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Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase.pdf (1 MB)

Protein Film Infrared Electrochemistry Demonstrated for Study of H2 Oxidation by a [NiFe] Hydrogenase.

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posted on 2019-06-18, 08:19 authored by Philip A. Ash, Ricardo Hidalgo, Kylie A. Vincent
Understanding the chemistry of redox proteins demands methods that provide precise control over redox centers within the protein. The technique of protein film electrochemistry, in which a protein is immobilized on an electrode surface such that the electrode replaces physiological electron donors or acceptors, has provided functional insight into the redox reactions of a range of different proteins. Full chemical understanding requires electrochemical control to be combined with other techniques that can add additional structural and mechanistic insight. Here we demonstrate a technique, protein film infrared electrochemistry, which combines protein film electrochemistry with infrared spectroscopic sampling of redox proteins. The technique uses a multiple-reflection attenuated total reflectance geometry to probe a redox protein immobilized on a high surface area carbon black electrode. Incorporation of this electrode into a flow cell allows solution pH or solute concentrations to be changed during measurements. This is particularly powerful in addressing redox enzymes, where rapid catalytic turnover can be sustained and controlled at the electrode allowing spectroscopic observation of long-lived intermediate species in the catalytic mechanism. We demonstrate the technique with experiments on E. coli hydrogenase 1 under turnover (H2 oxidation) and non-turnover conditions.


The work of K.A.V. and P.A.A. was supported by the European Research Council (EnergyBioCatalysis-ERC-2010-StG-258600), Engineering and Physical Sciences Research Council IB Catalyst award EP/N013514/1, and Biotechnology and Biological Sciences Research Council (BB/L009722/1 and BB/N006321/1). R.H. was supported by Ministerio de Ciencia y Tecnolog├Ča, Universidad de Costa Rica, and Lincoln College, Oxford. The authors acknowledge Mr. Charlie Jones, Mr. Charlie Evans and staff of the Mechanical Workshop (Department of Chemistry) for assistance in the design and manufacture of spectroelectrochemical cells used in this work.



Journal of Visualized Experiments, 2017 (130), e55858

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