Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase.
Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase.
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探究质子耦合电子转移至双氧的机制：牛血清胺氧化酶的氧化半反应。
DOI：
10.1021/bi981103l
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Klinman,JP
中科院分区：
文献类型：
--
作者：
Su,Q;Klinman,JP
牛血清胺氧化酶（BSAO）催化伯胺的氧化脱胺，同时通过乒乓机制将分子氧还原为过氧化氢。为分析二氧转化为过氧化氢的化学和动力学机制，已经制定了一套方案。稳态动力学表明，两个基团需要去质子化才能促进氧化半反应。vmax /Km（O2）随pH变化的pKa值分别为6.2±0.3和7.0±0.2。用紫外-可见分光光度法测定厌氧还原BSAO的pKaof为7.2±0.1。从还原酶滴定中获得的pka2与从稳态动力学中获得的pka2几乎一致，这表明第二个pka2是由还原的辅因子产生的。在厌氧条件下，EPR光谱检测到辅因子半醌信号形成的pH依赖性支持pKais的分配。为了解决氧化半反应中限速步骤的性质，确定了溶剂同位素效应、粘度效应和O-18同位素对Vmax/Km（O2）的影响。溶剂同位素效应与统一性难以区分，排除了质子转移作为速率决定步骤的可能性。使用葡萄糖作为溶剂粘原没有显示出粘度效应，这表明氧的结合不在速率决定步骤中。O-18的动力学同位素效应与pH无关，其平均值为18(V/K) = 1.0097±0.0010。这已经与计算出的平衡O-18同位素对各种双氧中间物种的影响进行了比较[Tian and Klinman (1993)J。点。化学。Soc. 115, 8891]，从而得出结论，第一电子转移到双氧或产物过氧化物从Cu(II)−OOH配合物的解吸可能是限速步骤。因此，通过停流实验和苯胺氧化dvandd （V/K）分析相结合的方法来分析稳态酶的分布。我们得出结论，在稳定状态下积累的主要物质是氧化的辅因子-底物希夫碱配合物和还原的氨基喹啉辅因子。这些数据排除了氨基醌形式的酶缓慢释放产物氢过氧化物的可能性，从而得出结论，从底物还原辅因子到二氧的第一个电子转移是氧化半反应的速率决定步骤。这一步也估计有40%的不朱率限制。从这项研究中得出的一个重要的机制结论是，双氧结合是形成超氧化物的限速电子转移步骤的一个单独步骤。根据最近确定的多态汉氏菌酵母胺氧化酶活性形式的x射线结构[Li et al. (1998) structure 6,293]，在还原辅因子的O-2位置附近确定了一个疏水空间，作为假定的双氧结合位点。在电子进一步从辅因子转移到超氧化物之前，超氧化物可能会从这个位点转移到活性位点的Cu（II）上。
Bovine serum amine oxidase (BSAO) catalyzes the oxidative deamination of primary amines, concomitant with the reduction of molecular oxygen to hydrogen peroxide via a ping-pong mechanism. A protocol has been developed for an analysis of chemical and kinetic mechanisms in the conversion of dioxygen to hydrogen peroxide. Steady-state kinetics show that two groups need to be deprotonated to facilitate the oxidative half-reaction. The pH dependence ofVmax/Km(O2) reveals pKa's of 6.2 ± 0.3 and 7.0 ± 0.2, respectively. A pKaof 7.2 ± 0.1 has been obtained from a titration of anaerobically reduced BSAO using UV−vis spectrophotometry. The near identity of the pKaobtained from the reduced enzyme titration with the second pKafrom steady-state kinetics suggests that this second pKaarises from the reduced cofactor. The assignment of pKais supported by the observed pH dependence for formation of the cofactor semiquinone signal, detected by EPR spectroscopy under anaerobic conditions. To address the nature of rate-limiting steps in the oxidative half-reaction, the solvent isotope effect, viscosity effect, and O-18 isotope effect on Vmax/Km(O2) have been determined. The solvent isotope effect is indistinguishable from unity, ruling out a proton transfer as a rate-determining step. Use of glucose as a solvent viscosogen shows no viscosity effect, indicating that binding of oxygen is not in the rate-determining step. The O-18 kinetic isotope effect is independent of pH with an average value of18(V/K) = 1.0097 ± 0.0010. This has been compared to calculated equilibrium O-18 isotope effects for various dioxygen intermediate species [Tian and Klinman (1993)J. Am. Chem. Soc. 115, 8891], leading to the conclusion that either the first electron transfer to dioxygen or the desorption of product peroxide from a Cu(II)−OOH complex could be the rate-limiting step. The distribution of steady-state enzyme species was, therefore, analyzed through a combination of stopped-flow experiments and analysis ofDVandD(V/K) for benzylamine oxidation. We conclude that the major species accumulating in the steady state are the oxidized cofactor−substrate Schiff base complex and the reduced, aminoquinol form of cofactor. These data rule out a slow release of product hydroperoxide from the aminoquinone form of enzyme, leading to the conclusion that the first electron transfer from substrate-reduced cofactor to dioxygen is the rate-determining step in the oxidative half-reaction. This step is also estimated to be 40% rate-limiting inkcat. An important mechanistic conclusion from this study is that dioxygen binding is a separate step from the rate-limiting electron-transfer step to form superoxide. On the basis of a recently determined X-ray structure for the active form of a yeast amine oxidase fromHansenula polymorpha[Li et al. (1998)Structure 6, 293], a hydrophobic space has been identified near the O-2 position of reduced cofactor as the putative dioxygen binding site. Movement of superoxide from this site onto the Cu(II) at the active site may occur prior to further electron transfer from cofactor to superoxide.