Inhibition of the Electron Transfer of Plant Cytochrome b561 by the Modification with Diethylpyrocarbonate: In Search of A Common Mechanism for the Transmembrane Electron Transfer from Ascorbate

Cytochromes b₅₆₁ constitute a novel class of ascorbate (AsA)-specific transmembrane electron transport proteins present in large variety of eukaryotic cells with a number of highly relevant common structural features including six hydrophobic transmembrane iquest-helices and two heme ligation sites. Of particular interest is the presence of a large number of plant homologues that encode proteins having possible AsA- and monodehydroascorbate (MDA) radical-binding sites proposed previously for mammalian cytochromes b₅₆₁. It is well established that pre-treatment of bovine cytochrome b₅₆₁ with diethylpyrocarbonate (DEPC) caused a significant inhibition of the electron transfer from AsA. In the present study, we conducted DEPC-modification studies employing Zea mays cytochrome b₅₆₁ heterologously-expressed in yeast Pichia pastoris cells. Pre-treatment of purified Zea mays cytochrome b₅₆₁ with DEPC in oxidized form caused a drastic inhibition of the electron transfer from AsA and such inhibition was protected by the presence of AsA during the treatment. Treatment of three site-specific mutants (K83A, Y71A, R72D) with DEPC showed a significant inhibitory effect more or less on their electron acceptance from AsA. These results suggested that plant cytochrome b₅₆₁ might perform an AsA-related transmembrane electron transfer by utilizing a very similar molecular mechanism (concerted H⁺/e_- transfer mechanism) with that of animal cytochrome b₅₆₁ and further that the specific N-carbethoxylation of the heme axial His residue(s) was the major cause of the inhibition of electron acceptance upon the DEPC-treatment.