Oriented Immobilization of a Membrane-Bound Hydrogenase onto an Electrode for Direct Electron Transfer
Oriented Immobilization of a Membrane-Bound Hydrogenase onto an Electrode for Direct Electron Transfer
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DOI：
10.1021/la200141t
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发表时间：
2011-05-17
期刊：
影响因子：
3.9
通讯作者：
De Lacey, Antonio L.
中科院分区：
文献类型：
--
作者：
Gutierrez-Sanchez, Cristina;Olea, David;De Lacey, Antonio L.
氧化还原酶与电极的相互作用对于研究氧化还原酶的催化机制和生物电子应用具有重要意义。由于后者蛋白质具有更高的结构复杂性和不稳定性，因此使用可溶性酶比使用膜酶在生物催化剂和电极之间进行有效的电子传输取得了更大的成功。在这项工作中，我们报告了一种将膜结合酶固定到金电极上的策略，其完全活性构象的方向受控。固定化氧化还原酶是来自 Desulfovibrio vulgaris Hildenborough 的 Ni-Fe-Se 氢化酶，它可逆地催化 H(2)-氧化，并通过脂质尾与细胞质膜相连。通过原子力显微镜 (AFM) 和电化学方法研究了用这种酶和磷脂修饰的金表面。综合研究表明，通过两步固定程序，氢化酶可以通过其脂质尾插入到金表面上形成的磷脂双层上，仅允许酶和电极之间介导的电子转移。然而，一步固定程序有利于在金表面上形成氢化酶单层，并将其脂质尾插入氢化酶分子顶部形成的磷脂双层中。后一种方法首次实现了天然构象的膜结合酶与电极之间的有效电子转移。
The interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble enzymes than with membrane enzymes because of the higher structural complexity and instability of the latter proteins. In this work, we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H(2)-oxidation reversibly and is associated with the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, allowing only mediated electron transfer between the enzyme and electrode. However, a one-step immobilization procedure favors the formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted into a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.