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

Cytochromes b₅₆₁ constitute a novel class of proteins in eukaryotic cells with a number of highly relevant common features including six transmembrane α-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 b₅₆₁ heterologously expressed in Pichia pastoris cells. Pre-treatment of cytochrome b₅₆₁ with diethylpyrocarbonate in oxidized form caused N-carbethoxylation of His⁸⁶, His¹⁵⁹ and Lys⁸³, 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 b₅₆₁ conducted an ascorbate-specific transmembrane electron transfer by utilizing a concerted H⁺/e⁻ transfer mechanism and that the specific N-carbethoxylation of haem axial His⁸⁶ that would inhibit the removal of a proton from the bound ascorbate was a major cause of the inhibition. On the other hand, Lys⁸³ might be important for an initial step(s) of the fast electron acceptance from ascorbate.