title: Inhibition of electron acceptance from ascorbate by the specific N-carbethoxylations of maize cytochrome b561: a common mechanism for the transmembrane electron transfer in cytochrome b561 protein family.
abstract: Cytochromes b(561) constitute a novel class of proteins in eukaryotic cells with a number of highly relevant common features including six transmembrane alpha-helices and two haem groups. Of particular interest is the presence of a large number of plant homologues having putative ascorbate- and monodehydroascorbate radical-binding sites. We conducted a diethylpyrocarbonate-modification study employing Zea mays cytochrome b(561) heterologously expressed in Pichia pastoris cells. Pre-treatment of cytochrome b(561) with diethylpyrocarbonate in oxidized form caused N-carbethoxylation of His(86), His(159) and Lys(83), leading to a drastic inhibition of the electron transfer from ascorbate. The activity was protected by the inclusion of ascorbate during the treatment. However, midpoint potentials of two haem centres did show only slight decreases upon the treatment, suggesting that changes in the midpoint potentials were not the major cause of the inhibition. Present results indicated that Zea mays cytochrome b(561) conducted an ascorbate-specific transmembrane electron transfer by utilizing a concerted H(+)/e(-) transfer mechanism and that the specific N-carbethoxylation of haem axial His(86) that would inhibit the removal of a proton from the bound ascorbate was a major cause of the inhibition. On the other hand, Lys(83) might be important for an initial step(s) of the fast electron acceptance from ascorbate.
authors.affiliation: Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Nakanishi
authors.forename: Md Motiur
authors.initials: MM
authors.lastname: Rahman
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Sakamoto
authors.forename: Masahiro
authors.initials: M
authors.lastname: Miura
authors.forename: Fusako
authors.initials: F
authors.lastname: Takeuchi
authors.forename: Sam-Yong
authors.initials: SY
authors.lastname: Park
authors.forename: Motonari
authors.initials: M
authors.lastname: Tsubaki
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000378:metabolism / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
chemical_list: D003573:Cytochrome b Group; D008565:Membrane Proteins; D011994:Recombinant Proteins; C023646:cytochrome b561; D004047:Diethyl Pyrocarbonate; D001205:Ascorbic Acid