Catalytic Turnover of [FeFe]-Hydrogenase Based on Single-Molecule Imaging

• Published in: Journal of the American Chemical Society 2012-01, Vol.134 (3), p.1577-1582
• Main Authors: Madden, Christopher, Vaughn, Michael D, Díez-Pérez, Ismael, Brown, Katherine A, King, Paul W, Gust, Devens, Moore, Ana L, Moore, Thomas A
• Format: Article
• Language: English
• Subjects: BASIC BIOLOGICAL SCIENCES; Clostridium acetobutylicum - chemistry; Clostridium acetobutylicum - enzymology; Clostridium acetobutylicum - metabolism; electrochemical; Electrochemical Techniques; FeFe; Hydrogen - metabolism; hydrogenase; Hydrogenase - chemistry; Hydrogenase - metabolism; Iron - chemistry; Models, Molecular; photoelectrochemical
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja207461t

Hydrogenases catalyze the interconversion of protons and hydrogen according to the reversible reaction: 2H+ + 2e– ⇆ H2 while using only the earth-abundant metals nickel and/or iron for catalysis. Due to their high activity for proton reduction and the technological significance of the H+/H2 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.