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 α-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 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.