Catalytic Turnover of [FeFe]-Hydrogenase Based on Single-Molecule Imaging
Catalytic Turnover of [FeFe]-Hydrogenase Based on Single-Molecule Imaging
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DOI：
10.1021/ja207461t
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发表时间：
2012-01-25
影响因子：
15
通讯作者：
Moore, Thomas A.
中科院分区：
文献类型：
--
作者：
Madden, Christopher;Vaughn, Michael D.;Moore, Thomas A.
氢化酶催化质子和氢的相互转化，根据可逆反应：2H(+) + 2e（-）可逆箭头H-2，而只使用地球上丰富的金属镍和/或铁进行催化。由于[FeFe]-加氢酶具有较高的质子还原活性和H+/H-2半反应的技术意义，因此利用生化和电化学技术表征[FeFe]-加氢酶的催化活性具有重要意义。在对乙酰丁酸梭菌（CaHydA）的[FeFe]-氢化酶进行了详细的电化学和光电化学研究之后，我们现在报告了对催化活性氢化酶制备进行的电化学和单分子成像研究。CaHydA酶是巴氏梭菌（Clostridium pasteurianum, CpI） [FeFe]-氢化酶的同源物（70%同源性），吸附在带负电荷的自组装单层（SAM）上，通过电化学扫描隧道显微镜（EC-STM）技术和宏观电化学测量进行了研究。EC-STM成像显示均匀的表面覆盖，具有足够的稳定性，可以进行STM尖端的重复扫描以及其他电化学研究。当电位被扫描到足够负时，循环伏安法产生一个特征阴极产氢信号。通过对Au-SAM表面上单个酶分布的直接观察，再加上从同一电极获得的宏观电化学测量，可以评估单个[FeFe]氢化酶分子的转换频率（TOF）作为电位的函数。
Hydrogenases catalyze the interconversion of protons and hydrogen according to the reversible reaction: 2H(+) + 2e(-) reversible arrow H-2 while using only the earthabundant metals nickel and/or iron for catalysis. Due to their high activity for proton reduction and the technological significance of the H+/H-2 half reaction, it is important to characterize the catalytic activity of [FeFe]-hydrogenases using both biochemical and electrochemical techniques. Following a detailed electrochemical and photoelectrochemical study of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaHydA), we now report electrochemical and single-molecule imaging studies carried out on a catalytically active hydrogenase preparation. The enzyme CaHydA, a homologue (70% identity) of the [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, was adsorbed to a negatively charged, self-assembled monolayer (SAM) for investigation by electrochemical scanning tunneling microscopy (EC-STM) techniques and macroscopic electrochemical measurements. The EC-STM imaging revealed uniform surface coverage with sufficient stability to undergo repeated scanning with a STM tip as well as other electrochemical investigations. Cyclic voltammetry yielded a characteristic cathodic hydrogen production signal when the potential was scanned sufficiently negative. The direct observation of the single enzyme distribution on the Au-SAM surface coupled with macroscopic electrochemical measurements obtained from the same electrode allowed the evaluation of a turnover frequency (TOF) as a function of potential for single [FeFe]-hydrogenase molecules.