Radical formation in cytochrome c oxidase.
Radical formation in cytochrome c oxidase.
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DOI：
10.1016/j.bbabio.2011.06.012
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发表时间：
2011-10
期刊：
影响因子：
--
通讯作者：
Gerfen GJ
中科院分区：
文献类型：
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作者：
Yu MA;Egawa T;Shinzawa-Itoh K;Yoshikawa S;Yeh SR;Rousseau DL;Gerfen GJ
在 O2 氧化还原化学和质子易位过程中，牛细胞色素 c 氧化酶 (bCcO) 中自由基的形成是一个尚未解决的争议问题。为了确定单周转条件下 bCcO 催化反应中是否形成自由基，在定制的快速冷冻猝灭 (RFQ) 装置中引发 O2 与酶的反应，并被抗坏血酸盐或连二亚硫酸盐还原，产物在 77 K 下被捕获，反应时间为 50 µs 至 6 ms。手工混合额外的样品以获得多种周转条件，并在几分钟的反应时间内淬灭。反应产物的 X 波段 (9 GHz) 连续波电子顺磁共振 (CW-EPR) 谱显示，两种还原剂均形成窄自由基。 D 波段 (130 GHz) 脉冲 EPR 谱可以确定 g 张量主值，并揭示当使用抗坏血酸盐作为还原剂时，主要自由基种类位于抗坏血酸基部分上，而当使用连二亚硫酸盐作为还原剂时，自由基是 SO2•− 离子。当从光谱中减去还原剂的贡献时，在 O2 与还原的 bCcO 的反应中没有发现基于蛋白质的自由基的证据。作为反应中间体上形成的自由基的替代物，通过使用 RFQ 装置在 50 µs 捕获产物来研究过氧化氢 (H2O2) 与氧化 bCcO 在 pH 6 和 pH 8 下的反应，以确定初始反应事件。为了进行比较，还检查了孵育几分钟后形成的自由基，并且 X 带和 D 带分析导致了 Tyr-244 和 Tyr-129 上自由基的鉴定。在 RFQ 测量中，过氧化氢 (R – O – O•) 物质可能是由 O2 与氨基酸基之间的反应形成的。据推测，Tyr-129 可能在质子易位过程中发挥核心作用，通过在自由基物质形成时喷射质子，然后在其还原过程中通过源自血红素 a3 丙酸基团区域的三个水分子链重新质子化。
The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 µs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO2•− ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O2 with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H2O2) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50 µs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (R – O – O•) species was formed, presumably by the reaction between O2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a3.