard. (2003). A phylogenetic study of cytochrome b561 proteins. Genome Biology 4: R38. 12801412
Verelst, W. and H. Asard. (2004). Analysis of an Arabidopsis thaliana protein family, structurally related to cytochromes b561 and potentially involved in catecholamine biochemistry in plants. J Plant Physiol. 161: 175-181. 15022831
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. 16996694