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

Cytochrornes 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 may cytochrome b(561) obtained by a heterologous expression system using yeast Pichia pastoris cells could utilize the 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 round to have a very important role(s) for the binding of ascorbate and the succeeding electron transfer via electrostatic interactions based oil 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. Oil the other hand, pulse radiolysis experiments Oil 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 Set 118 and Trp(122) do not have major roles For the redox events oil the Intravesicular side.