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Electrochemistry of Metalloproteins: Protein Film Electrochemistry for the Study of E. coli [NiFe]-Hydrogenase-1

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Metalloproteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1122))

Abstract

Protein film electrochemistry is a technique which allows the direct control of redox-active enzymes, providing particularly detailed information on their catalytic properties. The enzyme is deposited onto a working electrode tip, and through control of the applied potential the enzyme activity is monitored as electrical current, allowing for direct study of inherent activity as electrons are transferred to and from the enzyme redox center(s). No mediators are used. Because the only enzyme present in the experiment is bound at the electrode surface, gaseous and liquid phase inhibitors can be introduced and removed whilst the enzyme remains in situ. Potential control means that kinetics and thermodynamics are explored simultaneously; the kinetics of a reaction can be studied as a function of potential. Steady-state catalytic rates are observed directly as current (for a given potential) and non-steady-state rates (such as interconversions between different forms of the enzyme) are observed from the change in current with time. The more active the enzyme, the higher the current and the better the signal-to-noise. In this chapter we outline the practical aspects of PFE for studying electroactive enzymes, using the Escherichia coli [NiFe]-hydrogenase 1 (Hyd-1) as an example.

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Acknowledgements

This research was supported by the Biological and Biotechnological Sciences Research Council (Grants BB/H003878-1 and BB/I022309-1). We thank Elena Nomerotskaia for help with protein purification.

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Authors and Affiliations

  1. Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QR, UK

    Rhiannon M. Evans & Fraser A. Armstrong

Authors
  1. Rhiannon M. Evans
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  2. Fraser A. Armstrong
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Correspondence to Fraser A. Armstrong .

Editor information

Editors and Affiliations

  1. Metalloproteins Unit, Institut de Biologie Structurale (IBS), Grenoble, France

    Juan C. Fontecilla-Camps

  2. Metalloproteins Unit, Institut de Biologie Structurale (IBS), Grenoble, France

    Yvain Nicolet

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Evans, R.M., Armstrong, F.A. (2014). Electrochemistry of Metalloproteins: Protein Film Electrochemistry for the Study of E. coli [NiFe]-Hydrogenase-1. In: Fontecilla-Camps, J., Nicolet, Y. (eds) Metalloproteins. Methods in Molecular Biology, vol 1122. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-794-5_6

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  • DOI: https://doi.org/10.1007/978-1-62703-794-5_6

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-793-8

  • Online ISBN: 978-1-62703-794-5

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