Proton-Coupled Electron Transport in Two Distinct CYBASC Paralogs of Arabidopsis thaliana: A Comparative Characterization of Highly Conserved Tyrosine and Lysine Residues

Abstract

CYBASC proteins are ascorbate (AscH − ) reducible, diheme b-containing integral membrane cytochrome b 561 proteins (cytb 561 ), which are proposed to be involved in AscH − recycling and facilitation of iron absorption. Two distinct CYBASC paralogs from the plant Arabidopsis thaliana, Atcytb 561 -A (A-paralog) and Atcytb 561 -B (B-paralog), have been found to differ in their visiblespectral characteristics and their interaction with AscH − and ferric iron chelates. A previously determined crystal structure of the Bparalog provides the first insights into the structural organization of a CYBASC member and implies hydrogen bonding between the substrate AscH − and the conserved lysine residues at positions 77 (B-K77) and 81 (B-K81). The function of the highly conserved tyrosine at position 70 (B-Y70) is not obvious in the crystal structure, but its localization indicates the possible involvement in proton-coupled electron transfer. Here we show that B-Y70 plays a major role in the modulation of the oxidation−reduction midpoint potential of the high-potential heme, E M (b H ), as well as in AscH − oxidation. Our results support the involvement of the functionally conserved B-K77 in the stabilization of the dianion Asc 2− . These findings are supported by the crystal structure of the Bparalog, but a comparative biochemical and biophysical characterization of the A-and B-paralogs implied distinct and more complex functions of the corresponding residues A-Y69 and A-K76 in the A-paralog. Our results emphasize the need for a high-resolution crystal structure of the A-paralog to illuminate the differences in functional organization between the two paralogs.

The cysteine S–H stretching mode reports on long‐range conformational changes in pyruvate oxidase from E. coli

“…> 3 m m active sites). Before loading the cell, a redox mediator cocktail was added to the concentrated protein solutions to accelerate the otherwise slow redox reaction [47]. The final concentration of each mediator component was 25 μ m .…”

“…The thin‐layer spectroelectrochemical reflection cell was used as described elsewhere [29,47]. Before sample application, the working electrode was chemically coated by soaking the gold surface with 2 m m pyridine‐3‐carboxaldehyde thiosemicarbazone for at least 30 min, followed by rinsing with water.…”

“…With our spectroelectrochemical setup, it is in principle possible to perform stepwise potentiometric titrations [47], but here we focussed on the full two‐electron redox transition of the flavin. We triggered the FAD↔FADH 2 reaction (Fig.…”

Tonoplast cytochrome b561 is a transmembrane ascorbate‐dependent monodehydroascorbate reductase: functional characterization of electron currents in plant vacuoles

“…One CYBDOM isoform was experimentally localized on the plasma membrane (Picco et al ., 2015). Among CYB561s (also known as CYBASC for Asc‐reducible cytochromes b ; Preger et al ., 2005; Klein et al ., 2020), the isoform A (CYB561A) was localized on the tonoplast in Arabidopsis leaves (Griesen et al ., 2004) and in etiolated bean hypocotyls (Preger et al ., 2005), but on the plasma membrane in wild watermelon ( Citrullus lanatus ; Nanasato et al ., 2005). The intracellular location of the remaining three CYB561 isoforms (B‐D) is unknown but the crystal structure of CYB561B from Arabidopsis thaliana was solved (Lu et al ., 2014).…”

Comparison of insect and human cytochrome b561 proteins: Insights into candidate ferric reductases in insects

“…The well-characterized human duodenal cytochrome b561 (Dcytb) is a ferric reductase that facilitates iron uptake at the duodenal brush border, and it is also suspected to play other physiological roles [8][9][10]. The structurally well-characterized Arabidopsis thaliana (plant) cytochrome b561 B-paralog (AtCytb561-B) has in vitro ferric-chelate reductase activity, but its biological functions are unknown [11][12][13]. Other cytb561 family members with in vitro ferric reductase activity include mammalian stromal cell-derived receptor 2 (SDR2), tumor suppressor 101F6 (TScytb), chromaffin granule cytb561 (CGcytb), and lysosomal cytb561 (Lcytb), as well as Drosophila melanogaster (fly) CG8399, and A. thaliana AtCytb561-A [6,11,12,14,15].…”