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The Armstrong Research Group
Inorganic Chemistry Laboratory

Third floor ICL, rooms: T7–T12, T17
Phone: T12 (Fraser’s office): +44 (0)1865 272647
e-mail: fraser.armstrong@chem.ox.ac.uk




Jump to : Protein Film Electrochemistry  | Molecular Biology  | EPR Spectroscopy  |


Protein Film Electrochemistry
In a Protein Film Electrochemistry (PFE) experiment, the protein to be studied is typically adsorbed onto an electrode surface as a sub-monolayer (ca. 10‑12 mol cm‑2) under which conditions it ideally still functions as in vivo. Redox processes occurring at the protein cause a transfer of electrons between the electrode and adsorbed protein, setting up a potential difference at the electrode/solution interface. This potential difference is monitored by reading the potential difference between two electrodes: the one of interest (known as the working electrode), and a reference electrode, such as the standard hydrogen electrode (SHE) or standard calomel electrode (SCE), for which a precise value of the potential difference at the electrode/solution interface is known. A counter electrode (typically platinum) is also used to overcome the voltage drop caused by the resistance of the electrolyte solution to the passage of electrons between the working and reference electrodes.

Protein Film Electrochemistry offers many advantages over conventional solution electrochemical techniques. In addition to the low sample volumes necessary, the fact that the oxidation state of the whole sample can be altered instantaneously allows the kinetics of electron transfer reactions and activation/inactivation processes to be revealed.

Cyclic Voltammetry and Chronoamperometry

In a cyclic voltammetry experiment, the working electrode potential is swept linearly back and forth between two limits at the desired scan rate, whilst the current, i, is recorded as a function of the applied potential, E, to produce a cyclic voltammogram (also known as a 'scan'). Cyclic voltammetry is a useful technique for the rapid initial characterisation of many different proteins, and can reveal interesting features in the catalytic profile, inactivation/reactivation processes, and can also be used to evaluate the response of the protein to the presence of inhibitors. In a chronoamperometry experiment, the current is monitored at constant applied potential as a function of time. The electrode potential can be 'stepped' from one value to another extremely rapidly, and resulting changes in the catalytic current are monitored. Chronoamperometric experiments are very useful in probing the rates of inactivation/reactivation processes and in evaluating the effect of inhibitors on catalysis.
Molecular Biology
We use standard molecular biology techniques to tag enzymes for affinity purification; this allows the facile purification of pure protein for PFE and spectroscopic characterisation. Additionally, site-directed mutagenesis is being used to probe the effect of specific alterations in the amino acid sequence of hydrogenase enzymes. By comparing the electrochemical and spectroscopic characteristics of particular mutants with those of the wild-type enzyme, we are able to understand further how certain properties of a hydrogenase, such as catalytic rate, catalytic bias, substrate affinity, and sensitivity to inhibitors including oxygen, is related to the primary structure of the protein.
Electron Paramagnetic Resonance Spectroscopy
Electron paramagnetic resonance (EPR) is a powerful tool for obtaining structural information about systems with paramagnetic centres. In the complex enzymes we study, information can selectively be obtained about the environment around the paramagnetic centres (for example by measurement of hyperfine couplings) and long range order (up to ~60 Å) by distance measurements between electrons, which may be part of the active site or the 'naturally' paramagnetic metal clusters of metalloenzymes. Because of its ability to provide direct structural information, EPR perfectly complements the other physical and biological techniques that we use to study complex enzymes. We use both continuous wave and pulsed EPR techniques at X-band (9.5 GHz) and W-band (95 GHz) frequencies. More information about our EPR facilities can be found at the Oxford CAESR website.