Importance of the Conserved Lysine 83 Residue of Zea mays Cytochrome b561 for Ascorbate-Specific Transmembrane Electron Transfer As Revealed by Site-Directed Mutagenesis Studi

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• 作者：Nobuyuki Nakanishi ; Motiur Md. Rahman ; Yoichi Sakamoto ; Tadakazu Takigami ; Kazuo Kobayashi ; Hiroshi Hori ; Toshiharu Hase ; Sam-Yong Park ; Motonari Tsubaki
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：November 10, 2009
• 年：2009
• 卷：48
• 期：44
• 页码：10665-10678
• 全文大小：1048K
• 年卷期：v.48,no.44(November 10, 2009)
• ISSN：1520-4995

文摘

Cytochromes b₅₆₁, a novel class of transmembrane electron transport proteins residing in a large variety of eukaryotic cells, have a number of common structural features including six hydrophobic transmembrane α-helices and two heme ligation sites. We found that recombinant Zea mays cytochrome b₅₆₁ obtained by a heterologous expression system using yeast Pichia pastoris cells could utilize the ascorbate/mondehydroascorbate radical as a physiological electron donor/acceptor. We found further that a concerted proton/electron transfer mechanism might be operative in Z. mays cytochrome b₅₆₁ as well upon the electron acceptance from ascorbate to the cytosolic heme center. The well-conserved Lys⁸³ residue in a cytosolic loop was found to have a very important role(s) for the binding of ascorbate and the succeeding electron transfer via electrostatic interactions based on the analyses of three site-specific mutants, K83A, K83E, and K83D. Further, unusual behavior of the K83A mutant in pulse radiolysis experiments indicated that Lys⁸³ might also be responsible for the intramolecular electron transfer to the intravesicular heme. On the other hand, pulse radiolysis experiments on two site-specific mutants, S118A and W122A, for the well-conserved residues in the putative monodehydroascorbate radical binding site showed that their electron transfer activities to the monodehydroascorbate radical were very similar to those of the wild-type protein, indicating that Ser¹¹⁸ and Trp¹²² do not have major roles for the redox events on the intravesicular side.