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., 2006; Su 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)