Purification and Biochemical Analyses of Zea mays Cytochrome ь561 Heterologously Expressed in Pichia pastoris

Cytochromes
ь
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 α-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
ь
₅₆₁
. In the present study, we conducted a molecular cloning of cytochrome
ь
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
ь
₅₆₁
protein (WTZM
b₅₆₁
-H
₆
) showed characteristic visible absorption peaks very similar to those of bovine cytochrome
ь₅₆₁
. The results from a stopped-flow analysis indicated that
Zea mays
cytochrome
ь₅₆₁
utilizes ascorbate and, possibly, monodehydroascorbate radical as a physiological electron donor and acceptor, respectively. Pre-treatment of the purified
Zea mays
cytochrome
ь₅₆₁
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
ь₅₆₁
might perform an ascorbate-related transmembrane electron transfer reaction by utilizing a very similar molecular mechanism with that of bovine cytochrome
ь₅₆₁
. Our new system offers an improvement in yield and other advantages over existing insect and yeast cell systems for producing the recombinant cytochrome
ь₅₆₁
for the studies on structure and functions.