Monitoring catalysis of the membrane-bound hydrogenase from Ralstonia eutropha H16 by surface-enhanced IR absorption spectroscopy.
Monitoring catalysis of the membrane-bound hydrogenase from Ralstonia eutropha H16 by surface-enhanced IR absorption spectroscopy.
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DOI：
10.1002/anie.200802633
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发表时间：
2009-01
影响因子：
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通讯作者：
N. Wisitruangsakul;O. Lenz;M. Ludwig;B. Friedrich;F. Lendzian;P. Hildebrandt;I. Zebger
中科院分区：
文献类型：
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作者：
N. Wisitruangsakul;O. Lenz;M. Ludwig;B. Friedrich;F. Lendzian;P. Hildebrandt;I. Zebger
[NiFe] 氢化酶构成一类酶，可催化分子氢 (H2) 的异解分裂以及逆反应，即质子还原为 H2。[1]催化位点是由四个保守的半胱氨酸残基桥接的双金属镍/铁络合物。此外，一个 CO 和两个 CNÀ 配体作为相当不寻常的外源配体与铁结合。虽然大多数这些酶仅在严格的厌氧条件下才有催化活性，但拉尔斯通尼亚属物种的[NiFe]氢化酶具有显着的耐氧性，其起源在分子水平上尚未完全了解。[2-4]这种特殊的耐氧性在过去引起了相当大的研究成果，其动机是阐明这些酶的催化机制及其对生物技术能量储存和转换的潜在重要性。此类应用需要将酶固定在导电载体上，同时保留天然结构和功能。 [5]事实上，Ralstonia 种膜结合氢化酶的成功固定化的首次报道已被报道。这些酶附着在热解石墨电极上，构建简单的酶燃料电池，即使在空气中氢气浓度低于 3% 的情况下也能运行。[2, 3] 为了研究固定在表面上的酶的性能，非常需要开发一种能够原位探测活性位点的分子结构及其在催化过程中变化的实验方法。红外光谱是通过探测 Ni-Fe 活性位点的 CO 和 CNÀ 配体的拉伸模式来识别催化循环各种状态的主要技术之一。这些模式的频率敏感地反映了催化中心内由于金属氧化态、连接模式和辅因子-蛋白质相互作用的改变而引起的电子密度的变化。因此，红外光谱已被广泛用于表征本体溶液中氢化酶的酶促过程，并与EPR光谱结合，为酶促过程的机理提供了重要的见解。[6-8]传统红外光谱的主要缺点是灵敏度相对较低，不足以研究固定化酶。表面增强红外吸收（SEIRA）光谱有望克服这一限制，因为固定在金表面上的蛋白质的红外吸收可以增强两个数量级。[9, 10]SEIRA光谱已成功应用于监测固定条件下金电极上的蛋白质固定和蛋白质氧化还原连接的结构变化，最近，在时间分辨领域。[11-14]在此，该技术首次应用于氢化酶，即富养产碱杆菌 H16 (Re MBH) 的膜结合氢化酶，附着在金表面。与之前对辅因子-蛋白质复合物的研究不同，SEIRA对氢化酶的光谱分析不限于蛋白质结构的变化，而是允许在光谱窗口中直接观察催化位点的特征标记带，而不受蛋白质的红外吸收带的干扰。 SEIRA 实验是在 Kretschmann ATR 配置中进行的，使用通过无电沉积涂有金膜的半圆柱形硅晶体。 [15]随后，金表面被自组装单层次氮基三乙酸镍 (Ni-NTA)[11, 16] 覆盖，用于 Re MBH 的亲和结合，并进行了修饰……
[NiFe] hydrogenases constitute a class of enzymes that catalyze the heterolytic splitting of molecular hydrogen (H2) as well as the reverse reaction, the reduction of protons to H2.[1] The catalytic site is a bimetallic Ni/Fe complex bridged by four conserved cysteine residues. Moreover, one CO and two CNÀ ligand bind to iron as rather unusual exogenous ligands. Whereas most of these enzymes are catalytically active only under strictly anaerobic conditions, the [NiFe] hydrogenases of Ralstonia species have a remarkable oxygen tolerance, the origin of which is not yet fully understood on the molecular level.[2–4] This particular oxygen tolerance has prompted considerable research effects in the past that were motivated by the interest in elucidating the catalytic mechanism of these enzymes and by their potential importance for biotechnological energy storage and conversion. Such applications require the immobilization of the enzymes on electrically conducting supports under preservation of the native structure and function.[5] In fact, the first demonstrations of successful immobilization have been reported for the membrane-bound hydrogenases of Ralstonia species. These enzymes were attached to pyrolytic graphite electrodes to build simple enzymatic fuel cells that operate even with H2 concentrations lower than 3% in air.[2, 3] To investigate the performance of enzymes immobilized on surfaces, it is highly desirable to develop an experimental approach that is capable of probing the molecular structure of the active site and their changes during the catalytic processes in situ. IR spectroscopy is one of the main techniques used for the identification of the various states of the catalytic cycle by probing the stretching modes of the CO and CNÀ ligands of the Ni–Fe active site. The frequencies of these modes sensitively reflect changes of the electron density within the catalytic center caused by alterations of the metal oxidation state, ligation pattern, and cofactor–protein interactions. Therefore, IR spectroscopy has been widely used for characterizing the enzymatic process of hydrogenases in bulk solution and, in conjunction with EPR spectroscopy, it has provided important insights into the mechanism of the enzymatic process.[6–8]The main drawback of conventional IR spectroscopy is the relatively low sensitivity, which is not sufficient for studying immobilized enzymes. Surface-enhanced infrared absorption (SEIRA) spectroscopy promises to overcome this limitation, as the IR absorption can be enhanced by up to two orders of magnitude for proteins immobilized on gold surfaces.[9, 10] SEIRA spectroscopy has already been successfully applied to monitor protein immobilization on gold electrodes and redox-linked structural changes of proteins under stationary conditions, and more recently, in the timeresolved domain.[11–14] Herein, this technique is applied for the first time to a hydrogenase, namely the membrane-bound hydrogenase of Ralstonia eutropha H16 (Re MBH), attached to a gold surface. Unlike previous studies of cofactor–protein complexes, the SEIRA spectroscopic analysis of hydrogenases is not restricted to changes of the protein structure, but allows the direct observation of the characteristic marker bands of the catalytic site in a spectral window, without interference from IR absorption bands of the protein. SEIRA experiments were carried out in a Kretschmann ATR configuration using a semicylindrical silicon crystal coated with a gold film by electroless deposition.[15] The gold surface was subsequently covered by a self-assembled monolayer of nickel nitrilotriacetic acid (Ni-NTA)[11, 16] for affinity binding of the Re MBH, which was modified …