title: Stopped-flow analyses on the reaction of ascorbate with cytochrome b561 purified from bovine chromaffin vesicle membranes.
abstract: Cytochrome b(561) in adrenal chromaffin vesicle membranes conveys electron equivalents from extravesicular ascorbate to the intravesicular monodehydroascorbate radical. We conducted a stopped-flow study on the reaction of ascorbate with purified cytochrome b(561) in the detergent-solubilized state for the first time. The time course of the reduction of oxidized cytochrome b(561) with ascorbate could not be fitted with a single exponential but with a linear combination of at least four exponential functions. This result is consistent with the notion that cytochrome b(561) contains two hemes b, each having a distinct redox potential and a function upon reactions with ascorbate and monodehydroascorbate radical. The fastest phase, which was assigned to the first one-electron donation from ascorbate to heme b on the extravesicular side, was further analyzed by transient phase kinetics employing a two-step bi-uni sequential ordered mechanism. The result showed K(s) = 2.2 mM for ascorbate at pH6.0. At a region below pH5.5, there was a significant lag before the reduction of hemes b occurred. This time lag was interpreted as due to a pH-dependent transient state before the first electron transfer to take place. The fastest phase was completely lost by N-carbethoxylation of heme-coordinating histidyl residues (His88 and His161) and Lys85 upon treatment with diethylpyrocarbonate. The presence of ascorbate during the treatment inhibited the N-carbethoxylation of the histidyl residues and, thereby, restored the final reduction level of hemes b. But the reduction rate was still only one-twentieth of the native form. This result suggested an important role of the conserved Lys85 for the interaction with ascorbate.
authors.affiliation: Department of Life Science, Graduate School and Faculty of Science, Himeji Institute of Technology, Kamigoori-cho, Akou-gun, Hyogo 678-1297, Japan.
authors.forename: Tadakazu
authors.initials: T
authors.lastname: Takigami
authors.forename: Fusako
authors.initials: F
authors.lastname: Takeuchi
authors.forename: Masashi
authors.initials: M
authors.lastname: Nakagawa
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Hase
authors.forename: Motonari
authors.initials: M
authors.lastname: Tsubaki
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration
chemical_list: D003573:Cytochrome b Group; C023646:cytochrome b561; D001205:Ascorbic Acid