Inhibition of Electron Acceptance from Ascorbate by the Specific N-carbethoxylations of Maize Cytochrome b561: A Common Mechanism for the Transmembrane Electron Transfer in Cytochrome b561 Protein Family

Abstract

Cytochromes b(561) constitute a novel class of proteins in eukaryotic cells with a number of highly relevant common features including six transmembrane alpha-helices and two haem groups. Of particular interest is the presence of a large number of plant homologues having putative ascorbate- and monodehydroascorbate radical-binding sites. We conducted a diethylpyrocarbonate-modification study employing Zea mays cytochrome b(561) heterologously expressed in Pichia pastoris cells. Pre-treatment of cytochrome b(561) with diethylpyrocarbonate in oxidized form caused N-carbethoxylation of His(86), His(159) and Lys(83), leading to a drastic inhibition of the electron transfer from ascorbate. The activity was protected by the inclusion of ascorbate during the treatment. However, midpoint potentials of two haem centres did show only slight decreases upon the treatment, suggesting that changes in the midpoint potentials were not the major cause of the inhibition. Present results indicated that Zea mays cytochrome b(561) conducted an ascorbate-specific transmembrane electron transfer by utilizing a concerted H(+)/e(-) transfer mechanism and that the specific N-carbethoxylation of haem axial His(86) that would inhibit the removal of a proton from the bound ascorbate was a major cause of the inhibition. On the other hand, Lys(83) might be important for an initial step(s) of the fast electron acceptance from ascorbate.

Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase

“…This analysis also suggests that two key mutations from either side, K81A/R150A on the cytoplasmic side or F105W/H106E on the noncytoplasmic side, are sufficient for abrogating electron transfer from the corresponding side. Notably, an important role of the amino acid that corresponds to Lys81 of Cyt b 561 has previously been reported for ZmCyt b 561 and bovine CGCyt b 561 , where mutation or chemical modification of the corresponding residue was shown to affect electron acceptance from ascorbate in a negative manner (11,13,22).…”

“…Expression levels of DCyt b 561 in the duodenal mucosa cell membrane are closely associated with iron metabolism disorders, such as chronic anemia and iron-deficiency anemia (19). Similar to CGCyt b 561 , ZmCyt b 561 uses ascorbate and the monodehydroascorbate radical as the physiological electron donor and acceptor, respectively (11,21,22).Despite rigorous investigation, there is no detailed structural information for any member of the Cyt b 561 family. Consequently, the electron transfer path is yet to be identified and the molecular mechanisms of substrate recognition and catalysis remain largely mysterious.…”

“…Expression levels of DCyt b 561 in the duodenal mucosa cell membrane are closely associated with iron metabolism disorders, such as chronic anemia and iron-deficiency anemia (19). Similar to CGCyt b 561 , ZmCyt b 561 uses ascorbate and the monodehydroascorbate radical as the physiological electron donor and acceptor, respectively (11,21,22).…”

High-yield production, purification and characterization of functional human duodenal cytochrome b in an Escherichia coli system

“…This leads to the conclusion that ascorbate reacts more quickly at b H than b L . It is interesting to note that other members of the cyt b 561 family also show multi-phase ascorbate reaction kinetics [23,25,26]. Further study will be needed to determine if the kinetic selectivity between the b H and b L heme centers observed here for Dcytb is a general feature of cyt b 561 family members.…”

Properties of Human Tumor Suppressor 101F6 Protein as a Cytochrome b561 and Its Preliminary Crystallization Trials

“…It was proposed that, compared to insect cells (e.g., Sf9 cell) or other mammalian cultured cells, Pichia pastoris cells are much easier to handle, can be grown at lower cost, and can be expressed quicker in a large scale (Asada et al, 2011). Such successful examples for expressing eukaryotic membrane proteins were reported previously (Weiß et al, 1998;Wetterholm et al, 2008;Nakanishi et al, 2009a;Nakanishi et al, 2009b;Alisio & Mueckler, 2010;Ostuni et al, 2010;Mizutani et al, 2011).…”