Importance of the conserved lysine 83 residue of Zea mays cytochrome b(561) for ascorbate-specific transmembrane electron transfer as revealed by site-directed mutagenesis studies.
Cytochromes b(561), 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 alpha-helices and two heme ligation sites. We found that recombinant Zea mays cytochrome b(561) 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 b(561) as well upon the electron acceptance from ascorbate to the cytosolic heme center. The well-conserved Lys(83) 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 Lys(83) 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 Ser(118) and Trp(122) do not have major roles for the redox events on the intravesicular side.
Nakanishi
Nobuyuki
N
Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Rahman
Motiur Md
MM
Sakamoto
Yoichi
Y
Takigami
Tadakazu
T
Kobayashi
Kazuo
K
Hori
Hiroshi
H
Hase
Toshiharu
T
Park
Sam-Yong
SY
Tsubaki
Motonari
M
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000378:metabolism*; D015153:Blotting, Western; D017124:Conserved Sequence; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D003313:Zea mays / Q000201:enzymology*
D003573:Cytochrome b Group; D011994:Recombinant Proteins; C023646:cytochrome b561; D008239:Lysine; D001205:Ascorbic Acid