title: Catalytic turnover of [FeFe]-hydrogenase based on single-molecule imaging.
abstract: Hydrogenases catalyze the interconversion of protons and hydrogen according to the reversible reaction: 2H(+) + 2e(-) ⇆ H(2) 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(+)/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.
authors.affiliation: Center for Bioenergy and Photosynthesis, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Madden
authors.forename: Michael D
authors.initials: MD
authors.lastname: Vaughn
authors.forename: Ismael
authors.initials: I
authors.lastname: Díez-Pérez
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Brown
authors.forename: Paul W
authors.initials: PW
authors.lastname: King
authors.forename: Devens
authors.initials: D
authors.lastname: Gust
authors.forename: Ana L
authors.initials: AL
authors.lastname: Moore
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Moore
mesh_terms: D046969:Clostridium acetobutylicum / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D055664:Electrochemical Techniques; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000737:chemistry; D008958:Models, Molecular
chemical_list: D006859:Hydrogen; D007501:Iron; D006864:Hydrogenase