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

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6 Author(s)

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.

Published in:

Micro-NanoMechatronics and Human Science, 2007. MHS '07. International Symposium on

Date of Conference:

11-14 Nov. 2007