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

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 rea...

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Bibliographic Details
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
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Summary: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.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja207461t