Purification and Biochemical Analyses of Zea mays Cytochrome b561 Heterologously Expressed in Pichia pastoris

Cytochromes b ₅₆₁ constitute a novel class of transmembrane electron transport proteins present in large variety of eukaryotic cells, with a number of highly relevant common structural features including the six hydrophobic transmembrane alpha-helices and the two heme ligation sites. Of particular interest is the presence of a number of plant homologues that encode proteins having possible ascorbate- and monodehydroascorbate radical-binding sites proposed previously for mammalian cytochromes b ₅₆₁. In the present study, we conducted a molecular cloning of cytochrome b ₅₆₁ cDNA from corn plant, Zea mays, its functional heterologous expression in yeast Pichia pastoris, its purification, and its biochemical analyses. The purified recombinant Zea mays cytochrome b ₅₆₁ protein (WTZMb₅₆₁-H₆) showed characteristic visible absorption peaks very similar to those of bovine cytochrome b₅₆₁. The results from a stopped-flow analysis indicated that Zea mays cytochrome b₅₆₁ utilizes ascorbate and, possibly, monodehydroascorbate radical as a physiological electron donor and acceptor, respectively. Pre-treatment of the purified Zea mays cytochrome b₅₆₁ with diethylpyrocarbonate in the oxidized form caused a drastic inhibition of the electron transfer from ascorbate and such inhibition was protected by the presence of ascorbate during the treatment with diethylpyrocarbonate. These results suggested that plant cytochrome b₅₆₁ might perform an ascorbate-related transmembrane electron transfer reaction by utilizing a very similar molecular mechanism with that of bovine cytochrome b₅₆₁. Our new system offers an improvement in yield and other advantages over existing insect and yeast cell systems for producing the recombinant cytochrome b₅₆₁ for the studies on structure and functions.