Roles of conserved Arg^<72> and Tyr^<71> in the ascorbate-specific transmembrane electron transfer catalyzed by Zea mays cytochrome b_<561>(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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Cytochromes b_<561>, 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 b_<561> suggested that concerted proton/electron transfer mechanism was functioning in plant cytochromes b_<561> as well and that conserved Lys^<83> on a cytosollc 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>. Removal of a positive charge at Arg^<72> did not affect significantly on the final heme reduction level with ascorbate as reductant. However, characteristic pH-dependent initial time-lag upon electron acceptance from ascorbate was completely lost for R72A and R72E mutants. Substitution of Tyr^<71> with Ala or Phe affected both on the final heme reduction level and on the pH-dependent initial time-lag, causing acceleration of the electron transfer. These observations were interpreted as existence of specific interactions of Tyr^<71> and Arg^<72> with ascorbate. However, their mechanistic roles were distinctly different from that of Lys^<83>, as exemplified by K83A/Y71A double mutant, and might be related for expelling of monodehydroascorbate radical from the substrate-binding site to prevent a back-flow of electrons.
2013-05-00