Roles of conserved Arg(72) and Tyr(71) in the ascorbate-specific transmembrane electron transfer catalyzed by Zea mays cytochrome b561.

Cytochromes b561, novel transmembrane electron transport proteins residing in eukaryotic cells, have a number of common features including six transmembrane α-helices and two heme ligation sites. Our recent studies on recombinant Zea mays cytochrome b561 suggested that concerted proton/electron transfer mechanism was functioning in plant cytochromes b561 as well and that conserved Lys(83) on a cytosolic loop had important roles for ascorbate-binding and a succeeding electron transfer. In the present study, we conducted site-directed mutagenesis analyses on conserved Arg(72) and Tyr(71).

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.