Stopped-Flow Analyses on the Reaction of Ascorbate with Cytochrome b561 Purified from Bovine Chromaffin Vesicle Membranes

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• 作者：Tadakazu Takigami ; Fusako Takeuchi ; Masashi Nakagawa ; Toshiharu Hase ; and Motonari Tsubaki
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：July 15, 2003
• 年：2003
• 卷：42
• 期：27
• 页码：8110 - 8118
• 全文大小：133K
• 年卷期：v.42,no.27(July 15, 2003)
• ISSN：1520-4995

文摘

Cytochrome b₅₆₁ in adrenal chromaffin vesicle membranes conveys electron equivalents fromextravesicular ascorbate to the intravesicular monodehydroascorbate radical. We conducted a stopped-flow study on the reaction of ascorbate with purified cytochrome b₅₆₁ in the detergent-solubilized statefor the first time. The time course of the reduction of oxidized cytochrome b₅₆₁ with ascorbate could notbe 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 b₅₆₁ contains two hemes b, each having a distinctredox potential and a function upon reactions with ascorbate and monodehydroascorbate radical. Thefastest phase, which was assigned to the first one-electron donation from ascorbate to heme b on theextravesicular side, was further analyzed by transient phase kinetics employing a two-step bi-uni sequentialordered 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 dueto a pH-dependent transient state before the first electron transfer to take place. The fastest phase wascompletely lost by N-carbethoxylation of heme-coordinating histidyl residues (His88 and His161) andLys85 upon treatment with diethylpyrocarbonate. The presence of ascorbate during the treatment inhibitedthe 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 importantrole of the conserved Lys85 for the interaction with ascorbate.